InteractiveFly: GeneBrief

p53 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - p53

Synonyms - CG10873, Dmp53

Cytological map position - 94D12

Function - transcription factor

Keywords - cell cycle, apoptosis, DNA repair. oncogene, response to DNA damage

Symbol - p53

FlyBase ID: FBgn0039044

Genetic map position -

Classification - p53 family

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Di Cara, F., et al. (2015). The Hippo pathway promotes cell survival in response to chemical stress. Cell Death Differ [Epub ahead of print]. PubMed ID: 26021298
Summary:
Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. To identify novel players acting in stress response pathways, a cell culture RNA interference (RNAi) screen was conducted using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, how caffeine affects cell survival was evaluated when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, ten kinases and 4 phosphatases that are essential for cell survival were identified. Remarkably, the screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, it was shown that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, it was demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. These in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress.

Zhang, B., Rotelli, M., Dixon, M. and Calvi, B. R. (2015) The function of Drosophila p53 isoforms in apoptosis. Cell Death Differ [Epub ahead of print]. PubMed ID: 25882045
Summary:
The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. This study evaluated the apoptotic potential of multiple isoforms of the single p53 gene in Drosophila. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. This study shows that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoform-specific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, these data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions.

Chakraborty, R., Li, Y., Zhou, L. and Golic, K.G. (2015). Corp regulates P53 in Drosophila melanogaster via a negative feedback loop. PLoS Genet 11: e1005400. PubMed ID: 26230084
Summary:
The tumor suppressor P53 is a critical mediator of the apoptotic response to DNA double-strand breaks through the transcriptional activation of pro-apoptotic genes. This mechanism is evolutionarily conserved from mammals to lower invertebrates, including Drosophila melanogaster. P53 also transcriptionally induces its primary negative regulator, Mdm2, which has not been found in Drosophila. This study identified the Drosophila gene companion of reaper (corp) as a gene whose overexpression promotes survival of cells with DNA damage in the soma but reduces their survival in the germline. These disparate effects are shared by p53 mutants, suggesting that Corp may be a negative regulator of P53. Confirming this supposition, it was found that corp negatively regulates P53 protein level. It has been previously shown that P53 transcriptionally activates corp; thus, Corp produces a negative feedback loop on P53. It was further found that Drosophila Corp shares a protein motif with vertebrate Mdm2 in a region that mediates the Mdm2:P53 physical interaction. In Corp, this motif mediates physical interaction with Drosophila P53. These findings implicate Corp as a functional analog of vertebrate Mdm2 in flies.

Wylie, A., Jones, A. E., D'Brot, A., Lu, W. J., Kurtz, P., Moran, J. V., Rakheja, D., Chen, K. S., Hammer, R. E., Comerford, S. A., Amatruda, J. F. and Abrams, J. M. (2016). p53 genes function to restrain mobile elements. Genes Dev 30: 64-77. PubMed ID: 26701264
Summary:

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, this study shows that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53- germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, this study also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, these findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility (Wylie, 2015).

Slaidina, M. and Lehmann, R. (2017). Quantitative differences in a single maternal factor determine survival probabilities among Drosophila germ cells. Curr Biol [Epub ahead of print]. PubMed ID: 28065608
Summary:
Germ cell death occurs in many speciesand has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. This study focused on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. The decision to die was found to be a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. This study demonstrates that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, it is proposed that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, it was demonstrated that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, it is hypothesized that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage.
Tasnim, S. and Kelleher, E. S. (2017). p53 is required for female germline stem cell maintenance in P-element hybrid dysgenesis. Dev Biol [Epub ahead of print]. PubMed ID: 29294306
Summary:
Hybrid dysgenesis is a sterility syndrome resulting from the mobilization of certain transposable elements in the Drosophila germline. Particularly extreme is the hybrid dysgenesis syndrome caused by P-element DNA transposons, in which dysgenic female ovaries often contain few or no germline cells. Those offspring that are produced from dysgenic germlines exhibit high rates of de novo mutation and recombination, implicating transposition-associated DNA damage as the cause of germline loss. However, how this loss occurs, in terms of the particular cellular response that is triggered (cell cycle arrest, senescence, or cell death) remains poorly understood. This study demonstrated that two components of the DNA damage response, Checkpoint kinase 2 and its downstream target p53, determine the frequency of ovarian atrophy that is associated with P-element hybrid dysgenesis. It was further shown that p53 is strongly induced in the germline stem cells (GSCs) of dysgenic females, and is required for their maintenance. These observations support the critical role for p53 in conferring tolerance of transposable element activity in stem cells.
Contreras, E. G., Sierralta, J. and Glavic, A. (2018). p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development. PLoS One 13(4): e0194344. PubMed ID: 29621246
Summary:
Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, it is hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. The function of p53 was studied during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. p53 loss of function was shown to reduce animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, it was shown that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. This study has demonstrated that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals.
BIOLOGICAL OVERVIEW

The mammalian p53 protein functions as a tumor suppressor by controlling cell cycle progression and cell survival. p53 is often described as the 'guardian of the genome' because it is a critical component of the cellular mechanisms that respond to genotoxic stresses like DNA damage and hypoxia to maintain the genomic integrity in part by arresting cell-cycle progression or by inducing apoptosis. p53 plays no essential role in the normal cell cycle, since the p53 knock out mouse develops normally. However, these mice as well as the transgenic mice carrying mutant p53 alleles are highly prone to develop spontaneous and carcinogen-induced tumors (Somasundaram, 2000 and references therein).

Numerous studies have established that growth arrest and apoptosis are independent functions of p53. p53-dependent G1 arrest occurs largely through transcriptional induction of p21WAF1 (Drosophila homolog: Dacapo), which prevents entry into S phase by inhibiting G1 cyclin-dependent kinase activity. However, p21 is not required for p53-dependent apoptosis. In fact, p21 may protect against p53-induced apoptosis in at least some cell types (Ollmann, 2000 and references therein).

Induction of apoptosis by p53 is critical for the tumor suppressor function of p53. There appear to be multiple mechanisms through which p53 promotes apoptosis. For example, p53 can transcriptionally activate the proapoptotic genes Bax (see Drosophila death executioner Bcl-2 homolog, Fas, and IGF-BP3, as well as a set of genes that may promote apoptosis through the formation of reactive oxygen species. Furthermore, there is evidence that p53 can induce apoptosis in the absence of its transcriptional activation function (Ollmann, 2000 and references therein).

The recent discovery of two p53-related genes, p63 and p73, has revealed an additional level of complexity in the study of p53 function in mammals (Somasundaram, 2000). Both genes encode proteins with transactivation, DNA-binding, and tetramerization domains, and some isoforms of p63 and p73 are capable of transactivating p53 target genes and inducing apoptosis. It was initially thought that only p53 was induced in response to DNA damage and other stress signals. However, there is now evidence that p73 is also activated by some forms of DNA damage in a manner that is dependent on the c-Abl tyrosine kinase (see Drosophila Abl oncogene). These data suggest that p53-independent apoptotic pathways may be mediated by other p53 family members (Ollmann, 2000 and references therein).

Characterization of the single Drosophila p53 homolog (referred to here as Dmp53 or p53) was reported in back-to-back publications from two laboratories (Ollmann, 2000 and Brodsky, 2000a). Both identified the gene by homology searches of the expressed sequence tag database of the Berkeley Drosophila Genome Project.

To perturb the function of Dmp53 during Drosophila development, expression of dominant-negative forms in vivo were directed using the GAL4-UAS system. The effects of Dmp53 inactivation on the level of radiation induced apoptosis were tested. For these studies, transgenic strains were produced carrying a point mutantion in the DNA-binding domain [Dmp53(259H)] and the Dmp53(Ct) derivative possessing exclusively the C-terminal interaction domain. A similar C-terminal derivative has been used for tissue-specific inactivation of p53 in mice. The Dmp53 variants were specifically expressed in the posterior half of the developing Drosophila wing using an engrailed-GAL4 driver, line and effects on damage-induced apoptosis and cell cycle arrest were monitored after irradiation (Brodsky, 2000a).

The levels of apoptosis were tested in untreated and irradiated wing discs expressing dominant-negative Dmp53. In untreated wild-type discs, there are a small number of clustered apoptotic cells as visualized by staining with the vital dye acridine orange. In discs with engrailed-GAL4 driving expression of dominant-negative Dmp53, there is no substantial difference in the level of apoptosis in the anterior and posterior halves of the disc. Following irradiation, there is a massive increase in the amount of apoptosis throughout wild-type wing discs. However, in animals expressing dominant-negative Dmp53 in the posterior of the wing disc, radiation-induced apoptosis is greatly reduced in that region. This reduction is not due to minor differences in the age or handling of the discs since a robust radiation-induced apoptosis is observed in the anterior portion of the disc where dominant-negative p53 is not expressed. Together, these results indicate that Dmp53 is required for radiation-induced apoptosis in the wing, but not for the normal levels of cell death that occur in the absence of DNA-damaging agents (Brodsky, 2000a).

The effect of dominant-negative Dmp53 on radiation-induced arrest of cell cycle progression was tested. In mammals, p53 is required for radiation-induced G1/S arrest and has variable effects on radiation-induced G2/M arrest. The Drosophila wing exhibits a G2/M DNA damage checkpoint (Brodsky, 2000b) that is dependent on genes such as mei-41 (a homolog of the human ATM checkpoint gene) and grapes (a homolog of the yeast chk1 gene). Irradiation of wild-type wing discs blocks entry into mitosis; this block is not affected by expression of dominant-negative Dmp53. Thus, although Dmp53 is required for radiation-induced apoptosis, the data do not support a role for this protein during the G2/M checkpoint in the wing (Brodsky, 2000a).

In animals expressing dominant-negative Dmp53 under the control of engrailed-GAL4, the size and patterning of the adult wing is not noticeably altered. This result is consistent with the normal levels of mitosis and apoptosis in unirradiated wing discs expressing dominant-negative Dmp53. Similarly, widespread expression of dominant-negative Dmp53 using a tubulin-GAL4 driver does not generate any obvious adult phenotypes. These results suggest that, like human p53, Dmp53 is required in vivo to respond to certain cellular stresses, but may not be essential for normal development (Brodsky, 2000a). This role is unlike that of the mammalian homolog p63, which is required for limb development in both humans and mice (Celli, 1999; Mills, 1999; Yang, 1999).

The consequence of increased levels of wild-type Dmp53 was examined. Animals expressing wild-type Dmp53 using either the hsp70 or actin5C promoters do not survive to adulthood. Expression of Dmp53 using an eye-specific glass-dependent promoter leads to increased apoptosis in the eye imaginal disc and results in a rough, small eye phenotype; similar expression of Dmp53(259H) has no effect on eye morphology. Unlike radiation-induced apoptosis, apoptosis due to overexpression of Dmp53 is not suppressed by coexpression of the viral caspase-inhibitor p35; this observation suggests that overexpression of Dmp53 is sufficient to induce apoptosis, but that the response is either qualitatively or quantitatively different from the apoptotic response to DNA damage. Since the action of at least one Drosophila caspase is insensitive to p35 expression (Meier, 2000), overexpression of Dmp53 may activate apoptosis though a p35-resistant caspase (Brodsky, 2000a)

X irradiation of imaginal wing discs induces apoptosis that requires functional Dmp53. This poses the question of how Dmp53 activity is regulated in response to radiation. It is well established that mammalian p53 receives signals from a variety of cellular stresses such as various forms of DNA damage, nucleotide deprivation, incomplete DNA synthesis, and hypoxia. These signals are likely to work through a set of signaling pathways that activate and stabilize the p53 protein. Although understanding of the different gene products responsible for these various signaling pathways is still in its infancy, there is strong evidence that one pathway to p53, that induced by irradiation, requires functional ATM. The ATM protein kinase shares homology with other members of the PI3 kinase family, including the S. pombe DNA damage mediator kinase Rad3. Recently, two kinases that are downstream of ATM, CHK1, and CDS1/CHK2 (Drosophila homolog: loki) have been shown to phosphorylate and regulate human p53 (Chehab, 2000; Shieh, 2000). Given that many aspects of the DNA damage checkpoint response are conserved between yeast and mammals, it is possible that Dmp53 might be similarly regulated. Indeed, Drosophila homologs of ATM (mei41) and Chk1 (grapes) have been identified, and it will be interesting to determine their relationship to Dmp53. It should be mentioned here that while ATM has been clearly shown to regulate p53-mediated cell cycle arrest, there is evidence that apoptosis induced by p53 is independent of the function of ATM. Thus, the study of Dmp53-induced apoptosis in Drosophila may uncover new upstream regulators of p53 activity (Ollmann, 2000 and references therein).

Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, this study interfered with scox (the single Drosophila Sco orthologue) expression in the heart. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans (Martínez-Morentin, 2015).

Cardiomyopathies are a collection of myocardial disorders in which the heart muscle is structurally and functionally abnormal. In the past decade, it has become clear that an important proportion of cases of hypertrophic and dilated cardiomyopathies are caused by mutations in genes encoding sarcomeric or desmosomal proteins. In addition, cardiomyopathies (both hypertrophic and dilated) are frequently associated to syndromic and non-syndromic mitochondrial diseases. The importance of oxidative metabolism for cardiac function is supported by the fact that 25–35% of the myocardial volume is taken by mitochondria. The current view of mitochondrial involvement in cardiomyopathy assumes that ETC malfunction results in an increased ROS production, triggering a “ROS-induced ROS release” vicious circle which in turn perpetuates ETC dysfunction via damage in mtDNA and proteins involved in electron transport. Under this view, accumulated mitochondrial damage would eventually trigger apoptosis through mitochondrial permeability transition pore (mPTP) opening other mechanisms. Under normal circumstances, damaged mitochondria would be eliminated through mitophagy. Excessive oxidative damage is supposed to overcome the mitophagic pathway resulting in apoptosis. Nevertheless, although several potential mechanisms have been suggested, including apoptosis deregulation, oxidative stress, disturbed calcium homeostasis or impaired iron metabolism, the molecular basis of the pathogenesis of mitochondrial cardiomyopathy is virtually unknown (Martínez-Morentin, 2015).

Pathogenic mutations in human SCO1 and SCO2 have been reported to cause hypertrophic cardiomyopathy, among other clinical symptoms. However, the molecular mechanisms underlying this cardiac dysfunction have yet to be elucidated. This study reports the first cardiac-specific animal model to study human SCO1/2-mediated cardiomyopathy. Cardiac-specific scox KD in Drosophila provokes a severe dilated cardiomyopathy, as reflected by a significant increase in the conical chamber size, due to mitochondrial dysfunction. It presents a concomitant metabolic switch from glucose oxidation to glycolysis and an increase in ROS levels, leading to p53-dependent cell death. Interestingly, previous studies on patients and rat models have shown that mitochondrial dysfunction is associated with abnormalities in cardiac function and changes in energy metabolism, resulting in glycolysis optimization and lactic acidosis. Furthermore, in the Sco2KI/KO mouse model, where no evidence of cardiomyopathy has been described, partial loss of Sco2 function induces apoptosis in liver and skeletal muscle. In flies scox KD causes a significant reduction in FS and in the DI, as well as cardiac myofibril disorganization. This degenerative process is most likely due to mitochondrial dysfunction rather than to a developmental defect and moreover, the dilated cardiomyopathy developed by flies resembles that caused by mitochondrial fusion defects in flies (Martínez-Morentin, 2015).

The ETC is the major site of ROS production in cells, and aging and many neurodegenerative diseases have been linked to mitochondrial dysfunction that results in excessive oxidative stress. Interestingly, there is an increase in ROS formation associated with oxidative DNA damage in human Sco2−/− cells. Accordingly, it was found that cardiac-specific knockdown of scox increases oxidative stress, although it could not be distinguished whether this increase in free radical accumulation arises from the mitochondria or whether it comes from non-mitochondrial sources due to a loss of cellular homeostasis, as reported in yeast and in a neuro-specific COX-deficient Alzheimer disease mouse model (Martínez-Morentin, 2015).

Sco2 expression is known to be modulated by p53, a transcription factor that participates in many different processes, including cancer development, apoptosis and necrosis. p53 regulates homeostatic cell metabolism by modulating Sco2 expression and contributes to cardiovascular disorders. In addition, p53 activation in response to stress signals, such as increased oxidative stress or high lactic acid production, is well documented. Data from this study, showing that p53 is upregulated in response to scox KD, but not in response to KD of another Complex IV assembly factor, Surf1, suggest a specific genetic interaction between dp53 and scox. This is corroborated by the dramatic effects observed in the heart structure and function when dp53 is overexpressed in scox KD hearts. Furthermore, the functional and structural defects seen in scox KD hearts can be rescued in dp53-DN OE or dp53 null backgrounds, indicating that the scox-induced defects are mediated by increased p53 expression. Interestingly, opposed to scox KD, the heart structure defects induced by dp53 OE can be fully rescued by heart-specific Surf1 KD, further confirming the specificity of the genetic interaction between dp53 and scox (Martínez-Morentin, 2015).

It has recently been shown that SCO2 OE induces p53-mediated apoptosis in tumour xenografts and cancer cells. Furthermore, SCO2 KD sensitizes glioma cells to hypoxia-induced apoptosis in a p53-dependent manner and induces necrosis in tumours expressing WT p53, further linking the SCO2/p53 axis to cell death. In Drosophila, there is a dp53-mediated upregulation of Reaper, Hid and Grim in response to scox KD. This, coupled with the observation that Reaper overexpression in the adult heart enhances the structural defects caused by cardiac-specific scox KD, suggests that scox normally prevents the triggering of dp53-mediated cell death in cardiomyocytes in stress response. Indeed, it was found that there is massive cell death in the skeletal muscle and liver of Sco2KI/KO mice, supporting the hypothesis that Sco proteins might play this role also in mammals (Martínez-Morentin, 2015).

The study provides evidence that scox KD hearts exhibit partial loss of COX activity, with cardiomyocytes undergoing apoptosis. There is evidence from vertebrate and invertebrate models that partial inhibition of mitochondrial respiration promotes longevity and metabolic health due to hormesis. In fact, it has recently been shown that mild interference of the OXPHOS system in Drosophila IFMs preserves mitochondrial function, improves muscle performance and increases lifespan through the activation of the mitochondrial unfolded pathway response and IGF/like signalling pathways. This study speculates that cell death, rather than mitochondrial dysfunction itself, is likely to be the main reason for the profound heart degeneration observed in TinCΔ4-Gal4>scoxi flies. Expression of dominant negative dp53 in scox KD hearts rescues dysfunction and cardiac degeneration, and, most importantly, scox KD in dp53−/− animals causes no apparent heart defects, which could attribute the rescue observed to blockade of the p53 pathway. Indeed, inhibiting apoptosis by p35 or Diap1 OE almost completely rescues the morphological scox KD phenotype. As scox KD in the absence of dp53 causes no symptoms of heart disease, coupled with the inability of p35 and Diap1 to completely rescue the morphological phenotype, suggests that, in addition to inducing apoptosis, dp53 plays a key role in the development of cardiomyopathy (Martínez-Morentin, 2015).

The fact that heart-specific Surf1 KD neither upregulates p53 nor induces apoptosis supports the idea that the partial loss of scox function itself triggers dp53 upregulation and apoptosis, rather than it being a side effect of COX dysfunction and the loss of cellular homeostasis. In this context, it is noteworthy that SCO2 interference in mammalian cells induces p53 re-localization from mitochondria to the nucleus. It is therefore tempting to hypothesize that scox might play another role independent of its function as a COX assembly factor, perhaps in redox regulation as suggested previously and that it may act in conjunction with dp53 to fulfil this role. Another issue deserves further attention, the possibility of this interaction being a tissue-specific response. It may be possible that the threshold of COX deficiency tolerated by the heart might be lower than in other tissues, thus the scox/dp53 genetic interaction may be a tissue-dependent phenomenon or the consequence of a tissue-specific role of scox. In fact, mitochondrial dysfunction in mice is sensed independently from respiratory chain deficiency, leading to tissue-specific activation of cellular stress responses. Thus, more work is necessary to test these hypotheses and try to understand how the partial lack of scox induces cell death through dp53 (Martínez-Morentin, 2015).

Although the role of mitochondria in Drosophila apoptosis remains unclear, there is strong evidence that, as in mammals, mitochondria play an important role in cell death in flies. The localization of Rpr, Hid and Grim in the mitochondria is essential to promote cell death, and fly mitochondria undergo Rpr-, Hid- and Drp1-dependent morphological changes and disruption following apoptotic stimulus. Moreover, the participation of the mitochondrial fission protein Drp1 in cell death is conserved in worms and mammals. It has been proposed that p53 plays a role in the opening of the mPTP that induces necrotic cell death. According to this model, p53 translocates to the mitochondrial matrix upon ROS stimulation, where it binds cyclophilin D (CypD) to induce mPTP opening independent of proapoptotic Bcl-2 family members Bax and Bak, and in contrast to traditional concepts, independent of Ca2+ (Martínez-Morentin, 2015).

Apoptotic and necrotic pathways have a number of common steps and regulatory factors, including mPTP opening that is thought to provoke mitochondrial swelling and posterior delivery of necrotic factors, although Drosophila mPTP activation is not accompanied by mitochondrial swelling. Interestingly, although the p53 protein triggers mitochondrial outer membrane permeabilization (MOMP) in response to cellular stress in mammals, releasing mitochondrial death factors, MOMP in Drosophila is more likely a consequence rather than cause of caspase activation and the release of mitochondrial factors does not appear to play a role in apoptosis. Thus, in cardiac-specific scox KD flies, dp53 might induce mPTP opening to trigger cell death, which in the absence of mitochondrial swelling would result in apoptosis instead of necrosis, as occurs in mammals. Drosophila mPTP has been shown to be cyclosporine A (CsA)-insensitive in vitro, although CsA administration ameliorates the mitochondrial dysfunction with a severely attenuated ATP and enhanced ROS production displayed by collagen XV/XVIII mutants. Interestingly, mice lacking collagen VI display altered mitochondrial structure and spontaneous apoptosis, defects that are caused by mPTP opening and that are normalized in vivo by CsA treatment (Martínez-Morentin, 2015).

Small chaperons and autophagy protected neurons from necrotic cell death

Neuronal necrosis occurs during early phase of ischemic insult. However, knowledge of neuronal necrosis is still inadequate. To study the mechanism of neuronal necrosis, a Drosophila genetic model of neuronal necrosis was established by calcium overloading through expression of a constitutively opened cation channel mutant. This study performed further genetic screens and identified a suppressor of neuronal necrosis, CG17259, which encodes a seryl-tRNA synthetase. Loss-of-function (LOF) CG17259 activated eIF2alpha phosphorylation and subsequent up-regulation of chaperons (Hsp26 and Hsp27) and autophagy. Genetically, down-regulation of eIF2alpha phosphorylation, Hsp26/Hsp27 or autophagy reduced the protective effect of LOF CG17259, indicating they function downstream of CG17259. The protective effect of these protein degradation pathways indicated activation of a toxic protein during neuronal necrosis. The data indicated that p53 was likely one such protein, because p53 accumulated in the necrotic neurons and down-regulation of p53 rescued necrosis. In the SH-SY5Y human cells, tunicamycin (TM), a PERK activator, promoted transcription of hsp27; and necrosis induced by glutamate could be rescued by TM, associated with reduced p53 accumulation. In an ischemic stroke model in rats, p53 protein was also increased, and TM treatment could reduce the p53 accumulation and brain damage (Lei, 2017).

In a Drosophila model, neuronal necrosis was induced by the specific expression of a constitutively open glutamate receptor 1 channel (GluR1Lc) in neurons to overload calcium. By genetic screens using AG fly lethality, this study identified a novel suppressor of neuronal necrosis, LOF CG17259. CG17259 encodes a seryl-tRNA synthetase and functions in ligation of serine to its cognate tRNA. Therefore, LOF CG17259 may affect protein synthesis and induce cytoplasmic protein folding defects and/or ER stress. ER stress initiates through three distinct sensors in the ER membrane, including PERK, ATF6 and IRE1 (Deegan, 2013). Each signaling branch has both overlapping and distinct functions. For example, PERK phosphorylates eIF2α to reduce overall protein translation and promote cell survival. Whereas the IRE1 branch reduces protein synthesis by promoting the degradation of mRNA and activates JNK, which may, in turn, induce apoptosi. The current data demonstrated that the IRE1 branch was not activated in LOF CG17259, because transcription of Xbp1 sp and JNK pathway were not activated. In contrast, and the PERK/eIF2α branch was up-regulated in the LOF CG17259 flies. Consistent with these data, activation of the PERK/eIF2α signaling branch has been implicated in the treatment of various neurodegenerative diseases. For instance, treatment with salubrinal, an inhibitor of eIF2α dephosphorylation, can rescue neurodegeneration in α-synuclein transgenic mice or ischemic stroke in rats. Further, this study found that autophagy was activated in LOF CG17259. The coupling of the PERK/eIF2α signaling branch with autophagy has been well documented to protect neurons (Herz, 2014). The current research is consistent with these results from the literature. Additionally, this research provides an additional mechanism by which the eIF2α signaling pathway affects neuron survival (Lei, 2017).

The results showed that the rescue effect of CG17259 −/+ was abolished by the mutants of Hsp26/Hsp27, and overexpression of Hsp26 or Hsp27 was sufficient to rescue AG flies, suggesting Hsp26/Hsp27 are down stream of LOF CG17259. The small chaperones of Drosophila Hsp26/Hsp27 are likely to have a similar function to that of mammalian Hsp27, which is known to protect neurons under various pathological conditions, including ischemic stroke. The protective mechanisms of Hsp27 may involve the suppression of the formation of actin aggregates, activation of the NF-κB pathway, or direct inhibition of components in the apoptotic machinery. The mammalian Hsp27 may share the combined function of Drosophila Hsp26/Hsp27, because it localizes in both cytosol and nucleus upon phosphorylation; while, it mainly localizes in the nucleus upon dephosphorylation. The current data showed that the Drosophila Hsp26 and Hsp27 distributed in cytosol or nucleus, respectively. For functional study, these data suggest that Hsp26/Hsp27 and p53 may function in the same pathway, because the rescue effect of p53 and CG17259 −/+ was not additive and Hsp26/Hsp27 protein could pull down p53. Although the co-IP data was obtained under the Hsp26/Hsp27 overexpression condition, the interaction between Hsp26/Hsp27 and p53 has been reported by other studies (Lei, 2017).

The autophagy pathways can be further classified into autophagy (in this text macroautophagy refers to autophagy) and chaperone-mediated autophagy (CMA). Autophagy requires the formation of autophagosomes and the function of Atg genes. In contrast, the CMA pathway degrades proteins in lysosomes and does not require Atg genes (Todde, 2009). The current data suggested that autophagy was activated in the LOF CG17259 flies; up-regulation of autophagy rescued the AG lethality and down-regulation of autophagy had the opposite effect. Because LOF p53 rescued the enhancing death effect of LOF autophagy, it is possible that degradation of accumulated p53 was dependent on autophagy in the AG flies. Consistent with these data, the increase in the level of p53 protein has been observed in embryonic fibroblasts in Atg7 −/− or Atg5 −/− mice (Lei, 2017).

Function of p53 in apoptosis has been well documented. Upregulation of p53 has been linked to neuronal cell death in numerous models of injuries and diseases, including excitotoxicity. The absence of p53 protects neurons from a wide variety of toxic insults, including focal ischemia, ionizing radiation and MPTP-induced neurotoxicity. In response to various types of stress, p53 promotes apoptosis through either transactivation of specific target genes or transcription-independent pathways. As a transcription factor, p53 upregulates proapoptotic genes, such as Bax, Noxa and PUMA. In addition, p53 can interact with Bcl2 family proteins, such as Bax and Bak, to induce permeabilization of the outer mitochondrial membrane. Whether p53 is involved in neuronal necrosis is unclear. In support of its involvement in necrosis, p53 may physically interact with cyclophilin D (CypD), a component of the mitochondrial permeability transition pores and trigger the opening of the pores and necrosis. In addition, the formation of the p53-CypD complex occurs during brain ischemia/reperfusion insult. This study provides the genetic and cell biology evidence indicating that p53 is involved in neuronal necrosis. In SH-SY5Y cells, it was shown that p53 was accumulated upon cells treated with glutamate; and this accumulation was prohibited by TM treatment, which enhanced Hsp27 transcription. Similarly, the increased level of p53 in MCAO rat brain was down-regulated by TM treatment. Together, these results indicate conserved function of p53 in neuronal necrosis. In fact, protective effect of TM against neurodegeneration has been widely reported. The difference is that the current study evaluated potential down-stream function of TM to degrade p53 in neuronal necrosis. How does p53 trigger both apoptosis and necrosis? It is proposed that mild p53 accumulation likely induces apoptosis, whereas the additional accumulation of p53 promotes necrosis. This hypothesis requires further investigation however (Lei, 2017).

The inhibition of p53 transcriptional activity by pifithrin α or its mitochondrial targeting by pifithrin μ protects the brain in rodent models of stroke. However, p53 also benefits animal survival under hypoxic conditions. Thus, administration of pifithrins may interfere with the normal function of p53 and thereby produce side effects. An alternative way to target p53 may be to aim to reduce the accumulation of p53. This research suggests that the promotion of eIF2α signaling may activate endogenous mechanisms (activation of small chaperones and autophagy) to degrade p53 (Lei, 2017).

p53 and cyclin G cooperate in mediating genome stability in somatic cells of Drosophila

One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. This study identified Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. This study finds it in protein complexes together with p53, and shows that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor, it was shown that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity it is proposed that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila (Bayer, 2017).

Earlier work has shown that Drosophila CycG is important for the meiotic recombination checkpoint in the female germline. In cycG mutant germaria, DSB repair is delayed, and CycG protein is found in conjunction with the 9-1-1 complex suggesting that it may be involved in DSB sensing. This study extends this analysis to somatic tissue, where again problems were noted in DNA damage repair as detected by persistent γ-H2Av signals in irradiated cycG mutants. This indicates that in the absence of CycG, repair of double-strand breaks in the DNA is compromised. Accordingly, cycG mutants fail to repair DSBs with the fidelity of wild type, display more chromosomal aberrations upon irradiation, and are hypersensitive to genotoxic stress. No evidence was found for an involvement of CycG in DSB sensing in somatic cells, however. Instead, CycG appears to perform its role through modulating the activity of p53. Since a retardation and/or erroneous DNA repair was notice in the absence of CycG, it is proposed that CycG is required to resolve IR-induced DNA damage presumably as co-factor of p53. From genetic data it is concluded that CycG is a positive mediator of p53 activity, and indeed mutants in either gene resemble each other not only in life span but also in radiation sensitivity. The physical interaction of CycG and p53, however, strongly suggests that CycG directly promotes p53 activity, regardless of whether it may also regulate downstream or upstream components of the DNA damage repair machinery (Bayer, 2017).

Unlike in vertebrates, Drosophila cycG is not under the transcriptional control of p53. Instead a robust protein-protein interaction is seen in a yeast two-hybrid assay between p53 and CycG proteins, involving the cyclin repeats of CycG and the tetramerization domain of p53. Direct binding in vivo, however, required genotoxic stress. It is proposed that complex formation, rather than being permanent, occurs only in response to DNA damage and perhaps requires additional factors and/or protein modifications. With the help of a p53 biosensor this study showed that CycG is crucial for p53 mediated transcriptional response to genotoxic stress in the germline as well as in somatic tissue, suggesting that CycG may be involved in the activation or stabilization of p53 itself, or in the assembly of active transcriptional complexes (Bayer, 2017).

The CycG-p53 axis might have been expected given their close interrelationship in the mammalian system. Here, the two cyclin G homologues Ccng1 and Ccng2 have been involved in growth control to genotoxic stress. Ccng1 but not Ccng2 is a direct transcriptional target of p53. Both are found in complexes with protein phosphatase 2A, and together with Mdm2 Ccng1 is involved in Mdm2 mediated degradation of p53. As the two mammalian cyclin G proteins appear to act differently on cell proliferation, a lot of work has been invested to understand their respective roles. More recently it was proposed that observed discrepancies may arise from dose dependency of Ccng1. In fact, also Drosophila tissues and cells appear to respond differentially to the dose of CycG, as for example overexpression may impact the cell cycle in a dominant negative manner, and RNAi downregulation causes effects different from the gene deletion phenotypes. The role of Ccng1 in response to genotoxic stress has been analysed in quite some detail. Here, Ccng1 not only forms a complex with Mdm2, resulting in destabilizing p53. Moreover, it also interacts with ARF, thereby stabilizing and activating p53. It hence has been proposed that Ccng1 is required for a timely and proper response to genotoxic stress, first for the activation of p53 to allow for DNA damage repair, and then for p53 degradation to protect cells from apoptosis that have recovered from the initiating stress (Bayer, 2017).

Intensive searches in the Drosophila genome failed to uncover Mdm2 or ARF homologues to date. Recently, however, a Mdm2 analogue called Corp has been identified that shares several Mdm2 properties: Corp is a transcriptional target of p53 in response to genotoxic stress, it binds to p53 protein and results in reduced p53 protein levels presumably by proteolytic degradation. Hence, like Mdm2 Corp acts in a negative feed back loop on p53 activity. Whether Corp is likewise inactivated by phosphorylation and/or an ARF-like molecule remains to be shown. Moreover, it will be interesting to see, whether Corp can recruit CycG, and whether PP2A plays any role in its regulation. It is known already that Drosophila CycG also binds to the PP2A-B' subunit, similar to the two mammalian CycG proteins. Unlike in vertebrates, however, it acts negatively on PP2A activity by genetic means. Despite the similarity of the respective components and the processes they are involved in, there is not a 1:1 conformity when comparing flies and mammals. Perhaps, the manifold feed back loops weaved into the system, elude genetic analyses. Perhaps, as in mammals, Drosophila CycG forms protein complexes with disparate activities (i.e., repair or apoptosis) depending on tissue, cell cycle phase, or phase of response to DNA damage (Bayer, 2017).


REGULATION

Targets of Activity

The genomic region containing reaper, grim, and head involution defective is required for all cell death in Drosophila embryos, including radiation-induced apoptosis. rpr is transcriptionally induced in embryos following irradiation, and an 11 kb sequence upstream of the rpr start codon is sufficient to confer radiation responsiveness on a lacZ reporter transgene. To identify the minimal radiation-responsive cis-elements upstream of rpr, the ability of smaller fragments of this 11 kb regulatory region to activate lacZ transcription was tested. Each transgenic strain was tested for radiation-induced expression of beta-galactosidase. Multiple constructs containing sequences ~5 kb upstream of the rpr start codon show a robust radiation response. These experiments identify a discrete 150 bp enhancer that responds to radiation as strongly as the larger enhancer fragments tested. Since this enhancer retains radiation-responsiveness but does not recapitulate the developmental patterns of rpr expression seen with larger enhancer fragments, the results also indicate that cis-regulatory sequences responsible for damage-induced transcription of rpr can be isolated from others that respond to developmental cues (Brodsky, 2000a).

Within the 150 bp enhancer, a 20 bp sequence was identified that strongly resembles the consensus for human p53 DNA-binding sites. This 20 bp sequence is referred to as the p53 response element (p53RE) to reflect its response to Dmp53 in yeast. Like those found upstream of the human target genes mdm-2 and p21/WAF1, this putative p53 binding site upstream of rpr contains two tandemly arrayed 10mers, each of which matches the consensus motif at nine of ten positions. The two mismatches occur at the outer positions of the 20 bp element; the invariant core nucleotides of each 10mer motif match the consensus perfectly (Brodsky, 2000a).

Yeast one-hybrid assays were used to see whether Drosophila p53 interacts with the p53RE. For these studies, a reporter plasmid containing the p53RE upstream of the beta-galactosidase gene was integrated into the yeast genome to produce the p53RE bait strain. Next, this p53RE bait strain was transformed with test plasmids expressing either wild-type Dmp53 or Dmp53(259H) fused to the GAL4 activation domain. These strains were assayed for beta-galactosidase activity. Reporter expression in strains with Dmp53(259H) or the empty vector control (expressing the Gal4 activation domain alone) are indistinguishable from each other. Compared to these controls, each of the four independent transformants carrying the wild-type Dmp53 plasmid shows a substantial increase in beta-galactosidase levels. Based on these results, it has been concluded that the 150 bp radiation-responsive enhancer upstream of rpr contains a 20 bp binding site for Dmp53 (Brodsky, 2000a).

A test was performed to see whether the p53RE is sufficient to confer radiation-responsive transcriptional activation on a lacZ reporter construct in vivo. A transgene containing four copies of the p53RE and the minimal hsp70 promoter has showennegligible expression in untreated embryos but is substantially induced following irradiation. Therefore, the 20 bp Dmp53 binding site from the rpr locus is sufficient to mediate a transcriptional response to radiation and may define a minimal radiation responsive sequence. When analyzed in parallel to the 150-lacZ reporter, containing 150 bases surrounding the p53RE, the p53RE-lacZ reporter exhibits less robust and less uniform beta-galactosidase activity following irradiation. Reduced activity is often observed when DNA elements are tested in isolation from the normal flanking sequences and, in this instance, may reflect the influence of other factors that interact with the 150 bp enhancer sequence (Brodsky, 2000a).

Disruptions of development are often associated with excess apoptosis. For example, in a crumbs (crb) mutant background, abnormal epidermal development in the embryo leads to widespread apoptosis. This apoptosis is fully suppressed by deletions for the genomic region containing rpr, hid, and grim and is preceded by a dramatic induction of rpr expression, similar to that seen in irradiated embryos. A test was performed to see whether transcriptional activation mediated by p53RE represents a specific response to radiation damage or a common integration point for multiple pathways that lead to excess apoptosis. Beta-galactosidase expression was examined in wild-type and crb embryos carrying either the p53RE-lacZ or the 2kb-lacZ reporter constructs. In stage 12/13 wild-type embryos, expression of the 2kb-lacZ transgene is normally confined to the developing gut but, in similarly aged crb embryos, expression is induced throughout the epidermis. In contrast, the p53RE-lacZ transgene exhibits only basal expression in either wild-type or crb embryos. Thus, despite widespread apoptosis in crb embryos, there is no induction of reporter expression from the p53RE. These results indicate that the p53RE specifically responds to radiation damage, not generally to all proapoptotic signals. They also indicate that irradiation and disrupted development may activate rpr expression through distinct pathways (Brodsky, 2000a).

Drosophila p53 encodes a 385-amino acid protein with significant homology to human p53 in the region of the DNA-binding domain, and to a lesser extent the tetramerization domain. Although Drosophila p53 and human p53 share much sequence and biochemical homology, one major difference between Drosophila p53 and human p53 is that Drosophila p53 lacks the consensus box I sequence found in all vertebrate p53 proteins; this is located in the p53-MDM2 interaction region. Moreover, genome-wide searches in Drosophila have failed to identify an MDM2 homolog. Therefore, MDM2-mediated p53 degradation could be a later evolutionary event. Interestingly, there is a putative PEST region at the N terminus of Drosophila p53 but not human p53. These are P- (proline), E- (glutamate), S- (serine), and T- (threonine) rich sequences flanked by K (lysine) or R (arginine) but not interrupted by any basic amino acids (30), that act as protein degradation signals. It seems possible that Drosophila p53 protein stability is regulated through this PEST sequence instead of the more specific MDM2-p53 autoregulating loop in vertebrates. Purified Drosophila p53 DNA-binding domain protein has been shown to bind to the consensus human p53-binding site by gel mobility analysis. In transient transfection assays, expression of Drosophila p53 in Schneider cells transcriptionally activates promoters that contain consensus human p53-responsive elements. Moreover, a mutant Drosophila p53 (Arg-155 to His-155), like its human p53 counterpart mutant, exerts a dominant-negative effect on transactivation. Ectopic expression of Drosophila p53 in Drosophila eye disc causes cell death and leads to a rough eye phenotype. Drosophila p53 is expressed throughout the development of Drosophila with highest expression levels in early embryogenesis; this high level has a maternal component. Consistent with this, Drosophila p53 RNA levels were high in the nurse cells of the ovary. It appears that p53 is structurally and functionally conserved from flies to mammals. Drosophila will provide a useful genetic system to the further study of the p53 network (Jin, 2000).

The similarity between predicted structure of Drosophila p53 and the crystal structure of the human p53 DNA-binding domain prompted an exploration of whether Drosophila p53 is able to bind to the human p53 consensus binding site. Previous studies have shown that the DNA-binding domain plus N terminus and the DNA-binding domain alone of human p53 have similar affinities for the consensus DNA site as does the full-length protein. Therefore, the fragments of human p53 and Drosophila p53 containing the DNA-binding domains were purified to test their DNA-binding ability. The consensus site for human p53 is PuPuPuCA/T-T/AGPyPyPy-N0-13-PuPuPuCA/T-T/AGPyPyPy. A double-stranded oligonucleotide matching the consensus sequence (5'-TACAGAACATGTCTAAGCATGCTGGG-3') was end labeled and used for gel mobility-shift assay. Drosophila p53 forms a DNA-protein complex as does human p53. The specificity of the DNA-protein interaction was demonstrated by competition assays, in which the unlabeled specific oligonucleotide (SP, the consensus oligonucleotide itself) effectively competes with the labeled probe. The specificity of interaction was demonstrated by using a mutated p53 consensus oligonucleotide (5'-TACAGAAaATtTCTAAGaATtCTGGG-3'; mutation in consensus sequence shown in lowercase) as a probe in a similar gel shift assay. Both Drosophila p53 and human p53 proteins fail to form a complex with the mutated oligonucleotide. The gel mobility-shift analysis demonstrates that the binding affinity of Drosophila p53 to the oligonucleotide sequence is lower than that of human p53. It is not clear whether this was due to the specific oligonucleotide sequence chosen or that Drosophila p53 prefers a similar but a slightly different consensus site than that of human p53 (Jin, 2000).

MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but ascertaining the role of specific MKPs is hindered by functional redundancy in vertebrates. MKP function was characterized by examining the function of Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP, during embryonic and imaginal disc development. Puc is a key anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining basal JNK signaling. Furthermore, JNK signaling plays an important role in gamma-irradiation-induced apoptosis, and this study examined how JNK signaling fits into the circuitry regulating this process. Radiation upregulates both JNK activity and puc expression in a p53-dependent manner; apoptosis induced by loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream of both Reaper and effector caspases. JNK signaling directs normal developmentally regulated apoptotic events. However, if cell death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are key regulators of the delicate balance between proliferation, differentiation and apoptosis during development (McEwen, 2005).

Chk2 activates the apoptotic activity of p53

The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. In Drosophila, however, the regulation modes of p53 are still unknown. Overexpression of Drosophila p53 in the eye induces apoptosis, resulting in a small eye phenotype. This phenotype is markedly enhanced by coexpression with Drosophila Chk2 and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of Chk2. DN Chk2 also inhibits p53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant has no effect on p53-induced phenotypes. Chk2 also activates the p53 transactivation activity in cultured cells. Mutagenesis of p53 amino terminal Ser residues revealed that Ser-4 is critical for its responsiveness toward Chk2. Chk2 activates the apoptotic activity of p53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating p53. Chk2 may be the ancestral regulator of p53 function (Peters, 2002).

Various forms of cellular stress such as DNA damage or ionizing irradiation lead to activation and stabilization of the p53 tumor suppressor protein and to growth arrest and apoptosis. Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad 53 and the Schizosaccharomyces pombe Cds1 checkpoint genes, regulate p53 function in mammals in response to DNA damage. Chk2 is a protein kinase that acts downstream of the ataxia telangiectasia mutated (ATM) kinase and may induce cell cycle arrest. Loss of Chk2 in thymocytes results in failure to increase intracellular p53 levels in response to DNA damage, causing a defect in p53-mediated apoptosis. Chk1, an evolutionarily conserved protein kinase, implicated in cell cycle checkpoint control in lower eukaryotes, also has been suggested to play a role in p53 regulation. Chk1 also can phosphorylate p53, probably at the same sites as Chk2. Currently, the relative significance of p53 phosphorylation by Chk2 and/or Chk1 in the process of p53 activation is unclear. Several components involved in cell cycle checkpoint control pathways are conserved in Drosophila. Drosophila homologs of the ATM/ATR (mei-41), Chk1 (grapes), and Chk2 (Loki) kinases have been identified. Mei-41 mutant cells are sensitive to ionizing radiation, display high levels of mitotic chromosome instability, and do not arrest upon radiation treatment. Grapes (Grp) has been shown to be involved in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. The Drosophila maternal nuclear kinase (DMNK) protein is the homolog of the human Chk2 protein [referred to as Drosophila melanogaster Chk2 (Chk2)] and is highly expressed in Drosophila ovaries and functions in meiosis. The role of Chk2 or Grp in the regulation of Drosophila p53 (mp53) is unknown. It is also not clear, as to whether Chk2 functions in a cell cycle checkpoint pathway in vivo. Data from C. elegans suggest that Chk2 mutants are defective in meiosis but retain a DNA damage checkpoint in response to replication inhibition and ionizing radiation. Cloning and characterization of Drosophila p53 has shown an essential role for Dmp53 in radiation-induced apoptosis. Overexpressing of Drosophila p53 in the fly eye results in massive apoptosis and a small eye phenotype. These results have now been expanded by using genetic epistasis experiments; an essential role has been demonstrated for Drosophila Chk2 in the regulation of p53. The amino-terminal Ser at position 4 of the p53 molecule confers responsiveness toward Chk2. Interestingly, Grp is not involved in the regulation of Dmp53 in this system (Peters, 2002 and references therein).

Overexpression of human p53 in the Drosophila eye results in a striking reduction in eye size and a disruption of the ommatidia structure. Ommatidia are fused and exhibit a complete loss of bristles. Overexpression of Drosophila p53 produces a less severe phenotype, resulting in a reduced eye size with partial fusion of the ommatidia and some remaining bristles. Consistent with the known activity of the GMR promoter, the expression of both proteins was detected in the eye imaginal disc posterior to the morphogenetic furrow (Peters, 2002).

Expression of Drosophila or human p53 resulted in extensive apoptosis in the developing eye, as judged by acridine orange staining. The S-phase band posterior to the morphogenetic furrow, as visualized by BrdUrd immunohistochemistry, is present in both human p53 and Drosophila p53 transgenic flies. No considerable differences in cell cycle distribution or cell size could be discerned when human p53 or Drosophila p53-overexpressing flies were compared. These results show that overexpression of either human or Drosophila p53 resulted in apoptosis in the developing fly eye without detectable effects on cell cycle regulation (Peters, 2002).

To study the interaction between Chk2 and p53, the cDNA encoding the Drosophila homolog of human Chk2 was cloned. A kinase-dead form of Drosophila Chk2 was generated, in which the conserved aspartic acid at position 303 was mutated to Ala (DN-Chk2). Mutation of this amino acid has been shown to function as a DN mutation (Peters, 2002).

Drosophila Chk2 and DN-Chk2 were overexpressed in the Drosophila eye, under the control of the GMR promoter. No overt phenotype could be observed and no apoptotic cells were detected in the eye imaginal discs from third-instar larvae (Peters, 2002).

To study the genetic interaction between Drosophila p53 and Chk2, transgenic flies overexpressing p53 were crossed with transgenic flies overexpressing either wild-type or DN-Chk2. Coexpression of wild-type Chk2 and p53 results in a considerably more severe phenotype with almost complete loss of the eye compared to flies expressing p53 alone. Excessive apoptosis was detected in eye imaginal discs from third-instar larvae coexpressing Chk2 and p53. In contrast, coexpression of DN-Chk2 with p53 results in an almost complete rescue of the Drosophila p53-induced eye phenotype. Consistent with the observed eye morphology, no apoptosis could be found in eye imaginal discs in these animals. Analysis of p53 protein levels by immunohistochemistry in flies coexpressing wild-type or DN-Chk2 and p53 revealed no difference compared to flies expressing p53 alone, indicating that altered p53 protein levels do not account for the observed changes in eye phenotype (Peters, 2002).

A kinase assay was performed to confirm Drosophila Chk2 activity. Chk2 phosphorylates a synthetic Chk1/Chk2 peptide substrate. In the presence of increasing amounts of DN-Chk2, the kinase activity of the wild-type Chk2 is lost. These experiments show that the D303A mutant of Chk2 functions as a true DN protein, inhibiting the kinase activity of the wild-type protein (Peters, 2002).

Drosophila p53-mediated sensitivity to irradiation was investigated in wild-type and transgenic flies overexpressing a DN form of p53 (p53-D259H). The D259H point mutation corresponds to the human p53 mutational hotspot at position 273. In human p53, this amino acid directly contacts DNA and is required for DNA binding. Eye imaginal discs were dissected from third-instar larvae 4 h after gamma-irradiation, and apoptotic cells were visualized with acridine orange. Wild-type, unirradiated eye discs do not show apoptotic cells. In contrast, irradiated wild-type eye discs exhibit a high number of apoptotic cells both in the antenna discs and in the anterior and posterior part of the eye imaginal discs. Irradiated eye discs from flies overexpressing p53-D259H show abundant apoptotic cells only anterior to the morphogenetic furrow, where p53-D259H is not expressed. Posterior to the morphogenetic furrow, where p53-D259H is expressed, few apoptotic cells were present. These results show that p53 is implicated in gamma-irradiation-induced apoptosis in the Drosophila eye (Peters, 2002).

Irradiated eye imaginal discs overexpressing wild-type Chk2 posterior to the morphogenetic furrow show high numbers of acridine orange-positive apoptotic cells in this area. However, in DN-Chk2-overexpressing eye discs, there is almost a complete absence of apoptotic cells, comparable to discs overexpressing the DN form of p53. These results demonstrate a role for Chk2 in the regulation of gamma-irradiation-induced apoptosis in the developing eye in Drosophila (Peters, 2002).

The ability of Drosophila p53 to activate transcription in Drosophila S2 cells in the presence of wild-type or DN-Chk2 and Grp was analyzed by using a human p53 responsive CAT-reporter construct (PG13-CAT). Transfection of Drosophila p53 results in a dose-dependent increase in PG13-CAT reporter activity. Cotransfection of Drosophila Chk2 with Drosophila p53 causes a further increase in reporter activity. In contrast, expression of DN-Chk2 interfers with Drosophila p53-mediated transcription. In agreement with genetic studies, wild-type and DN Grp constructs have no influence on p53 transcriptional activity. p53 protein levels were unchanged by cotransfection with either wild-type or DN-Chk2 or Grp constructs, indicating that the observed effects are not caused by changes in Drosophila p53 protein levels. These results establish Chk2, but not Grp, as a regulator of p53 transcriptional activity in Drosophila (Peters, 2002).

Phosphorylation and acetylation have been implicated in regulating mammalian p53 stability and transcriptional activity after DNA damage. Sites of particular interest are the Ser-Glu amino acid pairs at positions 15 and 37 of human p53, which can be phosphorylated on the Ser residues by members of the ATM family of DNA damage-responsive kinases like ATM, Chk1, or Chk2. Based on homology, the nearby Ser-4-Glu-5 pair in Drosophila p53 might be a target for one of these kinases (Peters, 2002).

To define whether Drosophila p53 phosphorylation is part of the mechanism in the regulation of Drosophila p53 by Chk2, several point mutations (Drosophila p53-S4A, -S8A, -S16A, and -S20A) were introduced into the amino-terminal part of p53. Transfection of the various mutant Drosophila p53 molecules resulted in a similar increase in PG13-CAT-reporter activity as observed with the transfection of wild-type p53. Cotransfection of Chk2 with p53-S20A causes a strong increase in reporter activity similar to wild-type p53, whereas expression of DN-Chk2 interfers with Drosophila p53-S20A. Mutation of Ser-8 and Ser-16 also does not interfere with the transcriptional activation of p53 by Chk2 cotransfection. These results suggest that Ser-8, -16, and -20 are not required for the regulation of Drosophila p53 activity by Chk2 in Drosophila (Peters, 2002).

Mutating Ser-4 of p53, however, makes p53 unresponsive to Chk2. When Drosophila p53-S4A is cotransfected with wild-type or DN DmChk2, no increase or decrease of the PG13-CAT-reporter activity is observed; this indicates that Ser-4 might be crucial in the regulation of Drosophila p53 by Chk2. The D259H point mutation does not induce any reporter activity and is not affected by cotransfection with either wild-type or DN DmChk2 (Peters, 2002).

The analysis was extended into the Drosophila eye. Transgenic flies expressing some of the Ser mutants of Drosophila p53 were constructed. Overexpression of the Drosophila p53 mutants S4A and S20A in the fly eye results in a small eye phenotype identical to the overexpression of wild-type Drosophila p53. Genetic epistasis was performed crossing these flies to transgenic flies overexpressing either wild-type or DN-Chk2. In agreement with the results from S2 cells, Drosophila p53-S20A behaves indistinguishably from the wild-type Drosophila p53, whereas the S4A mutant results in a phenotype that is unaltered by coexpression with wild-type or DN-Chk2. These results confirm that Ser-4 of Drosophila p53 is important for its regulation by Chk2; mutating Ser-4 makes p53 unresponsive to Chk2 (Peters, 2002).

Upon DNA damage, cells respond with cell cycle arrest and activation of genes that coordinate DNA repair. If these mechanisms fail, genomic instability and predisposition for the development of cancer is the consequence. p53 is central to the execution of the DNA damage response. Regulation of p53 is coordinated mainly by two mechanisms: regulation of its stability and its activity. Ionizing irradiation induces phosphorylation of several amino-terminal amino acids of mammalian p53, a process that is mediated by both Chk1 and Chk2. Phosphorylation of human p53 at Ser-20 interferes with binding of murine double minute 2 protein (MDM2) to p53, thereby inhibiting degradation of p53 and leading to an increase in p53 protein levels. The mechanism underlying the regulation of p53 transcriptional activity is less clear, but phosphorylation of p53 could be involved in this process as well. In mammals, both regulatory mechanisms are tightly linked together and are difficult to differentiate. In Drosophila, genomewide searches have not identified an MDM2 homolog, allowing the investigation of the mechanism of p53-regulation independent of MDM2 (Peters, 2002).

This study demonstrates that in Drosophila, Chk2 is a potent activator of p53. Expression of both molecules in the Drosophila eye leads to a massive reduction in eye size caused by an increase in the number of apoptotic cells. Interestingly, the requirement for Chk2 to activate p53 is observed only upon overexpression or Drosophila p53 or after radiation treatment, since overexpression of Chk2 or DN-Chk2 alone does not show a phenotype. It has been shown that flies homozygously deleted for Chk2 are viable and lack obvious phenotype in the absence of irradiation. These observations suggest that endogenous p53 may function primarily in a DNA damage response. A kinase-dead, DN-Chk2 is able to almost fully rescue the p53-induced phenotype by inhibiting apoptosis in the eye imaginal discs. A functional interaction between Grp and p53 could not be detected, suggesting that Chk2 is the principal activator of Drosophila p53 in Drosophila. In an attempt to define the molecular mechanism of the observed interaction between p53 and Chk2, several point mutations were introduced into the amino-terminal part of the Drosophila p53 molecule and tested for their responsiveness to wild-type or DN-Chk2. The transcriptional activity and the eye phenotype caused by Drosophila p53-S4A mutant were unaffected by coexpression with either wild-type or DN-Chk2. These results indicate that Ser-4 of Drosophila p53 confers responsiveness to Chk2 (Peters, 2002).

It is hypothesized that the regulation of p53 by Chk2 in Drosophila could be mediated by direct phosphorylation. However, in vitro Chk2 kinase assays using wild-type or mutant glutathione S-transferase-p53 proteins as substrates did not detect any differences in phosphorylation between the various p53 molecules. One explanation for this phenomenon is that Chk2 does not directly phosphorylate Ser-4 and that a Chk2-regulated kinase is involved in this process. Alternatively, Chk2 may phosphorylate p53 at multiple sites, preventing discrimination between wild-type and Drosophila p53-S4A phosphorylation levels (Peters, 2002).

The mutationally altered p53 (p53-S4A) should be insensitive to activation by Chk2; however, p53-S4A overexpressing flies exhibit a phenotype similar to p53 wild-type transgenic flies. This finding is surprising given that DN-Chk2 almost completely rescues the Drosophila p53-induced small eye phenotype. There may be several reasons for this. Other signals not related to Chk2 that may or may not involve phosphorylation could be constitutively active, resulting in p53 stabilization and transcriptional activity. In this case, mutation of S4 could inhibit further activation of Drosophila p53 but would not prevent the constitutive activating signals present. Alternatively, introduction of the S4 mutation could introduce steric changes that confer increased stability to the p53 molecule. The increased stability could also account for the observed phenotype. A further possibility is that Chk2 additionally activates a kinase that phosphorylates Drosophila p53. In this situation, mutation of Drosophila p53 Ser-4 would prevent direct activation of p53 but would not interfere with indirect activation. This also would explain why DN-Chk2 could prevent Drosophila p53-mediated apoptosis (Peters, 2002).

This study has shown that radiation-induced cell death is inhibited by DN-Drosophila p53 and DN-Chk2 and that apoptosis resulting from Drosophila p53 overexpression can be inhibited by a kinase-dead form of Chk2. Chk2 regulation of Drosophila p53 activity is crucial for gamma-radiation-induced apoptosis, consistent with data showing that Chk2 functions upstream of Drosophila p53. In favor for this view, Chk2 null flies are refractory to apoptosis upon irradiation, suggesting that Chk2 controls p53-induced apoptosis. However, a scenario in which p53 and Chk2 make independent contributions to radiation-induced cell death, functioning in separate pathways cannot be ruled out (Peters, 2002).

Since Drosophila most likely does not have an MDM2 gene, the MDM2-mediated p53 degradation pathway could have emerged at a later point in evolution. The data presented here suggest that MDM2-independent regulation of Drosophila p53 is mediated by Chk2 and implies that Drosophila Chk1 does not function in this process. If MDM2-independent regulation of p53 is more ancient in evolutionary terms, then Chk2 may be the ancestral regulator of p53 activation. Thus, MDM2 and Chk1 probably emerged as regulators of p53 at a later evolutionary time (Peters, 2002).

The ability of Chk2 to regulate transcriptional activity of Drosophila p53 suggests that in mammals Chk2 might be involved in control of both p53 stability and activity. Generation of mouse 'knock-in' mutants for individual amino-terminal p53 phosphorylation sites should aid in resolution of apparent multiple roles of Chk2 in p53 regulation (Peters, 2002).

Genetic and microarray analyses have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).

p53 is involved in ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selDptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selDptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. This study investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. It was found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, it is proposed that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology (Dichtel-Danjoy, 2012)

The discovery of multiple p53 isoforms raises the question of their functional specificity in the spectrum of p53-mediated biological responses. In Drosophila, as the first and only p53 isoform identified in almost a decade, the truncated DΔNp53 isoform was initialy presumed responsible for all p53 activities. The identification of the full-length Dp53 isoform that contains a full N-terminal transactivation domain challenged this presumption. Using gain-of-function studies, this study examined the role of these two isoforms in apoptosis and apoptosis-induced proliferation. Both Dp53 isoforms were found to activate apoptosis but preferentially activate different DIAP antagonists (Rpr or Hid) for caspase activation DΔNp53 promotes wg expression and cell proliferation, independently of endogenous p53, whereas Dp53 is unable to do so. Dp53 was also found to be primarily responsible for damage-induced transcriptional activation of rpr, whereas DΔNp53 is the p53 isoform dedicated to promoting apoptosis-induced proliferation (Dichtel-Danjoy, 2012)

DΔNp53 binds a DNA damage response element in the rpr regulatory region, which is responsible for the induction of apoptosis in response to irradiation. This study showed that in wing imaginal discs, Dp53 is a stronger inducer of rpr expression than DΔNp53. Moreover, it was shown that DΔNp53 strongly induced hid expression, whereas Dp53 was only a weak inducer. Together, these observations suggest that the transcriptional competence of DΔNp53 differs from that of Dp53, and is consistent with a previous study showing that hid is transcriptionally induced by DΔNp53 in eye and wing imaginal discs. These results also suggest that some intrinsic ability to distinguish its activity for rpr and hid expressions is embedded in the N-terminus of the full length Dp53. Therefore, it is proposed that Dp53 is responsible for the damage-mediated activation of rpr for apoptosis, whereas DΔNp53 promotes apoptosis by inducing expression of hid. The physiological consequences of this functional segregation in apoptosis regulation by p53 isoforms remain to be determined (Dichtel-Danjoy, 2012)

Previous works have shown that apoptotic cells secrete morphogens that induce proliferation of surrounding cells. Although more clearly detected in 'undead cells', mitogen gene expression and extra proliferation have also been detected in genuine apoptotic cells. It was proposed that the initiator caspase Dronc leads to Dp53 expression, which in turn activates mitogen gene expression, but the specific roles of Dp53 and DΔNp53 remain to be established. This study showed that DΔNp53 is a potent inducer of wg expression both in the 'undead cell' and genuine apoptotic cell model. Specifically, this study showed that DΔNp53 induced wg expression independently of dronc. This indicates that DΔNp53 acts downstream of the apoptotic pathway to induce proliferation via the expression of wg. Thus, like JNK, DΔNp53 promotes proliferation independently of the apoptotic cascade. Further analysis will be required to determine the relationship between JNK and p53 isoforms in the induction of proliferation (Dichtel-Danjoy, 2012)

It has been proposed that in the apoptosis-induced proliferation process, there is a feedback loop that activates wg expression in 'undead cells' via Dronc and Dp53. The current results are consistent with such a feedback mechanism in which Dp53 and DΔNp53 induce apoptosis via rpr and hid, which in turn amplifies DΔNp53 via Dronc to promote wg expression. The results also suggest that the feedback loop not only functions in 'undead cells' but also in genuine apoptotic cells. Together, it is proposed that p53 isoforms act both upstream and downstream of the apoptotic pathway to promote wg expression and proliferation (Dichtel-Danjoy, 2012)

The results show that DΔNp53 is a potent inducer of wg expression in both wild-type and p53-null wing discs. In contrast, Dp53 only weakly increased wg expression in wild-type but not in p53-null flies. Therefore, the weak induction of wg expression by Dp53 in wild-type disc is likely dependent on the endogenous p53 gene. Further investigations will be required to determine if DΔNp53 is the only p53 isoform regulating wg expression or if another isoform such as Dp53ΔC or the one encoded by the recently annotated p53-RD transcript contribute as well to the regulation of wg expression (Dichtel-Danjoy, 2012)

One of the most intensely debated questions regarding Drosophila ΔNp53 isoforms is whether they have their own biological activity or exert a dominant negative activity on p53. The fact that DΔNp53 induced Wg expression independently of endogenous p53 gene indicates that DΔNp53 does not require p53 for this function. In vertebrate studies, zebrafish Δ113p53 and human Δ133p53 do not act exclusively in a dominant-negative manner toward p53 but differentially regulate p53 target gene expression to modulate p53 function. Similarly, the current results show that Drosophila p53 isoforms have the capacity to use distinct targets to orchestrate their biological functions; Dp53 promotes rpr expression, whereas DΔNp53 activates Hid and Wg expression in wing epithelium. Overall, it is proposed that balancing apoptosis and apoptosis-induced proliferation may represent one primordial function of the TP53 gene family, and that this function requires the expression of Dp53 and DΔNp53 isoforms in a tightly controlled manner. In vertebrate, this primordial functional capacity may be differently exploited by TP53, TP63 and TP73 to regulate specific aspects of death/proliferation in the equilibrium, depending upon tissues and physiological contexts (Dichtel-Danjoy, 2012).

Low levels of p53 protein and chromatin silencing of p53 target genes repress apoptosis in Drosophila endocycling cells

Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. This study shows that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. It was also found that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, the data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, these results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals (Zhang, 2014).

This study used Drosophila as a model system to define the molecular mechanisms for tissue-specific apoptotic responses to genotoxic stress. The data suggest that Drosophila endocycling cells repress the apoptotic response in two ways: low level expression of the p53 transcription factor and epigenetic silencing of the p53 target genes at the H99 locus (see Model for tissue-specific apoptotic responses in Drosophila). In mitotic cycling B-D cells, the major p53 protein isoform is p53A, and no expression was detected of the other predicted p53 protein isoforms. In endocycling salivary glands (SG) and fat body (FB) cells, all of the p53 protein isoforms, including p53A, were below the level of detection. The data suggest that, similar to human p53, Drosophila p53A is ubiquitinated and degraded by the proteasome in endocycling cells. Over-riding this proteolysis by forced expression of p53A did not activate H99 gene transcription or apoptosis in endocycling cells. These results suggest that downstream chromatin silencing of the H99 locus represses apoptosis in endocycling cells even when p53A protein is abundant. In contrast, over-expression of the longer p53B isoform was found to induced H99 gene expression and apoptosis in endocycling cells. However, the normal physiological expression of p53B protein and binding to the H99 locus was undetectable in endocycling cells, suggesting that the low level of expression of this isoform also contributes to the repression of apoptosis. In the absence of genotoxic stress, a paused RNA Pol II was found at the H99 gene promoters in both mitotic cycling and endocycling cells. In endocycling cells, this paused RNA Pol II complex is activated only when the longer p53B isoform is highly over-expressed. This result implicates polymerase activation as one step that is blocked after DNA damage or p53A over-expression. In mitotic cycling cells, both paused RNA pol II and p53A protein are bound to H99 promoters in the absence of stress, which may prepare cells for a rapid apoptotic response to DNA damage. In addition, the data suggest that p53A protein levels are regulated in mitotic cycling cells, which likely ensures that apoptosis occurs only in response to stress. Together, these results have revealed new mechanisms by which different cells in development modulate their apoptotic response (Zhang, 2014).

Previous evidence suggested that Drosophila p53 is regulated primarily by Chk2 phosphorylation and not protein stability. Consistent with this, it was found that in mitotic cycling cells p53A protein levels do not increase during the early response to radiation, a time when H99 genes are highly induced. At later times after irradiation, p53A protein levels increased only 2-3 fold, a magnitude that is proportional to the increase in p53 mRNA levels, as has been previously reported. Therefore, there is no evidence that the protein stability of p53A or other p53 isoforms changes in response to genotoxic stress. Both with and without genotoxic stress, the cellular levels of p53A protein were relatively low in mitotic cycling cells, and it was observed that the epitope tag on p53-Ch increased the abundance of p53A protein in p53 mutant but not p53 wild type cells. A cogent model is that the epitope-tag on p53-Ch partially interferes with p53A proteolysis in mitotic cycling cells, and that untagged p53 can promote the degradation of tagged p53-Ch in the same tetramer. Dampening of p53 protein levels may be critically important to prevent inappropriate apoptosis in the absence of stress. Consistent with this idea, it was found that elevated levels of p53A or p53B protein were sufficient to induce apoptosis in mitotic cycling cells even in Chk2 null animals. It is proposed that regulation of p53 protein levels in mitotic cycling cells tunes a threshold level of p53 protein that is poised to rapidly activate H99 gene expression when phosphorylated by activated Chk2 in response to DNA damage (Zhang, 2014).

In endocycling cells, however, no p53 protein isoforms were detected using a variety of methods. This tissue-specific regulation of p53 protein abundance is post-transcriptional because mRNA levels were similar between mitotic cycling and endocycling cells. This low level of p53 protein suggests that either its translation is repressed and/or that it is more efficiently proteolyzed in endocycling cells. A model is favored wherein it is p53 proteolysis that is regulated in endocycling cells (see Model for tissue-specific apoptotic responses in Drosophila). In support of this model, compromising proteasome function elevated p53A protein levels in salivary glands. Moreover, p53A is ubiquitinated in endocycling cells, and these modified forms increase when proteasome function is compromised, which is consistent with previous data that p53 turnover is regulated by ubiquitination in Drosophila S2 cells (S. Chen, 2011). In contrast, the longer p53B isoform remained undetectable when the proteasome function was reduced. Given that proteasome function was only partially compromised, the inability to detect p53B may reflect a more efficient degradation of this longer isoform. This idea is consistent with the known correlation between transactivation domains and ubiquitin-mediated proteolysis for mammalian p53 and other proteins (Zhang, 2014).

Although the results suggest that at least the p53A isoform is modified and targeted for degradation by a ubiquitin ligase, the identity of this ligase is unknown. The Drosophila genome does not have an obvious ortholog of the ubiquitin ligase MDM2, which targets p53 for degradation in mammalian cells. It remains possible that another family of ubiquitin ligases mediate p53 degradation in endocycling cells. Nonetheless, the results indicate that regulation of p53 is more similar between flies and humans than previously suspected, a finding that is interesting in the context of growing evidence for conserved p53 functions in flies and humans, including the response to hyperplasia (Zhang, 2014).

The data suggest that apoptosis in endocycling cells is repressed in part through chromatin silencing of the pro-apoptotic genes at the H99 locus. The evidence for silent chromatin marks H3K9me3 and H3K27me3 at H99 are consistent with cytogenetic observations that the H99 chromosome region (75C) is a highly-condensed constriction on salivary gland polytene chromosomes, and genome-wide studies that showed that H3K27me3 is enriched at H99 relative to other loci in salivary glands. Although genetic data indicate that knockdown of the writers and readers of H3K9me3 and H3K27me3 results in salivary gland apoptosis, it remains possible that knockdown of these regulators causes other types of stress that triggers apoptosis. It is important to note, however, that the results in endocycling cells are also consistent with a previous analysis that indicated that chromatin silencing at H99 dampens the apoptotic response during late embryogenesis (Zhang, 2014).

It was previously shown that the chromatin organization at the H99 locus impedes its DNA replication in endocycling cells. As a result, DNA at this locus is not duplicated every endocycle S phase, resulting in a final lower DNA copy number relative to euchromatic loci. This 'under-replication' is not the cause of apoptotic repression because it was found that in Suppressor of Underreplication (Su(UR)) mutants, in which the H99 locus is almost fully replicated, endocycling SG cells still did not apoptose in response to DNA damage (Zhang, 2014).

The data suggest that the apoptotic response to genotoxic stress is repressed in endocycling cells because paused RNA Pol II is not activated at rpr and hid genes. One possibility is that chromatin silencing in endocycling cells restricts recruitment of transcription elongation factors to H99 promoters. This study found that over-expressed p53A and p53B were similar in binding and recruitment of acetylation to rpr and hid promoters, but only p53B activated transcription and apoptosis in endocycling cells. This difference between p53A and p53B isoform activity is attributable to an additional 110 AA amino- terminal transactivation domain in p53B that is somewhat conserved with human p53. The N-terminus of over-expressed p53B, therefore, may bypass silencing of the H99 genes in endocycling cells by activating this paused RNA polymerase to promote transcriptional elongation. The normal biological function of these paused RNA pol II complexes may be to coordinate a rapid response to developmental signals that trigger apoptosis and autophagy of endocycling larval tissues during metamorphosis (Zhang, 2014).

It is proposed that low levels of p53 protein and downstream silencing of its target genes both prevent endocycling cell apoptosis. It has been proposed that the apoptotic response to genotoxic stress must be tightly repressed in polyploid endocycling cells because they have constitutive genotoxic stress caused by under-replication of heterochromatic DNA. Consistent with a possible linkage between the endocycle program and apoptotic repression, it was recently found that experimentally-induced endocycling cells (iECs) repress apoptosis independent of cell differentiation. It is clear that low levels of p53 protein is not the only mechanism of repression because over-expression of p53A resulted in abundant protein in endocycling cells, but failed to induce H99 transcription or apoptosis. Notably, over-expressed p53 had lower occupancy at H99 promoters in SG than B-D cells, another possible mechanism by which chromatin organization represses apoptosis downstream of p53. Moreover, the complete absence of endocycling cell apoptosis in response to IR suggests that both p53-dependent and p53-independent apoptotic pathways are repressed through silencing of the H99 locus, a point where these pathways intersect. These data, however, do not rule out the possibility that endocycling cells may use other mechanisms to repress the apoptotic response to DNA damage to ensure their survival despite the continuous genotoxic stress caused by under-replication (Zhang, 2014).

In mitotic cycling cells, the p53 protein and paused RNA Pol II were bound to rpr and hid gene promoters in the absence of stress. This suggests that Chk2 phosphorylation of p53 pre-bound to these promoters activates the paused RNA Pol II to elicit a coordinated and rapid transcriptional response to genotoxic stress. This is consistent with previous evidence that p53-dependent activation of rpr and hid transcription is readily detectable within 15 minutes of ionizing radiation. This strategy to rapidly respond to stress appears to be conserved to humans where it has been shown that p53 activates paused RNA Pol II at some of its target genes, by indirect or direct physical interaction of p53 with elongation factors. Together, these results suggest that mitotic cycling cells in Drosophila are poised to respond to stress by tuning a threshold level of p53 protein that is bound to H99 promoters with a stalled RNA Pol II (Zhang, 2014).

The data raise the question as to whether similar mechanisms repress apoptosis in mammalian polyploid cells. The transcriptome signatures of fly endocycles is very similar to that of polyploid cycles of mouse liver, megakaryocytes, and placental Trophoblast Giant Cells (TGCs), suggesting a conservation of cell cycle regulation. It is also known that mouse TGCs do not apoptose in response to UV. Moreover, evidence suggests that p53 protein levels decline when trophoblast stem cells switch into the endocycle and differentiate into TGCs, suggesting that the endocycle repression of apoptosis may be a theme conserved to mammals. The ubiquitin ligase that targets p53 for degradation in TGCs has not been identified, and it is possible that in both Drosophila and mouse the same family of ubiquitin ligases targets p53 for degradation in endocycling cells. In addition to developmentally-programmed endocycles, recent evidence suggests that cells can inappropriately switch from mitotic cycles into endocycles, and that this cell cycle switch contributes to genome instability and oncogenesis. Similar to developmental endocycles, apoptosis may be repressed in these endocycling cancer cells. In support of this idea, recent evidence showed that pro-apoptotic p53 target genes are epigenetically silenced in polyploid cancer cells. Therefore, the mechanisms that repress apoptosis in Drosophila endocycling cells may be conserved to humans and relevant to tissue-specific radiation therapy response and oncogenesis (Zhang, 2014).

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. This study establishes evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. Involvement was establised of two novel embryonic miRNAs-miR-981 and miR-317-for spatiotemporal regulation of cyclin B. In summary, the results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (Mei-41 and Grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, these results have established a novel role of Ago-1 as a regulator of the cell cycle (Pushpavalli, 2013).

The present study identified the role of Ago-1 in regulating cyclins, Cdk1 inhibitors, and p53 in Drosophila embryos. In the rapidly dividing cells of the Drosophila embryo, Ago-1 mutation led to severe mitotic disruption, as evidenced by chromosome fragmentation, missegregation, and abnormal mitosis during the precortical syncytial cycles. The present results demonstrate that Ago-1 modulated developmental arrays associated with establishing the cell cycle control, seeing that Ago-1 mutation down-regulated Cyc A, CycB3, p53, mei-41, and grp, but upregulated CycB transcripts. The reduction in grp and mei-41 levels suggests that the replication and DNA damage checkpoints are perturbed, allowing progression of mitosis before completion of DNA replication or DNA repair, which shows that the embryonic lethality is associated with Ago-1 mutation. These results are consistent with earlier findings that, in Drosophila, DNA replication checkpoint genes are activated to delay cell cycle progression during late cleavage stages (Pushpavalli, 2013).

The present study identified the role of Ago-1 in regulating cyclins, Cdk1 inhibitors, and p53 in Drosophila embryos. In the rapidly dividing cells of the Drosophila embryo, Ago-1 mutation led to severe mitotic disruption, as evidenced by chromosome fragmentation, missegregation, and abnormal mitosis during the precortical syncytial cycles. The present results demonstrate that Ago-1 modulated developmental arrays associated with establishing the cell cycle control, seeing that Ago-1 mutation down-regulated Cyc A, CycB3, p53, mei-41, and grp, but upregulated CycB transcripts. The reduction in grp and mei-41 levels suggests that the replication and DNA damage checkpoints are perturbed, allowing progression of mitosis before completion of DNA replication or DNA repair, which shows that the embryonic lethality is associated with Ago-1 mutation. These results are consistent with earlier findings that, in Drosophila, DNA replication checkpoint genes are activated to delay cell cycle progression during late cleavage stages. In the syncytial blastoderm, the essential replication checkpoint function is to prevent DNA damage and ensure proper repair by delaying the cell cycle (37). The reduced mei-41 or grp levels in the Drosophila embryo due to Ago-1 mutation may cause rapid progression from the S phase to mitosis, even before replication is complete (Pushpavalli, 2013).

The syncytial blastoderm stage in Drosophila involves only the S/M cycles and the expression patterns of cell cycle proteins; for example, mitotic cyclins are necessary for entry into and exit from mitosis. CycB is localized to microtubules during the blastoderm stage of Drosophila, and increased Cdk1/CycB activity causes shorter microtubules with a decreased metaphase and longer anaphase duration that leads to defective mitosis. The effect of miRNAs on CycB was also observed: Ago-1 affects the biogenesis of miRNAs that regulate CycB, leading to the increased expression of CycB. The elevated CycB levels found in the Ago-1 mutants showed that the microtubules were less stable and probably did not produce enough force to push the nuclei into the cortex, resulting in the observed decrease in pole cell formation. Thus, Ago-1 is necessary to ensure proper assembly of the mitotic spindle by controlling the timing of CycB expression, a prerequisite for proper nuclear migration during embryonic development. Moreover, less stable microtubules require a longer time to form proper metaphase structures. It is a well-established fact that PH3 staining indicates Cdk1 activity. In Ago-1 embryos, the PH3 signal often persists over the entire chromosome through the anaphase, whereas it is restricted to the telomeric regions during the wild-type anaphase, indicating the reminiscence of Cdk1 activity. In Drosophila wee1, a Cdk1 inhibitory kinase, functions downstream of mei-41 and is necessary for regulating the activity of Cdk1. Ago-1 mutant embryos reduced maternal wee1 transcript and hence reduced inhibitory phosphorylation of Cdk1, leading to rapid mitosis. Mutants with reduced maternal wee1 cause premature entry into mitosis, spindle fiber defect, and chromosome condensation defect (Pushpavalli, 2013).

The embryonic phenotypes such as mitotic asynchrony, mitotic catastrophe, and disruption of the actin cytoskeleton that are associated with Ago-1 mutation were restored to a normal pattern in the presence of one copy of mutant CycB, indicating the role of CycB in mitotic progression. From these results, it as confirmed that Ago-1 is necessary to ensure proper mitotic progression by controlling the timing of Cdk1/CycB expression, a prerequisite for proper microtubule assembly and nuclear migration during embryonic development (Pushpavalli, 2013).

The cell cycle checkpoint proteins control the timing of the regulatory pathways, such as DNA replication and chromosome segregation, with high fidelity. As in Drosophila, mammalian Atr and Chk1 are essential during embryogenesis One of the reasons for the observed segregation defects in these mutations in Drosophila is that damaged DNA or incompletely replicated DNA fails to trigger metaphase-to-anaphase delay. Recent data in mice indicate that depletion in the miRNA processing factors down-regulates a large number of cell cycle genes, including CycB1 (Ccnb1), implying that miRNAs positively regulate cell-cycle genes. In the current study, miRNAs, such as miR-774, miR-1186, and miR-466d-3p, activated CycB1 and regulated the cell cycle. Surprisingly miRNA down-regulated CycB1 during early embryogenesis in Drosophila was observed in the presence of wild-type Ago-1. The data clearly indicate that Ago-1 functions as a mitotic regulator by spatiotemporal regulation of Cdk1-CycB1, Chk1 (grp), and mei-41 (Pushpavalli, 2013).

In Drosophila, p53 has no role in damage-induced cell cycle arrest, but is absolutely necessary for genomic stability, which is achieved by its apoptotic rather than cell cycle function. It is speculated that decreased levels of p53 in the Ago-1 mutant may be associated with genomic instability in the early embryos when subjected to stress. Both mei-41 and grp function in the same genetic pathway and maternal mei-41 and grp are necessary for wild-type cell cycle delays during the late syncytial blastoderm stage. The reduction in maternal mei-41 and grp caused mitotic defects during the later syncytial divisions, indicating that gene expression defects in the late embryos are secondary consequences of the mitotic errors (Pushpavalli, 2013).

Recent studies have identified that noncoding miRNAs act as regulators of gene expression in multicellular eukaryotes and have been implicated in various diseases. miRNAs control cell cycle progression by regulating the cyclin-dependent kinases, cyclins, andcyclin-dependent kinase inhibitors. Mutation in miRNA-processing factors (Ago-1 and Dcr-1) up-regulate the levels of CycB mRNA and protein, which indicates their involvement in CycB regulation. This study has identified the miRNA-dependent regulatory circuit that up-regulates CycB expression. It is therefore suggested that expression of miR-981 in Drosophila embryo and its ability to fine tune CycB make it an optimal mechanism for maintaining a balanced level of CycB expression. To date, no mammalian homologue of miR-981 has been identified. The miRNAs miR-981 and miR-317 are also Ago-1-associated miRNAs, with greatly reduced expression under Ago-1 knockdown conditions in S2 cells. The in silico prediction of miR-317 in the red flour beetle (insect class) indicates that components of cytoskeleton are its target. This study found strong homology between Drosophila and the red flour beetle in the miR-317 mature sequence, and it is postulated that downregulation of miR-317 in Drosophila might have affected the normal functioning of the cytoskeleton, as well as CycB, in the Ago-1 mutant embryos (Pushpavalli, 2013).

In the case of mammals, it has been reported that in several tumor cell lines, the level of Ago-1 is significantly lower than in nontumor cells. Wilms' tumor exhibits the deletion of a region of human chromosome 1 that harbors the Ago-1 gene and is also associated with neuroectodermal tumors. The haploinsufficient maternal Ago-1 mutant, with all its mitotic defects, survives to develop into the adult only if zygotic transcription of Ago-1 occurs at about stage 9, in the absence of which it dies during the late embryonic stage (Pushpavalli, 2013).

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans

How a p53 enhancer transmits regulatory information was examined in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, this study found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restore target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation (Link, 2013).

This study present in vivo functional evidence that a single enhancer region can specify regulation of multiple targets in cis and in trans. Using tailored deletions, it was found that a p53 regulatory element controlled stimulus-dependent induction of multiple genes, with effects on targets that range from 4 kb to 330 kb throughout the Drosophila Reaper region. In these studies, the p53RE also regulated xrp1, a genetically unlinked target residing across the centromere. Furthermore, when transplanted to ectopic locations, contacts with target sites were re-established and regulation of some target genes was restored. Together, these functional studies offer compelling evidence that an enhancer transmits regulatory activity in trans through direct physical contact (Link, 2013).

In principle, long-range regulation of xrp1 by the native p53RE could involve local induction of an activator that subsequently induces distant genes, but this type of expression cascade would not explain the data. First, no correlation exists between the timing of RIPD gene induction and proximity to the p53RE. Second, cis targets in the Reaper interval encode products with no known function in the nucleus or in transcription. Third, conventional expression cascades would not account for the restoration of regulation and contacts by a transgenic rescue fragment. Therefore, the idea is favored that long-range regulation by the p53RE involves chromosomal architectures that link this enhancer to target genes regardless of whether they are in cis or in trans (Link, 2013).

Using either 3C or direct visualization, suggestive chromatin links between enhancers and distant genomic sites in trans have been reported. Few have been genetically tested, and, where functionally studied, detectable effects were not seen. the current finding that productive looping contacts can be assembled from a foreign site suggests that determinants of long-range chromatin interactions are modular and probably specified through sequence motifs, secondary structures, and epigenetic features that occur in vivo. It is further noted that the presence of contacts is not sufficient for target induction. For example, despite loops between the native p53RE and sites near grim or contacts between the ectopic p53RE and sites near rpr and skl, transcriptional induction was not seen. Therefore, elements that map outside of the rescue fragment or constraints imposed by flanking chromatin may also be important (Link, 2013).

Given that p53 enhancers in both flies and humans share a common sequence motif, mechanisms by which these response elements form long-range interactions in trans may be conserved. It will be interesting to see whether other enhancer regions share this property. Likewise, it will be important to determine whether these contacts are mediated through complexes involving proteins such as Cohesin, Mediator, Ldb1, Polycomb, or CTCF. If broadly generalized, the precedent established here could offer a framework that helps explain genetic disease alleles mapping to noncoding sequences (Link, 2013).

Functional Characterization

Yeast one- and two-hybrid assays were used to examine Dmp53 biochemical functions. For each assay, five Dmp53 derivatives were tested: full-length, i.e., Dmp53; N-terminal fragment, Dmp53(Nt); central DNA-binding fragment, Dmp53(Db); C-terminal fragment, Dmp53(Ct); and full-length with a point mutation in the DNA-binding domain, Dmp53(259H). The 259H point mutation corresponds to the human p53 mutational hotspot at position 273. In human p53, this amino acid directly contacts DNA and is required for DNA binding. To assay for DNA binding, the activation domain of GAL4 was fused to Dmp53 derivatives and they were tested in directed one-hybrid assays. Two reporter constructs were tested for each GAL4-Dmp53 hybrid: the negative control plasmid (pLacZi) contains a minimal promoter upstream of the lacZ gene; the tester plasmid (p53BLUE) contains three copies of a 20 bp consensus binding site for human p53 upstream of the minimal promoter. Full-length Dmp53 is able to activate transcription from the reporter containing human p53 binding sites, but not from the negative control reporter. None of the individual domains (Nt, Db, Ct) were able to activate transcription; significantly, the 259H mutation specifically eliminates activation. These results indicate that Dmp53 can interact with a consensus binding site for human p53 and that a residue required for sequence specific binding in human p53 plays a similar role in Dmp53 (Brodsky 2000a).

To test for transcriptional activation, the DNA-binding domain of GAL4 was fused to Dmp53 derivatives. Three GAL4-dependent reporter constructs were present: the lacZ gene under control of the GAL7 promoter, the ADE2 gene under control of the GAL2 promoter, and the HIS3 gene under control of the GAL1 promoter. While full-length Dmp53 is unable to mediate detectable transcriptional activation of any reporter, Dmp53(Nt) confers modest transcriptional activation of the lacZ reporter construct. However, this derivative does not activate sufficient transcription from the ADE2 and HIS3 constructs to allow growth on plates without adenine and histidine. The weak transcriptional activation due to the N terminus does not provide a strong conclusion about its in vivo function (Brodsky 2000a).

To test for oligomerization, a two-hybrid assay was used with the same reporters as described for the activation assay. Dmp53(Nt) was not tested with the lacZ reporter because it gives a weak positive signal in the activation assay; all other Dmp53 derivatives were tested against themselves in the two-hybrid assay since, tested alone, these fusions are unable to activate the GAL4-dependent reporters. For all three reporters, oligomerization activity is strongest with the Dmp53(Ct) fusions. Full-length Dmp53 and Dmp53(259H) exhibits weaker oligomerization activity in this assay. These results suggest that, despite the lack of sequence similarity in the putative tetramerization domain, the C-terminal region of Dmp53 does contain sequences that can mediate oligomerization (Brodsky 2000a).

The ability of wild-type and mutant forms of Dmp53 to activate transcription in Drosophila S2 cells was tested. Two variants with point mutations in the DNA-binding domain were also used. Dmp53(259H) contains a point mutation that directly disrupts DNA binding. Dmp53(155H) contains a mutation equivalent to the hotspot mutation 175H in human p53; this residue does not directly contact DNA and the mutation may disrupt p53 function by partially unfolding the DNA-binding domain. For each transfection, the localization of Dmp53 was examined using polyclonal antibodies raised against the C terminus of the protein. Wild-type Dmp53 and the two point mutant derivatives localize to the nucleus of transfected cells. In contrast, Dmp53(Ct) is found in both the cytoplasm and the nucleus (Brodsky 2000a).

Two reporter constructs that originally established the transcriptional activity of human p53 were used. One construct, PG13-CAT, contains a multimer of a human p53 binding site upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. A second construct, MG15-CAT, contains a multimer of a mutated site that is not bound by human p53. Transfection of these reporters into S2 cells does not cause increased CAT activity relative to control cells. Cotransfection of PG13-CAT and a vector expressing Dmp53 results in a 10-fold increase in CAT activity. Cotransfection of MG15-CAT and Dmp53 results in only a 2-fold increase in CAT activity. Expression of either point mutant or the C-terminal fragment does not increase activation of the PG13-CAT reporter over background levels. These results demonstrate that wild-type, but not mutant, Dmp53 can activate transcription from a promoter containing binding sites for human p53. Thus, the sequence conservation between human p53 and Dmp53 reflects functional conservation of DNA binding and transcriptional activation (Brodsky 2000a).

Most p53 mutants in human tumors can act as dominant-negative forms, typically leaving the tetramerization domain intact but disrupting DNA binding. Such variants are thought to suppress activity of the wild-type protein through the formation of inactive complexes. A test was performed to see whether the transcriptionally inactive forms of Dmp53 could inhibit transcription mediated by wild-type Dmp53 in S2 cells. Cotransfection of a 3-fold excess of Dmp53(155H) reduces transcription by wild-type Dmp53 by roughly 50% whereas cotransfection of either Dmp53(259H) or Dmp53(Ct) in similar amounts reduces transcription by wild-type Dmp53 to near background levels. Therefore, like their human counterparts, these Dmp53 variants can act as dominant-negative forms that partially or completely block activity of the wild-type protein. The dominant-negative activity of Dmp53(Ct) is consistent with the observation in yeast assays that this domain contains an oligomerization domain (Brodsky 2000a).

To determine whether the sequence similarity of Dmp53 and human p53 may reflect a conserved function as a DNA binding transcription factor, a test was performed to see whether Dmp53 can bind to a double-stranded DNA molecule containing a p53 recognition site using an electrophoretic mobility shift assay. Dmp53 binds specifically to oligonucleotides containing p53 binding sites from the human p21 and GADD45 genes, demonstrating that both DNA binding and target site specificity have been conserved through more than 500 million years of evolution. This interaction is specific, since addition of unlabelled wild-type GADD45 oligoduplex DNA competes for Dmp53 binding, whereas unlabelled mutant GADD45 oligoduplex DNA does not. Moreover, an anti-Dmp53 polyclonal antibody prevents DNA binding by Dmp53, and an anti-Dmp53 monoclonal antibody supershifts the Dmp53/DNA complex. It is interesting that human p53, which was expressed and tested in an identical assay, binds p53 binding sites only in the presence of the activating antibody PAb421. PAb421 is thought to act by associating with a region in the carboxyl terminus of p53 that normally negatively regulates DNA binding. The ability of Dmp53 to bind DNA without any activating treatments may indicate that a similar negative regulatory element does not exist in Dmp53 (Ollmann, 2000).

Dmp53 was expressed in Drosophila larval eye discs using glass-responsive enhancer elements. The glass-Dmp53 (gl-Dmp53) transgene expresses Dmp53 in all cells posterior to the morphogenetic furrow. The morphogenetic furrow marks the front of a wave of cellular differentiation that sweeps from the posterior to the anterior of the eye disc during larval development. Thus, gl-Dmp53 larvae express Dmp53 in all eye disc cells as they differentiate as well as in a subset of cells behind the furrow that undergo a final round of cell division before terminal differentiation. Expression of Dmp53 from the gl-Dmp53 transgene produces viable adults that have small, rough eyes with fused ommatidia. TUNEL staining of gl-Dmp53 eye discs shows that this phenotype is due, at least in part, to widespread apoptosis in cells expressing Dmp53. Similar results are seen when apoptotic cells are detected by acridine orange or Nile Blue. TUNEL-positive cells appear within 15-30 cell diameters of the furrow. Given that the furrow is estimated to move approximately five cell diameters per hour, this indicates that cells initiate apoptosis within 3-6 hr after Dmp53 is expressed (Ollmann, 2000).

The ability of p53 to induce apoptosis in some vertebrate cell types can be inhibited by overexpression of p21. The precise mechanism(s) through which p21 inhibits apoptosis is unknown, but direct inhibitory interactions with procaspase 3 and apoptosis signal-regulating kinase 1 have been reported. To determine if expression of human p21 can similarly suppress Dmp53-induced apoptosis, Dmp53 and p21 were co-expressed in the developing eye disc. p21 expression dramatically suppresses Dmp53-induced apoptosis in the disc as well as the adult rough-eye phenotype. This suppression does not appear to involve reduction of p53 protein levels, since matched disc samples from larvae expressing gl-Dmp53 or gl-Dmp53 plus gl-p21 show similar levels of anti-Dmp53 antibody staining. These data suggest that p53-related proteins in flies and vertebrates trigger apoptosis through similar p21-suppressible pathways. Surprisingly, similar inhibition of apoptosis could not be achieved by coexpression of the baculovirus p35 protein, a universal substrate inhibitor of caspases. Given that p35 inhibits human p53-induced apoptosis in lepidopteran and Drosophila cells, the lack of p35 suppression of apoptosis may reflect different rates and/or levels of Dmp53 and p35 protein accumulation (Ollmann, 2000).

In addition to its ability to affect cell death pathways, mammalian p53 can induce cell cycle arrest at the G1 and G2/M checkpoints. In the Drosophila eye disc, the second mitotic wave is a synchronous, final wave of cell division posterior to the morphogenetic furrow. This unique aspect of development provides a means to assay for similar effects of Dmp53 on the cell cycle. Transition of these cells from G1 to S phase in wild-type discs can be detected by bromodeoxyuridine (BrdU) incorporation into DNA. This transition from G1 to S phase is not blocked or delayed by Dmp53 overexpression from the gl-Dmp53 transgene. In contrast, expression of human p21 or a Drosophila p21 homolog, Dacapo, under control of the same Glass-responsive enhancer element completely blocks DNA replication in the second mitotic wave. However, overexpression of Dmp53 does affect M phase in the eye disc. In wild-type discs, an M phase-specific anti-phospho-histone antibody typically stains a distinct band of cells within the second mitotic wave. In gl-Dmp53 larval eye discs, this band of cells is present but is significantly broader and more diffuse, suggesting that Dmp53 alters the entry into and/or duration of M phase (Ollmann, 2000).

Examination was made of whether loss of Dmp53 function affected apoptosis or cell cycle arrest in response to DNA damage. In order to examine the phenotype of tissues deficient in Dmp53 function, dominant-negative Dmp53 alleles were expressed as transgenes under the control of tissue-specific promoters. Coexpression of Dmp53R155H with wild-type Dmp53 suppresses the rough eye phenotype that normally results from wild-type Dmp53 overexpression, confirming that this mutant protein has dominant-negative activity in vivo. The same result was obtained by expressing the Dmp53H159N protein. Unlike wild-type Dmp53, overexpression of the dominant-negative alleles using the glass enhancer or a constitutive enhancer (arm-GAL4) has no visible effect on normal development (Ollmann, 2000).

In mammalian systems, p53-induced apoptosis plays a crucial role in preventing the propagation of damaged DNA. DNA damage also leads to apoptosis in Drosophila. To determine if this response requires the action of Dmp53, dominant-negative Dmp53 transgenes were overexpressed in the posterior compartment of the wing disc. Wild-type wing discs show widespread apoptosis detectable by TUNEL staining 4 hr after X irradiation. When either dominant-negative allele of Dmp53 is expressed in the posterior compartment of the wing disc, apoptosis is blocked in the cells expressing Dmp53, whereas the anterior compartment displays a normal amount of X ray-induced cell death. Thus, induction of apoptosis following X irradiation requires the function of Dmp53. This proapoptotic role for Dmp53 appears to be limited to a specific response to cellular damage, because developmentally programmed cell death in the eye and other tissues is unaffected by expression of either dominant-negative Dmp53 allele (Ollmann, 2000).

Although the data strongly suggest that Dmp53 function is required for X ray-induced apoptosis, it does not appear to be necessary for the cell cycle arrest induced by the same dose of irradiation. In the absence of irradiation, a random pattern of mitosis is observed in third instar wing discs of Drosophila. Upon irradiation, a cell cycle block in wild-type discs leads to a significant decrease in anti-phospho-histone staining. This cell cycle block is unaffected by expression of dominant-negative Dmp53 in the posterior of the wing disc. Several time points after X irradiation were examined, and all gave similar results, suggesting that both the onset and maintenance of the X ray-induced cell cycle arrest is independent of Dmp53 (Ollmann, 2000).

An intergenic regulatory region mediates Drosophila Myc-induced apoptosis and blocks tissue hyperplasia

Induction of cell-autonomous apoptosis following oncogene-induced overproliferation is a major tumor-suppressive mechanism in vertebrates. However, the detailed mechanism mediating this process remains enigmatic. This study demonstrates that dMyc-induced cell-autonomous apoptosis in the fruit fly Drosophila relies on an intergenic sequence termed the IRER (irradiation-responsive enhancer region). The IRER mediates the expression of surrounding proapoptotic genes, and an in vivo reporter of the IRER chromatin state was used to gather evidence that epigenetic control of DNA accessibility within the IRER is an important determinant of the strength of this response to excess dMyc. In a previous work, it was shown that the IRER also mediates P53-dependent induction of proapoptotic genes following DNA damage, and the chromatin conformation within IRER is regulated by polycomb group-mediated histone modifications. dMyc-induced apoptosis and the P53-mediated DNA damage response thus overlap in a requirement for the IRER. The epigenetic mechanisms controlling IRER accessibility appear to set thresholds for the P53- and dMyc-induced expression of apoptotic genes in vivo and may have a profound impact on cellular sensitivity to oncogene-induced stress (Zhang, 2014).

p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex

The THO complex (THO; see Thoc5) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. This study examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When the endogenous p53 level was examined in the testis, it was found to be sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, whether the PI3K/AKT signaling is enhanced in the mutant flies was also examined. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together, these results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO (Moon, 2013).

A previous report showed the reduced life span and the increased susceptibility to the environmental stresses in mutant flies for Drosophila THO subunits. To understand the underlying mechanisms of these defects, this study investigated genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT pathways (Moon, 2013).

The following evidence suggests that defects in the function of THO cause up-regulation of p53 in a cell autonomous manner. First, endogenous level of p53 was sustained much longer during spermatogenesis in the male germline lacking THO compared to the control germline. FRT/FLP-based clonal analysis showed that p53 level was cell-autonomously sustained in the mutant clone. Second, mutations in the THO subunit genes elevated the level of overexpressed p53 in the eye, showing an increased sensitivity to p53-mediated apoptosis. Third, the sensitivity to p53-mediated apoptosis was directly correlated with the genetic background; the more severe defect in THO the genetic background had, the greater the sensitive to p53-mediated apoptosis was (Moon, 2013).

Why p53 is up-regulated in the flies with impaired THO? The fact that the nucleolar integrity was severely disrupted in flies lacking THO let to a postulate that disruption of nucleolus might be a good candidate to answer for this question. It has been known that disruption of nucleolus mediated stabilization of p53 in response to DNA damage and other stresses in mammalian cells. In addition, it has been reported that genetic disruption of nucleolus by knocking out murine TIF-1A gene caused p53 to be stabilized by dissociating it from MDM2. However, it is doubtful that the same is true in the Drosophila model. First, mutations in THO subunits caused nucleolar disruption only in certain types of cells including male germline and salivary gland cells. Moreover, this study failed to find any signs of nucleolar disruption in the THO-deficient eyes which were sensitized to p53-mediated apoptosis. Second, it has recently been reported that p53 level was not significantly increased in the eyes of viriato mutant in which nucleolar architecture was severely compromised. Finally, the Drosophila homolog of MDM2, which plays a key role in nucleolar disruption-mediated p53 stabilization in mammalian cells, has not been found to date. Consistent with these facts, in Drosophila, it has been shown that posttranslational modification rather than abundance was sufficient to activate p53 signaling in response to DNA damage. For these reasons, it is speculated that nucleolar disruption is not directly involved in the up-regulation of p53 in the THO mutant flies. An alternative possibility is that the phenotype of condensed chromatin structure in thoc5 might represent genomic instability, and the genomic instability could lead to activation of MNK, Drosophila homolog of CHK2, which activates p53 following DNA damage. But the fact that DNA damage activates p53 without significant changes in protein level is inconsistent with the current findings which show obvious changes of p53 level in the testis. Another alternative explanation for these ambiguities is that upregulation of p53 in the THO mutant may be restricted to certain limited types of cells, and the mechanisms underlying this may also be different depending on cell types. To clarify these issues further studies are certainly required (Moon, 2013).

In addition to p53 signaling, PI3K/AKT signaling was examined in the THO mutant flies. It has been well established that PI3K/AKT signaling pathway is a key player in regulating life span as well as body size in Drosophila. Combined with the previously reported lifespan reduction, increased body size in the THO mutant flies compared to control is well matched with the known phenotypes of mutant flies with defects in PI3K/AKT signaling pathway. Although no global increase in PI3K/AKT signaling was detected in the THO mutant flies, a cell-autonomous elevation of endogenous p-AKT level in the mutant male germline provided a piece of evidence for the relevance of Drosophila THO with PI3K/AKT signaling (Moon, 2013).

Another interesting finding in this report is that the levels of both p53 and p-AKT are very high in the wild-type male germ germline. If p53 is important for spermatogenesis, why p53-null flies are not sterile? With regard to the female germline, it has recently been reported that DNA double strand breaks formed during meiotic recombination provoked activation of p53, and unrepaired DNA breaks during meiotic recombination led to sustained p53 activity. But it has been known that meiotic recombination is very rare in the Drosophila male germline, and this study showed that p53 was detected only in the pre-meiotic germline. Thus, it is unlikely that the role of p53 in male germline is similar to that in female germline. Certainly these issues will be a good topic for future study (Moon, 2013).

Taken together, this study found the significant genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT signaling pathways. Both signalings were up-regulated by THO dysfunction in a cell autonomous manner. However, it seems unlikely that THO generally plays a major role in regulating these signaling pathways, because not only Western blot analysis of whole-fly extract, but also FRT/FLP-mediated clonal analysis in the imaginal discs showed no significant changes in the endogenous levels of both p53 and p-AKT in the THO mutants. It seems rather likely that the effect of THO dysfunction on these two signaling pathways is different depending on cell types; it might be generally mild in most cells except certain types of cells such as germline (Moon, 2013).

Protein Interactions

Drosophila UTX coordinates with p53 to regulate ku80 expression in response to DNA damage

UTX is known as a general factor that activates gene transcription during development. This study demonstrates an additional essential role of UTX in the DNA damage response, in which it upregulates the expression of ku80 in Drosophila, both in cultured cells and in third instar larvae. UTX mediates the expression of ku80 by the demethylation of H3K27me3 at the ku80 promoter upon exposure to ionizing radiation (IR) in a p53-dependent manner. UTX interacts physically with p53, and both UTX and p53 are recruited to the ku80 promoter following IR exposure in an interdependent manner. In contrast, the loss of utx has little impact on the expression of ku70, mre11, hid and reaper, suggesting the specific regulation of ku80 expression by UTX. Thus, these findings further elucidate the molecular function of UTX (Zhang, 2013).

To understand the mechanism underlying UTX function in tumorgenesis, this study explored whether UTX is involved in DNA damage response in Drosophila. This study found that UTX plays an essential role in DNA damage response by upregulation of ku80, which is uniquely required for p53 activated ku80 expression. In addition, the gene activity of utx is correlated with loss of histone demethylation at H3K27, suggesting that UTX could function as a histone demethylase and serve a gene-specific co-activator of p53 gene activation. This study therefore provides an example that p53 target genes expression may be regulated at the level of histone modifications (Zhang, 2013).

It is clear that p53 plays a pivotal role in the DNA damage response (DDR). One of the functions of p53 is to activate its target gene after DNA damage as transcription factor. For instance, p53 has been best characterized in regualting expression of cell cycle genes and apoptosis gene. However, the precise regulation mechanism of p53 is still not clear. It is interesting that in Drosophila ku80 upregulation mediated by p53 requires UTX, but not other genes in related to DNA repair and apoptosis. However, reduced H3K27me3 levels were found in apoptotic genes, which raises the possibility that there could be additional histone demethylases participating in DDR pathways that coordinate with p53 regulating expression of hid and reaper after DNA damage, and remaining to be determined in further studies. In contrast, reduced H3K27me3 levels were not detected in the ku70 promoter region following IR treatment. Further analysis revealed that the H3K27me3 level in the ku70 promoter region was lower than at the ku80 promoter. The expression of ku70 is independent of UTX, possibly due to the extremely low levels of H3K27me3 in the ku70 promoter region, which might not require demethylation for the expression of ku70 to occur. Thus, the data demonstrate the complexity of the function of p53 in the activation of target genes in response to DNA damage, particularly in terms of histone modification and the action of different demethylases (Zhang, 2013).

UTX has been reported to participate in many biological processes, including cell fate determination and animal development, largely depending on the transcriptional regulation of the target genes of UTX. UTX appears to play an important role in orchestrating several histone markers, including acetylation at H3K27 and ubiquitination at H2A, and mediates derepression of polycomb (Pc) target genes, such as HOX genes, by affecting Pc recruitment. These roles are consistent with UTX being a histone demethylase specific for H3K27. However, sporadic mutations of UTX have been linked to many types of human cancers and it remains to be elucidated whether this is also sufficiently explained by its enzymatic activity. Indeed, several studies have proposed a role of UTX independent of its demethylase activity in chromatin remodeling and embryonic development. This study found UTX is also involved in DDR by upregulation of ku80 in Drosophila after IR. Although there are no available data demonstrating that ku80 mRNA levels are increased following DSBs in human cells, the current data provide evidence that UTX functions to maintain genome stability and sheds light on the mechanism underlying the function of UTX in human cancer. Recent studies suggest that loss of polycomb-mediated silencing might promote the upregulation of DNA repair genes and facilitate the recovery of cells from genotoxic insults. UTX might therefore be required for various cell defense mechanisms under environmental stress, thereby contributing to tumor suppression (Zhang, 2013).


DEVELOPMENTAL BIOLOGY

Embryonic

The expression of Dmp53 transcripts during embryogenesis was examined to assess potential roles for Dmp53 during Drosophila development. Dmp53 RNA is maternally loaded into oocytes and is abundant until cellularization of the blastoderm. Zygotic expression of Dmp53 begins at cellularization and is initially ubiquitous. At midembryogenesis, Dmp53 RNA levels are highest in the mesoderm and gut, with only low levels of RNA detectable in the epidermal and neural cell layers. As development proceeds, the expression of Dmp53 becomes progressively more restricted and falls dramatically in all tissues except for the primordial germ cells and a small patch of hindgut cells. Although one must use caution when inferring function from expression data, the high levels of Dmp53 RNA in germ cells is likely to be significant because germline p53 expression is a common feature in species ranging from clam to human. This conservation of expression suggests an important function for p53 in germline development (Ollmann, 2000).

A developmental profile of Drosophila p53 RNA levels shows that p53 is present throughout development. RNA levels seem to be highest during early embryogenesis and in females, suggesting a maternal contribution. Consistent with this notion, p53 mRNA is found in cells of the egg chamber that provide the maternal contribution, the nurse cells, but p53 mRNA is undetectable in the somatic follicle cells of the egg chamber. Additionally, p53 RNA is expressed ubiquitously in early embryogenesis. The staining inside the blastoderm embryo probably stems from the maternal contribution (Jin, 2000).

Insects and mammals diverged ~150 million years ago in evolution. The striking conservation of p53 in the two systems suggests that p53 is an early-evolved gene and its functions are under strong selection pressure. p53 expression in Drosophila exactly mirrors that of Xenopus and is also very similar to that of mice in early embryonic development. The expression pattern in mice may reflect the function of p53 as teratogenesis suppressor, as shown by the observation that p53-null mice had a higher teratogenesis rate and lower abortion rate upon gamma-irradiation than wild-type mice. This conserved expression pattern in all species examined to date suggests that one major function of p53 might be protecting the genomic integrity of early embryos and that of the germ-line cells. This would of course be critical to ensure proper development of an individual organism, eliminating embryos with DNA damage and genetic defects. Therefore this function is strongly selected and maintained during evolution. The tumor suppressor function of p53 in differentiated somatic cells might be a more recently evolved adaptation. As organisms appeared with long lifespans, activating dividing cells in the adult, the selection pressure to eliminate somatic mutation concomitantly increased. Although p53 minus mice appear to develop normally, it would be interesting to see if p53 minus flies have an elevated degree of developmental defect and germ-line instability (Jin, 2000).

Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity

The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. This paper provides evidence that Drosophila p53 has a previously unidentified role in coordinating the compensatory growth response to tissue damage. The sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. These processes are regulated by p53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. These results indicate that once induced, p53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. These data suggest that p53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage (Wells, 2006; full text of article).

The repair of tissue after cellular damage can be critical to the survival of the animal. Previous studies demonstrated that undead cells stimulate the proliferation of neighboring cells, providing a model for how damaged and dying cells contribute to the replacement of lost tissue. With this model, it was found that the wing imaginal disc responds to this damage as a whole by deploying a multi-step process that ends with compensatory growth. p53 functions in a dronc-dependent manner at each step of the tissue-replacement process. Furthermore, p53 and the initiator caspase dronc may be generally required for tissue recovery in imaginal discs, because it was found that blastema formation was significantly impaired during regeneration induced in either p53 or dronc mutant leg discs (Wells, 2006).

The data suggest that p53 is induced and becomes functional in undead cells by a mechanism that does not require DNA-damage sensing or activation of the stress kinase AMPK. Rather, Dronc, an initiator caspase homologous to caspase-9, is necessary and sufficient to induce all aspects of the growth regulation by p53. It is not known how Dronc activity results in p53 expression and activity in these cells, but many caspase substrates are not directly involved in apoptosis. As an example, one of the first caspase substrates identified was the cytokine IL-1β, which regulates many aspects of the inflammatory response. Induction of p53 mRNA in undead cells is prevented in dronc mutant discs, and thus it is possible that a regulator of p53 is cleaved by Dronc, leading to its expression and ultimately to its ability to regulate the compensatory growth response in the imaginal discs. Regardless of the molecular mechanism, the data argue for direct communication between Dronc and p53 in response to tissue damage (Wells, 2006).

Collectively, these experiments imply that p53 serves as a master coordinator of tissue repair in imaginal discs, regulating both cell-autonomous and non-cell-autonomous cell-cycle arrests, the expression of the pattern-regulating genes wg and dpp, and compensatory cell proliferation and growth. Based on these results, it is suggested that cellular damage activates Dronc, which in a nonapoptotic role causes the induction of p53 mRNA and leads to p53 activity. It is proposed that p53 then acts as an overall damage monitor, in a role that includes its conserved functions in apoptosis (here, induction of hid and rpr expression) and growth arrest (by repression of stg/cdc25), but also allows for induction of signals that promote compensatory growth of the disc. The results suggest that p53 monitors tissue damage through a feed-forward loop with Dronc and the pro-apoptotic genes hid and rpr, which both amplifies and sustains the growth-regulating signal (Wells, 2006).

An intriguing puzzle left unanswered by these results is why the growth response to undead cells occurs only several days after they are generated: both HhGal4 and EnGal4 drive expression of Hid or Rpr from early embryonic stages, yet even with careful observation no growth phenotype was detected until the middle part of the third instar. Caspases are active in cells expressing Hid or Rpr + P35 at early time points, indicating that these cells are not immune to the apoptotic response early in development. The genes involved in the apoptotic response are subject to many levels of control, including that by micro-RNAs (miRNAs). Hid protein expression, for example, is suppressed by Bantam, a miRNA highly expressed early in imaginal disc development, but declining as development progresses. It is likely that rpr is also regulated by miRNA gene silencing. Hence, the delay of the growth response in discs with undead cells may reflect a requirement for threshold levels of these factors to fully activate the feedback loop. At the very least it emphasizes that the regulation of growth and cell death during wing disc development is complex and has multiple inputs, many of which are poorly understood (Wells, 2006).

Activity thresholds appear to play an important role in the processes induced by undead cells. Dronc, for instance, is haploinsufficient for its effect in compensatory proliferation. It is possible that the apoptotic functions of Dronc require a relatively low activity level, but that high Dronc activity allows activation of the p53-dependent tissue-damage response. Regulation of Dronc by critical activity thresholds could provide the animal some regenerative capacity and increase its chances for survival when conditions are appropriate for tissue repair (Wells, 2006).

As expected given its role in coordinating many cellular behaviors, p53 modulates the activity or expression of myriad effectors. Regulatory effectors of Drosophila p53 are only beginning to be identified, and these data add stg/cdc25 to the list. One of the first detectable disc responses to undead cells is G2 arrest, mediated by loss of stg mRNA. Cdc25 is also regulated by vertebrate p53 but is inhibited post-transcriptionally by p53-dependent 14-3-3 activity (Levine, 2006). Experiments with irradiated p53 mutant animals have not revealed a cell-cycle arrest role. However, recent work indicates that dp53 also regulates a G1 checkpoint under conditions of metabolic stress; thus, like vertebrate p53, Drosophila p53 can activate both a G1 and a G2 checkpoint in response to tissue stress. Other effectors and targets involved in the compensatory proliferation process remain unknown, although expression profiling experiments from irradiated p53 mutants identified several potential targets, several of which do not have obvious roles in cell death or DNA repair (Wells, 2006).

How does Drosophila p53 control the signaling that leads to compensatory proliferation? The events observed — G2 arrests in two different cell populations, ectopic expression of wg, and compensatory growth — are all regulated by p53. It is possible that p53 directly and coordinately controls each of these processes by regulating the expression of specific effectors. However, because the response is both cell autonomous and non-cell autonomous, the idea is favored that these processes are interdependent, but sequentially activated. It is envisioned that as a result of Dronc activation in undead cells, p53 induces loss of stg, leading to G2 arrest, and hid and rpr expression, initiating the feedback loop. It is postulate that cells then synthesize factors that stimulate their survival and proliferation. The non-cell-autonomous arrest in the anterior compartment may be a secondary effect of undead cells in the posterior. High levels of TUNEL activity was observed in the anterior cells of these discs, which could feasibly activate p53 in those cells. However, no p53 mRNA was detected in anterior cells. One possibility is that the DNA fragmentation resulting from dying anterior cells could activate ATM and Chk2 in those cells. Consistent with this, although loss of either of these kinases did not affect undead cell induction of Wg expression or compensatory growth, the cell-cycle arrest in anterior cells was reduced in a fraction of atm and chk2 mutants (Wells, 2006).

What is the growth-stimulating signal induced by undead cells? While its identity is still unclear, both Wg and Dpp have been implicated in this role. This makes sense, because Wg and Dpp are the major pattern organizers of all imaginal discs and are also involved in regulating their growth, and furthermore they are known to be induced in disc regeneration. However, although wg and dpp are ectopically expressed in undead cells, it was found that targets of both are sharply downregulated, specifically in the undead cells. These data also show that undead cells are able to proliferate and contribute to the compensatory growth. Thus, although the nonautonomous stimulation of growth (anterior cells near the A/P boundary) could be due to increased Dpp signaling, it is suspected that the autonomous growth stimulation is due to other, unidentified factors (Wells, 2006).

This study identified a growth-regulatory role for p53 that seems counter to its role as a tumor suppressor in vertebrates. However, it is speculated that the ability of p53 to sense and respond to tissue damage and promote compensatory proliferation and regeneration in Drosophila reflects an ancestral function, aspects of which have been appropriated for developmental processes and distributed among p53, p63, and p73 during vertebrate evolution. Although p63 and p73 initially were proposed to have evolved as duplications of p53, reanalysis of the phylogenetic relationship between the three family members has suggested that p63 may be the ancestral gene. p63 and p73 are structurally similar to p53 but contain an additional SAM domain. p53 is the sole member of the family encoded in the Drosophila genome, and although dp53 does not contain a SAM domain, based on the sequence of the DNA binding domain, the most highly conserved region of p53, it is more related to vertebrate p63 than to p53. After irradiation, cell-cycle arrest is not p53 dependent in either Drosophila or the nematode C. elegans, and therefore it has been proposed that the ancestral p53 function is apoptosis, rather than a “repair, then death” response when damage cannot be repaired. The experiments argue that as in vertebrates, p53 plays a role in cell-cycle arrest after tissue damage. The additional functions of p53 in promoting cell proliferation may have been conserved in p63, which regulates progenitor cell renewal in the epidermis. Other processes that require cell renewal may also be regulated by p53. For example, p53 mutants are reported to have fertility defects, so it is tempting to speculate that stem cell renewal in the gonad requires this previously unappreciated role of Drosophila p53 (Wells, 2006).

p53 activity is selectively licensed in the Drosophila stem cell compartment

Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, this study found that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the Ras(V12) oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53(-) stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis (Wylie, 2014).


EFFECTS OF MUTATION

Mutant alleles of Dmp53 analogous to the R175H (R155H in Dmp53) and H179N (H159N in Dmp53) tumor-derived mutations in human p53. These mutations in human p53 produce proteins with dominant-negative activity, presumably because they cannot bind DNA but retain a functional tetramerization domain. Thus, DNA binding by any tetramer that incorporates the mutant protein is disrupted. Both Dmp53R155H and H159N proteins inhibit binding of wild-type Dmp53 to a p53 binding site, although they do not bind to DNA themselves. These mutant forms of Dmp53 are useful tools to test the function of wild-type Dmp53 in vivo (Ollmann, 2000).

The similarity between Drosophila and human proteins prompted an exploration of the role Drosophila p53 plays in vivo. It is known that p53 exerts its role as a tumor suppressor partially through initiation of apoptosis. Consistently, expression of human p53 in the fly eye initiates apoptosis. It was reasoned that overexpression of wild-type Drosophila p53 might trigger an ectopic cellular response, thereby revealing some of its in vivo function. Using the UAS/GAL4 binary expression system, Drosophila p53 was expressed under the control of a photoreceptor specific promoter, gmr-GAL4. One of five transgenic fly lines tested shows a rough eye phenotype. At least 2-fold higher p53 RNA levels were observed in the line that shows the phenotype than in any of the other lines. Because the eyes of flies overexpressing Drosophila p53 are smaller than those of wild-type controls, it seemed likely that the observed phenotype was partially caused by ectopic apoptosis. To test this possibility, third-instar eye imaginal discs from animals overexpressing p53 were subjected to an acridine orange staining to visualize cell death. Overexpression of Drosophila p53 in the developing retina causes an increase in cell death. This observation is in accordance with an apoptosis-inducing function of p53. Moreover, ubiquitous expression in transgenic Drosophila results in a high percentage of lethality. This observation is consistent with an induction of extensive apoptosis in essential tissues of the fly, thereby reducing viability (Jin, 2000).

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).

Wee1 kinases may play a role in regulating genome stability as evidenced by a genetic interaction with Drosophila p53. In humans, the p53 tumor suppressor promotes apoptosis in cells that have suffered DNA damage. Overexpression of Drosophila p53 in the eye promotes extensive cell death by apoptosis, resulting in extremely defective eyes. There is significant suppression of the p53 overexpression eye phenotype by coexpression of either GMR-Dwee1 or GMR-Dmyt1, suggesting that these Cdk1 inhibitory kinases can negatively regulate p53-induced apoptosis. Since Cdk1 activity has been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition. Other reports relevant to this issue are somewhat contradictory, however. In human cell culture, Wee1 can inhibit granzyme B-induced apoptosis; furthermore, Wee1 appears to be downregulated through a p53-dependent mechanism, suggesting that p53 regulation of Wee1 might normally occur during this process. In contrast, Wee1 activity can actually promote apoptosis in a Xenopus oocyte extract system. Further studies are clearly needed to establish the physiological significance of any purported roles for Wee1 or Myt1 in regulating apoptosis, p53-dependent or otherwise (Price, 2002).

p53 protects the Drosophila retina during a developmentally regulated DNA damage response

Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. The UV response by cells of the Drosophila retina have been explored. The retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells include morphology changes and apoptotic cell death; these defects can be completely accounted for by DNA damage. Cell death, but not morphological changes, is blocked by the caspase inhibitor p53. Utilizing genetic and microarray data, evidence is provided for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, Reaper has no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV-mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue (Jassim, 2003).

Previous work demonstrates that dominant-negative versions of p53 suppress ionizing radiation-induced cell death in larvae, and p53 is able to bind to an upstream P53 consensus binding site within the reaper locus. Surprisingly, flies lacking both functional copies of p53, due to an introduced stop codon, consistently display an enhanced retinal sensitivity to UV light and an increase in apoptotic cell death. It was not possilbe to assess the effect of overexpressing wild-type P53 on UV-mediated damage, since overexpression alone leads to extensive cell death in the retina. The protective effect of p53 is likely mediated by its transcriptional activity: retina-targeted overexpression of two different dominant-negative forms of p53 that lack DNA binding activity (p53259 and p53CT) leads to a similar increase in retinal sensitivity (Jassim, 2003).

Most previous studies have found that P53 acts to promote cell death, in contrast to the current observation. Potential explanations for the protective effect of p53 following irradiation include: (1) p53 is required for cell cycle arrest to provide the time required by cells to repair; or (2) p53 helps direct DNA damage repair. The first possibility is unlikely in the current experimental paradigm, as nearly all of the cells in a 24 h APF pupal retina have been post-mitotic for >14 h. Consistent with this view, BrdU staining of untreated or irradiated retina at 24 h APF indicates no cell divisions (other than the normal, few cell divisions that complete the interommatidial bristle organules). Therefore the photoreactivation repair system was utilized to determine whether loss of p53 compromises DNA repair (Jassim, 2003).

Light-mediated photorepair of genotypically p53-null mutants for 2 h results in a complete reversal of the retinal phenotype. Reducing the dose of photorepair to 30 min still fully restores wild-type retinae, but is less efficient at restoring p53 mutants. This suggests that p53 mutants are more sensitive to UV damage because DNA repair is impaired. Because full rescue of the retina requires intact photorepair and nucleotide excision repair, this result suggests that one of these pathways is deficient in a p53 mutant; alternatively, p53 could function at a downstream step, for example preventing Diap1 degradation. To explore further the potential connection between p53 and DNA repair, a single mutant copy of p53 and of the nucleotide excision repair mutant xpg/mus201D1 were placed in trans to determine whether they demonstrate a dominant genetic interaction. Irradiation of flies containing a single mutant copy of either of these genes by themselves yielded a phenotype similar to irradiated wild-type retinae. Combining a single copy of p53 and xpg/mus201D1 in trans results in a strong enhancement of the UV phenotype. A similar genetic interaction was also observed between p53 and mei-9. Taken together with the repair data, this suggests that p53 promotes DNA repair and cell viability, primarily by acting to enhance nucleotide excision repair (Jassim, 2003).

UV irradiation directs both morphology changes and cell death in the developing retina. Both classes of defects are fully rescued by reversal of DNA damage and exacerbated by removing DNA repair genes, indicating that DNA damage is the primary or sole source of the cells' response. It is presumed the widespread apoptosis upon irradiation is a direct response to DNA damage, although it cannot be rule out that death as a secondary response to cells' release from the apical surface ('anoikis'). In either case, cell death -- but not morphology defects -- is fully blocked by p53, indicating that apoptotic death is caspase dependent. Based on genetic evidence, Dronc, which is not fully inhibited by p53, may play a role in these morphology changes. If so, this activity must diverge before it reaches downstream p53-sensitive caspases (Jassim, 2003).

The major inhibitor of caspase activity in Drosophila is Diap1. Stability of Diap1 is the central point of cell death regulation in the developing retina and this is also true during UV irradiation in the retina. Genetic and microarray results further suggest that the retina requires Hid as a primary regulator of Diap1 stability during UV irradiation. Hid may represent the primary regulator of Diap1 during UV (versus ionizing) irradiation response by the fly. Alternatively, the retina utilizes Hid as its major RHG factor during its development, and this preference may simply extend to its response to UV; other tissues may exploit different Diap1 regulators that reflect their use during development (Jassim, 2003).

This close similarity between periods of developmental cell death and sensitivity to DNA damage is also seen in the developing mammalian CNS. The extreme sensitivity of the developing CNS to irradiation has limited the usefulness of radiation therapy as a treatment for pediatric CNS tumors. Although clear links have been made between the status of a cell in the cell cycle and its response to DNA damage, this study, performed on a developing post-mitotic nervous system, suggests a mechanism behind this radiosensitivity. Hid is both necessary and sufficient to confer radiation sensitivity to at least the interommatidial cells, mirroring its requirement during normal retinal development. Recent work in the mammalian nervous system indicates that the functionally related RGH family member Smac is capable of conferring cell death sensitivity to neurons. The factor has yet to be identified that confers a similar sensitivity to, for example, the photoreceptor neurons in irradiated Drosophila retinae (Jassim, 2003).

Reaper, also an inhibitor of Diap1 function, is thought to be of central importance during the larval wing disc's response to ionizing radiation; however, no evidence was found for its use in the retina. This suggests that p53 -- which is active in this experimental paradigm -- can act in a manner independent of any regulation of Reaper. p53 has been shown to be capable of targeting sequences upstream of reaper, and it is not known if p53 is required for the observed upregulation in hid expression in irradiated retinae; the results suggest its primary targets may be DNA repair enzymes (Jassim, 2003).

Remarkably, although all the cells in the pupal retina are sensitive to UV during the 18-25 h APF window, only the interommatidial cells are rescued when Hid activity is removed. Reaper has been ruled out as a regulator of the retina's UV response, leaving open the question regarding what factor(s) acts to destabilize Diap1 within the ommatidial core. An equally intriguing question is why the ommatidial cores demonstrate a window of UV competence identical to the interommatidial cells; no cell death occurs within this cell population at any stage of normal development (Jassim, 2003).

Mammalian P53 can arrest proliferation to permit repair or it can promote cell death, depending on the cellular context. In the fly retina a different result was observed: p53 is required to prevent cell death following UV irradiation, but its role is unrelated to cell cycle regulation as these retinal cells are post-mitotic. Nor is it likely to be linked directly to caspase stability, as in the case of Diap1. Instead, genetic and photorepair evidence is presented that p53 functions to promote DNA repair and viability. There is growing data that supports the idea that P53 can direct repair of DNA damage; the current work provides in situ support for this proposal. Recent work has reported that p53 mutant larvae are more sensitive to ionizing radiation; this effect was ascribed to a block in the death of severely damaged cells. An alternative interpretation is proposed: the cells of irradiated larvae can not repair DNA damage proficiently, leading to an increased likelihood of cell death as well as the observed increase in mutation rates. The connection between DNA repair, p53 and transcription of repair enzymes remains to be elucidated; future experiments comparing upregulated transcripts in wild-type and p53-null tissues should help address this issue (Jassim, 2003).

Together, these results identify two points of regulation during a retinal cell's response to UV irradiation. The early step involves pyrimidine dimers, and requires proper repair from factors that include XPG and p53. The second step involves activation of caspases and requires regulation of Diap1 stability; interommatidial cells utilize Hid at this step, and the remaining cells employ a different (unknown) regulator. One challenge will be to connect these two points of regulation. Multiple signaling pathways are suggested by the microarray data. These include EGFR/Ras1 signaling (a central regulator of Hid), JNK pathway signaling and TGFß pathway signaling. The role of these factors is not known, but understanding them may help to connect early and late events (Jassim, 2003).

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. The Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene (alternative name: ATM). In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. It is proposed that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus, 2004).

In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence. This analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres (van Steensel, 1998; Takai, 2003; Wong, 2003). Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM (Oikemus, 2004).

Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae

Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. It is concluded that DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. It is proposed that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes (Jaklevic, 2004).

In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell cycle as needed. Central components of this checkpoint in mammals include four kinases: ATM, ATR, Chk1, and Chk2. Homologs of these exist in other eukaryotes and assume similar roles where examined. Human patients with ATM mutations, as well as their cells, show a dramatic sensitivity to killing by ionizing radiation. The importance of checkpoints in cellular survival to DNA damaging agents is presumed to be due to the role of checkpoints in cell cycle regulation. This is because mutants in the budding yeast gene rad9, the first checkpoint gene to be characterized, fail to arrest the cell cycle following damage and show increased radiation sensitivity; the latter phenotype is rescued by experimental induction of cell cycle delay. Consequently, cell cycle delay is thought to allow time for DNA repair and thereby ensure survival (Jaklevic, 2004 and references therein).

Components of the DNA damage checkpoint are found to activate DNA repair and to promote programmed cell death, which would cull cells with damaged DNA. For example, phosphorylation of NBS (a component of the Mre11 repair complex) by human ATM is of functional importance, while ATM knockout mice show a reduction in radiation-induced cell death in the CNS. Therefore, the essential role of checkpoints in conferring survival to genotoxins may be due to DNA repair and cell death responses in addition to or instead of cell cycle regulation. Furthermore, what is important for survival at the cellular level may not be so in a multicellular context. For instance, the failure to arrest the cell cycle by checkpoints may be detrimental to individual cells, but removal of these by cell death and replacement via organ homeostasis may make cell cycle regulation inconsequential for survival of multicellular organs (Jaklevic, 2004).

To address how DNA damage checkpoints operate in the context of multicellular organisms in vivo, the effect of ionizing radiation on Drosophila melanogaster is being studied. In Drosophila, mei-41 (ATR homolog) and grp (Chk1 homolog) are required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. Thus, mei-41 and grp play similar roles to their homologs in other systems. Moreover, mei-41 mutants are deficient in DNA repair. The role of mei-41 and grp in radiation-induced cell death has not been tested, but mei-41 is dispensable for cell death after enzymatic induction of DNA double-strand breaks (Jaklevic, 2004 and references therein).

Mutants in mei-41, grp, p53, and okra, a homolog of budding yeast RAD54 that functions in repair of DNA double-strand breaks (DSB) have been used to address the relative importance of cell cycle regulation, cell death, and DNA repair to the ability of a multicellular organism to survive ionizing radiation. The three responses are affected to different degrees in these mutants: wild-type larvae regulate S and M phases and repair DNA; grp mutants are unable to regulate the cell cycle but are able to repair DNA; okra mutants are able to regulate the cell cycle but are deficient in DNA repair; and mei-41 mutants are unable to regulate the cell cycle and are also deficient in DNA repair. All genotypes with the exception of p53 mutants are proficient in radiation-induced cell death, suggesting that mei-41 and grp do not contribute to this response. Under these conditions, it is found that while mei-41 and okra mutants are highly sensitive to killing by ionizing radiation, p53 mutants show reduced but significant survival and grp mutants resemble wild-type. These results suggest that cell death is neither sufficient nor absolutely necessary, DNA repair is essential, and optimal cell cycle regulation is dispensable for surviving ionizing radiation in Drosophila larvae (Jaklevic, 2004).

The effects of DNA damage by ionizing radiation on the maintenance and survival of Drosophila larvae was studied. Despite an extensive loss of cells to radiation-induced cell death, organ size and morphology are maintained remarkably well, and larvae survive to produce viable adults. Surprisingly, optimal cell cycle regulation by checkpoints is neither necessary (as in grp mutants) nor sufficient (as in okra mutants) to ensure organ homeostasis and organismal survival. p53-dependent cell death is also largely dispensable in this regard. Instead, DNA repair appears to be of paramount importance as might be expected (Jaklevic, 2004).

Drosophila MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage

Genetic and microarray analysis have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member Eiger. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).

Previous studies have established that Drosophila p53 mediates X-irradiation-induced apoptosis and expression of rpr and skl. This study characterized the pathway that transduces the DNA damage signal to the apoptosis and cell cycle machineries. The results indicated that a number of genes in this pathway are largely specific to the cell cycle or apoptotic response. Both cellular assays and transcriptional profiling suggest that Drosophila p53 is required for IR-induced regulation of apoptosis but is not required for G2 arrest. In contrast, mei-41, mus304, and grps were required for cell cycle arrest, but not induction of apoptosis. The biochemical experiments suggested that mnk, which encodes a conserved damage-activated kinase, is required for phosphorylation of p53 following IR. All IR-induced transcription required both mnk and p53. The absence of genes that required mnk or p53 only was consistent with a linear signaling pathway of MNK activating p53, which acts as a global regulator of IR-induced transcription (Brodsky, 2004).

Although mnk and p53 mutant animals have similar defects in IR-induced transcription, mnk also acts in p53-independent pathways. In animals with mutations in double-strand break repair enzymes, unrepaired breaks formed during meiotic recombination activate an mnk-dependent checkpoint signal that disrupts oocyte patterning and nuclear morphology. Induction of the meiotic checkpoint differs from IR-induced transcription in at least two respects: (1) activation of mnk during meiosis requires mei-41, the Drosophila homolog of ATR; (2) p53 is not required for this damage response pathway. In a different damage response pathway, mnk, but not p53, is required for damage-induced inactivation of centrosomes. In this study, IR was found to induced a p53-independent decrease in RNA levels of at least 17 genes, including many developmental regulators. Although this observation could indicate a transcriptional repressor that is regulated by mnk, a model is favored in which an mnk-dependent cell cycle delay following IR has a secondary effect on the developmental induction of these genes. Together, these results and previous studies indicate that mnk regulates multiple signaling pathways in addition to p53-dependent induction of gene expression (Brodsky, 2004).

In mammals, Chk2 and other checkpoint kinases block Mdm2-mediated turnover and inhibition of p53. Several lines of evidence suggest that this regulatory mechanism is not conserved in Drosophila. (1) Simple sequence searches have not revealed an obvious Mdm2 homolog in the Drosophila genome. (2) The Drosophila p53 protein sequence does not contain a conserved binding site for Mdm2. (3) p53 protein levels were not dramatically altered following IR. p53 did exhibit an IR-induced change in gel mobility due to mnk-dependent phosphorylation. Thus, these results provide a clear example of damage-induced activation of p53 without changes in p53 protein levels (Brodsky, 2004).

Phosphorylation of p53 by Chk2 may represent an important step in the evolution of DNA damage responses in multicellular animals. Checkpoint pathways regulating cell cycle control and DNA repair have been highly conserved in eukaryotes, including unicellular organisms such as yeast. In contrast, induction of apoptosis during development or in response to cellular stress is confined to multicellular organisms. p53 phosphorylation by Chk2/MNK was found to be a conserved molecular link between DNA damage detection and the core apoptotic machinery in metazoans. Mdm2 adds an additional layer of complexity to the regulation of mammalian p53 compared to Drosophila p53. Regulation of p53 turnover by Mdm2 may provide mammalian cells with greater control of the levels or timing of p53-dependent transcription (Brodsky, 2004).

Microarray analysis was used to perform a comprehensive analysis of p53 targets following exposure to IR. The number of genes identified in these experiments was substantially smaller than the number of p53 targets identified in mammals. In part, this observation may reflect underlying differences in the damage response pathway in flies and mammals. For example, induction of p21 by mammalian p53 mediates G1 arrest following damage. IR-induced G1 arrest has not been described in Drosophila, consistent with the observation that the Drosophila p21/p27 homolog dacapo is not induced by IR. However, the smaller number of targets identified in Drosophila also reflects experimental differences. Expression changes induced by IR were examined during a defined window of embryonic development. In contrast, targets of mammalian p53 have been identified in many different cell types following different types of DNA damage or simply overexpression of p53. It is likely that additional targets of Drosophila p53 will be identified using other types of cellular stresses in different cell types or developmental stages. For example, UV irradiation of Drosophila embryos has been shown to induce Apaf1 through either E2F or mei-41, depending on the developmental stage (Brodsky, 2004).

The most prominent group of p53 targets identified in this study regulates two apoptotic pathways that are also targeted by mammalian p53. hid, rpr, and skl are part of a group of genes that induce apoptosis by blocking the caspase-inhibiting activity of IAP proteins. Recent experiments have confirmed that HTRA2, a functional homolog of these genes, is a target of mammalian p53. The Drosophila p53 target Eiger is a member of the TNF ligand family and can induce apoptosis when overexpressed. In mammals, FAS and DR5/Killer are p53 targets that can regulate apoptosis by acting as receptors for TNF ligand family members. Thus, two examples of mammalian and Drosophila p53 regulating common signaling pathways have been identified. Combined with the many other proapoptotic targets of mammalian p53, these results support the general hypothesis that multiple components of proapoptotic signaling pathways can be targets for transcriptional regulation following stresses such as DNA damage (Brodsky, 2004).

Although FAS and DR5/Killer are targets of mammalian p53 and act in the extrinsic apoptosis pathway, it is unclear what role they play in DNA damage-induced apoptosis. Analysis of deletion mutations in the Drosophila p53 target Eiger indicates that this gene is not required to initiate IR-induced apoptosis. This negative result is not due to redundancy with a related molecule, since Eiger is the only TNF-related gene in the Drosophila genome sequence. It is possible that the conserved activation of the TNF pathway by p53 is required for the induction of apoptosis under specific conditions not tested in these experiments. Alternatively, induction of Eiger may activate other cellular responses to DNA damage. Further characterization of Eiger function should reveal how cell-cell signaling contributes to survival or genomic stability following DNA damage in multicellular organisms (Brodsky, 2004).

Analysis of the remaining proapoptotic targets of p53 indicates that they are part of a dosage-sensitive mechanism that regulates IR-induced apoptosis. In contrast to Eiger, the proapoptotic genes in the genetic region containing hid, rpr, and skl are both sufficient and necessary for apoptosis. Animals with deletions that include genes in this region are defective in IR-induced apoptosis. Because these proapoptotic genes act, at least in part, by inhibiting a common target (IAP1/Thread), it has been proposed that they contribute to a rheostat-like mechanism in which the added activity of all proapoptotic proteins present must pass a threshold before a cell undergoes the irreversible decision to undergo programmed cell death. Following the observation that three of these genes are induced following DNA damage, the effect of lowering the dose of all proapoptotic genes in this region by half was tested. It was found that deletions in this region were haploinsufficient for IR-induced apoptosis. Dose sensitivity may represent an important feature of damage-induced apoptosis. Animals heterozygous for these deletions exhibit apparently normal morphology and fertility, suggesting that they are not haploinsufficient for developmentally regulated apoptosis. One possible interpretation of these results is that the apoptotic signal in many developmental contexts is well past the threshold required to commit to apoptosis, while the apoptotic signal following DNA damage is closer to that threshold. A lower apoptotic signal following DNA damage may allow cells to monitor DNA repair and block apoptosis if repair is successful. Haploinsufficiency of some tumor suppressor genes, including p53, has been proposed to contribute to cancer development. If stress-induced apoptosis in mammals is sensitive to the dose of p53 target genes, haploinsufficiency of these genes may also contribute to suppression of apoptosis, particularly in cells with extensive aneuploidy (Brodsky, 2004).

Analysis of animals heterozygous for deletions that removed a subset of genes has revealed that loss of one copy of hid is sufficient to reduce IR-induced apoptosis. A greater reduction was observed in larger deletions, indicating that additional genes in this region, likely rpr and skl, also contribute to IR-induced apoptosis. Previous analysis of animals heterozygous for two overlapping deletions [Df(3L)H99 and Df(3L)xr38] that remove both copies of rpr demonstrate reduced levels of IR-induced apoptosis. The current results indicate that part of that reduction is due to haploinsufficiency of hid and other genes in this region. Although the induction of hid RNA was lower than that observed for rpr and skl, hid may exhibit a greater absolute difference in RNA and protein levels following IR. Because null mutations in hid are embryonic lethal, the effects of completely removing hid function were not investigated. The dose-sensitive effects of hid suggest that total loss of hid would completely block IR-induced apoptosis. However, even in animals with normal levels of hid, increased levels of rpr and skl may be required to pass the proapoptotic signaling threshold required for a full DNA damage response. The Ras pathway and a micro-RNA in the bantam locus regulate hid expression. These and other pathways regulating hid may help determine which cells in the developing wing are most sensitive to DNA damage (Brodsky, 2004).

The other class of p53 targets identified in these experiments includes components of the Ku and Mre11 DNA repair complexes. Both of these complexes participate in repair of double-strand DNA breaks by nonhomologous end joining (NHEJ). Compared with homologous recombination, NHEJ is a potentially error-prone mechanism for DNA repair. Mutagenic DNA repair mechanisms are a prominent feature of the SOS response in bacteria that apparently promotes cell survival following DNA damage at the expense of genomic integrity. The ability of multicellular animals to eliminate damaged cells by apoptosis might suggest that low-fidelity mechanisms of DNA repair would not be favored following damage. However, the induction of NHEJ components by p53 suggests that mechanisms such as apoptosis or cell cycle arrest that are presumed to prevent mutations following DNA damage may compete with mechanisms that promote cell survival and prevent aneuploidy by error-prone DNA repair. The previous demonstration that an isoform of Ku86 is also a target of mammalian p53 suggests that this is an evolutionarily conserved response to DNA damage in metazoans that may modulate mutagenesis following DNA damage (Brodsky, 2004).

Role of Drosophila p53 in the maintenance of histone H3 acetylation and response to UV-light irradiation

It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. This work explores if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, K9 and K14 H3 acetylation patterns were analyzed in situ after UV irradiation of wild-type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild-type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3-K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3-K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans (Rebollar, 2006).

The results presented here show that there are some similarities and differences between fly and human cells. For instance, in wild type third instar larvae there is an increase in the acetylation of K9 and K14 in the histone H3 in response to UV light irradiation. This observation is similar to previous reports in mammalian cells. Other similarity between both systems is that mutations in p53 affect the increase in the K9 acetylation after DNA damage, but not the acetylation of K14 in H3. In contrast, both human p53 and Dm p53 are required to maintain the basal histone H3 acetylation levels. In the case of human cells, the basal K9 acetylation level seems to be preferentially diminished when human p53 is mutated. In the case of Drosophila, K14 basal acetylation is dramatically reduced by the absence of Dm p53. Several scenarios may explain these differences. The first is that since not all p53 functions are conserved between human p53 and Dm p53 and the only region with significant identity between both proteins is the DNA binding domain, it is possible that the interactions with factors involved in histone modifications are different. Another possibility is that cancer cells deficient in human p53 could have other mutations. Usually they are aneuploid and therefore a mutated human p53 may interact with other mutated genes producing a phenotype on histone modifications. It is relevant to mention that in the Dm p53 null fly used in this study only Dm p53 is affected and therefore the effects that were observed in H3 acetylation are due only to this mutation (Rebollar, 2006).

The fact that a deficiency in Dm p53 produces a phenotype in basal H3 acetylation levels and in the increase of histone acetylation after UV light irradiation, indicates there is an important cross-talk between chromatin modifiers, Dm p53 and the nucleotide excision repair machinery in the fly. A similar network has been suggested to exist in human cells and therefore the fly becomes an interesting model to study the mechanisms that operate between DNA damage, p53 and chromatin dynamics. In contrast, the reduction in the basal levels of K14 acetylation in H3, does not have any effect in viability and fertility of the Dm p53 null flies. However, Dm p53 null organisms are very sensitive to UV light irradiation and a short life span. During development, the organism is exposed to genotoxic stress as consequence of the cell metabolism. Dm p53 may participate in the DNA repair during development and it is possible that the reduction in K14 basal acetylation in the Dmp53 null fly is product of a deficient DNA repair mechanism (Rebollar, 2006).

This work opens several interesting avenues that can be explored exploiting Drosophila genetics. For instance different mutant backgrounds in genes involved in genome stability, including Dm p53 can be used for the analyses of different histone modifications after DNA damage. It can also be interesting to find out if these histone modifications are different depending on the chromatin state. Also, since there are two pathways in nucleotide excision repair, transcription coupled repair and global genome repair it will be interesting to know if the increase in histone acetylation after DNA damage is higher in transcribed regions. However it is difficult to determine differences in histone modifications in specific sequences with this kind of analysis. These questions will be eventually answered by doing chromatin inmunoprecipitations and genetics (Rebollar, 2006).

Ionizing radiation induces caspase-dependent but Chk2- and p53-independent cell death in Drosophila melanogaster

Ionizing radiation (IR) can induce apoptosis via p53, which is the most commonly mutated gene in human cancers. Loss of p53, however, can render cancer cells refractory to therapeutic effects of IR. Alternate p53-independent pathways exist but are not as well understood as p53-dependent apoptosis. Studies of how IR induces p53-independent cell death could benefit from the existence of a genetically tractable model. In Drosophila, IR induces apoptosis in the imaginal discs of larvae, typically assayed at 4-6 hr after exposure to a LD50 dose. In mutants of Drosophila Chk2 or p53 homologs, apoptosis is severely diminished in these assays, leading to the widely held belief that IR-induced apoptosis depends on these genes. This study shows that IR-induced apoptosis still occurs in the imaginal discs of chk2 and p53 mutant larvae, albeit with a delay. This phenomenon is a true apoptotic response because it requires caspase activity and the chromosomal locus that encodes the pro-apoptotic genes reaper, hid, and grim. Chk2- and p53-independent apoptosis is IR dose-dependent and is therefore probably triggered by a DNA damage signal. It is concluded that Drosophila has Chk2- and p53-independent pathways to activate caspases and induce apoptosis in response to IR. This work establishes Drosophila as a model for p53-independent apoptosis, which is of potential therapeutic importance for inducing cell death in p53-deficient cancer cells (Wichmann, 2006).

The Drosophila homologs of Chk2 and p53 are required, not for induction of apoptosis, but for timely induction of apoptosis in response to irradiation. Radiation-induced cell death still occurs in chk2 and p53 mutants, albeit with a delay. Four lines of evidence support the idea that this delayed cell death is apoptosis rather than necrosis: (1) it is detected by staining with AO, which has been shown to stain apoptotic but not necrotic cells; (2) it accompanies activation of caspases, a hallmark of apoptosis but not necrosis; (3) it requires caspase activity, which is required for apoptosis but not necrosis, and (4) it requires the chromosomal locus encoding the proapoptosis genes rpr, hid, and grim, whose protein products are required to inhibit DIAP1 and activate caspases. These results indicate that there is a Chk2-/p53-independent pathway that commits damaged cells to apoptosis and utilizes many of the same downstream components as the Chk2-/p53-dependent apoptosis pathway (Wichmann, 2006).

Two lines of evidence support the idea that DNA damage is the signal that induces Chk2-/p53-independent apoptosis after exposure to ionizing radiation. First, the amount of Chk2-/p53-independent apoptosis appears to increase with IR dose. This dose dependence suggests that the amount of DNA damage is what induces Chk2-/p53-independent apoptosis but does not rule out the contribution of other damages that result from IR. Second, higher levels of Chk2-/p53-independent apoptosis are observed when the ability to repair DNA is compromised, as in mei-41, p53 double mutants. Collectively, these data suggest that DNA damage caused by x-rays induces Chk2-/p53-independent apoptosis (Wichmann, 2006).

IR-induced apoptosis in chk2 and p53 mutants shows a temporal delay. IR-induced apoptosis is also delayed in H99 heterozygotes, possibly because H99 heterozygotes contain half the gene dose of the proapoptotic Smac/Diablo orthologs and it may take longer for the proapoptotic gene products to accumulate to the point of an apoptosis-stimulating threshold. IR induced increase in the transcripts of rpr and hid, two of the H99-encoded genes, still occurred in chk2 (rpr and hid) and p53 (hid) mutants, but to lower levels (for rpr) and after a delay. Therefore, apoptosis may be delayed in chk2 and p53 mutants because proapoptotic gene products take longer to accumulate to a threshold in the absence of Chk2 or p53 regulation. The data showing that IR-induced apoptosis is further delayed in a chk2, H99/+ double mutant, compared with a chk2 single mutant, support this claim. Furthermore, the results suggest the existence of at least another signaling pathway that does not operate through Chk2 or p53, but nonetheless links the same signal (DNA damage) to a similar outcome (accumulation of H99-encoded gene products) (Wichmann, 2006).

RT-PCR experiments revealed interesting differences in the identity and onset of induction of proapoptotic genes in chk2 and p53 mutants. rpr and hid are induced at 4 hr after irradiation in chk2 mutants, whereas hid and skl are induced between 12 and 18 hr after irradiation in p53 mutants. The basis for these differences is not understood. More detailed time courses as well as deletion analysis of the H99 locus to determine the contribution of each proapoptotic gene to Chk2-/p53-independent apoptosis needs to be performed to address these issues (Wichmann, 2006).

The data presented in this study establish Drosophila as a model for studying p53-independent apoptosis. p53 is the most commonly mutated gene in human cancers. Loss of p53 poses an immense clinical problem because p53-deficient cancer cells no longer stimulate p53-dependent apoptosis in response to radiation or genotoxic chemotherapy drugs. In this scenario, p53-independent apoptotic pathways become key for inducing cancerous cells to die because they provide potential therapeutic targets. In mammals, a p53-independent apoptosis pathway that is mediated by p73, another member of the p53 family, has been identified. In Drosophila, Dmp53 is the only known p53 family member. Therefore, the p53-independent apoptosis that was identified and characterized in this article is likely to represent a previously unknown process. An important goal in the future will be to dissect the Chk2-/p53-independent pathway that links DNA damage to the proapoptotic genes of the H99 locus (Wichmann, 2006).

Several candidates were tested and eliminated as regulators of Chk2-/p53-independent cell death. Mei-41 (ATR) is not required for Chk2-/p53-independent cell death because mei-41, p53 double mutants actually exhibit more cell death than p53 alone. Recent work showed that ectopic induction of eiger, a TNF ligand homolog, can induce apoptosis in Drosophila. Chk2-/p53-independent cell death still occurs in p53, eiger double mutants, suggesting that the TNF pathway is not involved in the induction of cell death characterized in this study. Work in mammalian cells showed that overexpression of c-Myc can induce p53-independent apoptosis. Chk2-/p53-independent apoptosis still occurs in Dmyc, p53 double mutants, indicating that Dmyc is not required for this response (Wichmann, 2006).

A classical genetic screen may identify components of the Chk2-/p53-independent apoptosis pathway, as well as testing more candidates, such as the transcription factor de2f1, grapes (DmChk1), DmATM, and genes required for autophagy. Autophagic cell death, in which a cell lyses itself, occurs during Drosophila metamorphosis to lyse polyploid tissues such as the salivary glands and the fat body and provide nutrients for diploid cells of the imaginal discs; autophagy has been described in larvae only in the polyploid cells and only in response to starvation. Nonetheless, autophagy shares characteristics with apoptosis, including being detectable by AO staining and being dependent on caspases and the H99 locus, and for this reason remains a formal possibility (Wichmann, 2006).

In conclusion, studies have shown that IR-induced apoptosis in two key models for apoptosis, C. elegans and Drosophila, depends on p53. This study has provided evidence that, contrary to the accepted view, Chk2 and p53 are not required for radiation-induced cell death in Drosophila. Furthermore, normal timing of apoptosis that depends on Chk2 and p53 is also not required for ensuring survival after irradiation. Radiation-induced cell death that is independent of Chk2 and p53 depends on radiation dose, has characteristics of apoptosis and is likely to rely on a novel mechanism(s) because no other members of the p53-family are known in Drosophila. This work is the first to establish Drosophila as a model for p53-independent apoptosis. Identification of genes required for Chk2-/p53-independent cell death in Drosophila is of potential therapeutic value because protein products of their human homologs may represent novel targets that can be activated clinically to eliminate p53-deficient cancer cells (Wichmann, 2006).

Dmp53 activates the Hippo pathway to promote cell death in response to DNA damage

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size. Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki). This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although some of the events downstream of the Hpo kinase are understood, its mode of activation remains mysterious. This study shows that Hpo can be activated by Ionizing Radiations (IR) in a p53-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or p53 ectopic expression (Colombani, 2006).

Hpo is the ortholog of the Mammalian Sterile Twenty-like (MST) kinases, which belong to the Ste20 family of kinases. MSTs are highly similar to Hippo (Hpo) in their N-terminal serine/threonine kinase domains as well as in the C-terminal Salvador (Sav) binding region (or SARAH domain). MST1 functions both downstream and upstream of caspases to promote chromatin condensation and nuclear fragmentation, as well as activation of the JNK (Jun N-terminal kinase) and p38 pathways. Like most Ste20 family kinases, MST1/2 auto- or trans-phosphorylates at a number of residues. One of these, T183 in the activation loop, has been shown to be required for full kinase activity and has been used as a useful marker of MST1 activation in cultured cells. In order to study events upstream of Hpo, antibodies that have previously been shown to recognize MST1/2 phosphorylated on T183 were tested for their ability to cross-react with Hpo on the equivalent residue (T195). Interestingly, it was found antibodies that specifically recognized the phosphorylated form of Hpo upon treatment with staurosporine (sts), a known activator of MST1/2. This signal is abolished by RNAi-mediated Hpo depletion and disappears upon phosphatase treatment. Moreover, the antibodies recognize overexpressed tagged Hpo before immunoprecipitation. By contrast, the antibodies did not recognize a nonphosphorylable (T195A) Hpo mutant protein. Myc-tagged wild-type and T195A Hpo were immunoprecipitated and their auto-kinase activity and their activity on an exogenous substrate (Histone H2B, not shown) were measured in both the presence and absence of sts. As has been observed for MST1/2, overexpression of Hpo leads to its activation, presumably via trans-phosphorylation. Sts treatment potently stimulates Hpo kinase activity (5-fold). By contrast, the T195A mutant is severely compromised both in its unstimulated and stimulated activities, suggesting that T195 phosphorylation is crucial to normal Hpo kinase activity. Thus, these phospho-specific antibodies can be used as readouts of Hpo pathway activity (Colombani, 2006).

In the course of testing stimuli that would activate Hpo in tissue culture, it was observed that γ-irradiation potently and rapidly induced Hpo activation. The fly p53 ortholog has been shown to mediate cell death upon ionizing radiation (IR)-induced DNA damage. Although the pro-apoptotic genes reaper (rpr), hid, and sickle are p53 transcriptional targets, removal of these three proteins via chromosomal deficiencies only partially suppresses the cell-death effects of IR in embryos, suggesting that additional death signals act downstream of p53. This prompted an examination of whether the Hpo pathway could function downstream of Drosophila p53 in the response to IR (Colombani, 2006).

Initially, wing imaginal discs (the larval precursors of the adult wing) containing clones of hpo, wts, and sav mutant cells were treated with γ-rays and cell death was examined by staining for activated caspases. Interestingly, although caspase activation was efficiently induced in wild-type tissue or control discs, cell death was severely reduced in hpo, wts, and sav mutant clones and in p53 mutant discs. Quantification of the caspase staining indicated that apoptosis was reduced by 2- to 3-fold in hpo, wts, and sav clones compared to wild-type tissue. This was also true in eye imaginal discs (Colombani, 2006).

Overexpression of p53 in the posterior portion of late larval eye imaginal dics was sufficient to induce apoptosis. Loss of function of hpo, wts, and sav decreased cell death in this context, although the effect was less pronounced in sav clones, perhaps as a reflection of the weaker phenotype of the sav mutants. This suggests that the Hpo complex may function as an effector in the p53-mediated response to IR. To test this hypothesis, Hpo activation was measured in cultured cells treated with γ-rays in the presence or absence of dsRNAs directed against p53. Excitingly, depletion of Dmp53 markedly reduced Hpo phosphorylation by IR. The residual level of Hpo activation observed in p53-depleted cells can probably be explained by the fact that the dsRNA-mediated p53 depletion was never complete, as measured by RT-PCR. To check that the increased Hpo phosphorylation observed corresponded to increased activity, IP kinase assays were performed on cells expressing ectopic Hpo. It was observed that IR treatment potently induced Hpo kinase activity. Furthermore, p53 expression alone, in the absence of IR, was sufficient to activate Hpo phosphorylation. Finally, it was determined whether p53-dependent Hpo activation could be observed in vivo by taking advantage of the fact that p53 is not required for viability. Dissected ovaries from p53 mutant and wild-type flies were treated with γ-rays and examin Hpo activity was examined by Western blotting. Interestingly, although γ-rays potently activated Hpo in wild-type flies, this response was abolished in p53 mutant animals. p53 expression in the ovaries was able to induce apoptosis, ovary degeneration, and total loss of fecundity. It is concluded that Hpo is activated as part of a p53-dependent DNA-damage response both in cultured cells and in vivo (Colombani, 2006).

MST1 and 2 are known to be activated by caspase 3 through proteolytic cleavage. Therefore, the possibility exists that the Hpo activation observed is merely a by-product of Rpr-dependent caspase activation. Several lines of evidence suggest that this is not the case. First, reaper overexpression in S2 cells did not increase Hpo activity. Second, depletion of DIAP1 from cultured cells, which potently induces caspase activation, fails to trigger detectable Hpo activation. Third, the phospho-Hpo signal detected corresponds to full-length Hpo rather than a caspase-cleaved fragment. In fact, the caspase cleavage site present in the MSTs is not thought to be conserved in Hpo, and no evidence was seen of Hpo cleavage upon apoptotic stimuli. Fourth, treatment of cultured cells with caspase inhibitors did not affect Hpo activation by IR. Thus, it is unlikely that Hpo is stimulated via p53-dependent caspase activation (Colombani, 2006).

The time course of Hpo activation by IR (2–3 hr for maximal activation) suggests that transcription may be required for this response. Indeed, treatment of cells with IR in the presence of the transcription inhibitor Actinomycin D (ActD) abolishes Hpo activation. Thus, Hpo activation in response to IR requires new gene transcription, which could be mediated, at least in part, by p53. Hpo activity is induced by p53 expression, but Hpo protein itself does not appear to be a target of p53 because Hpo levels are not detectably upregulated when p53 is expressed in the posterior portion of the eye imaginal disc or in Dmp53-expressing clones in the wing disc. Future studies will be aimed at determining the exact mechanism through which Dmp53 promotes Hpo activation (Colombani, 2006).

This study has demonstrate by genetic and biochemical approaches not only that the Hpo pathway is required for the full apoptotic response induced by γ-ray irradiation but also that DNA damage triggers Hpo kinase activity in a p53-dependent manner both in vivo and in vitro. The apoptosis induced by p53 overexpression is strongly affected in hpo, wts, and sav mutant clones and p53 does not modulate Hpo levels. This study constitutes the first description of an upstream activating signal of the Hpo complex in vivo and during organism development (Colombani, 2006).

It is noted that the blockage of p53-induced apoptosis is not complete in hpo clones; this incomplete blockage likely reflects the role of other pro-apoptotic proteins, such as Reaper, Hid, and Sickle, in this process. Thus, it is proposed that, after exposure to ionizing radiations, the ATM, Chk2, p53 signaling pathway is activated and induces apoptosis by targeting expression of pro-apoptotic effectors such as Reaper, as well as by activating the Hpo pathway. This cell-death response to irradiation requires the caspase DRONC and leads to upregulation of JNK activity in a p53-dependent manner. Because Hpo has been shown to induce JNK activation when overexpressed in vivo, it will be interesting to determine whether Hpo is necessary for IR-induced JNK activation (Colombani, 2006).

Several reports have suggested that the mammalian homologs of members of the Hpo pathway might behave as tumor suppressors in humans. In addition, mice lacking the Wts homolog mLats1 are more sensitive to tumor-inducing agents. The current data suggest that one effect of mutations in Hpo-pathway members may be to protect these cells from DNA-damage-induced apoptosis and thus promote tumor progression and the accumulation of additional mutations. Further work on the Hpo pathway should further understanding of the DNA-damage response and its role in the transformation process (Colombani, 2006).

Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling

In Drosophila, p53 (Dmp53) is an important mediator of longevity. Expression of dominant-negative (DN) forms of Dmp53 in adult neurons, but not in muscle or fat body cells, extends lifespan. The lifespan of calorie-restricted flies is not further extended by simultaneously expressing DN-Dmp53 in the nervous system, indicating that a decrease in Dmp53 activity may be a part of the CR lifespan-extending pathway in flies. This report shows that selective expression of DN-Dmp53 in only the 14 insulin-producing cells (IPCs) in the brain extends lifespan to the same extent as expression in all neurons and this lifespan extension is not additive with CR. DN-Dmp53-dependent lifespan extension is accompanied by reduction of Drosophila insulin-like peptide 2 (dILP2) mRNA levels and reduced insulin signaling (IIS) in the fat body, which suggests that Dmp53 may affect lifespan by modulating insulin signaling in the fly (Bauer, 2007).

Expression of DN-Dmp53 constructs in the adult nervous system extends lifespan. This lifespan extension is not additive to CR (Bauer, 2005), suggesting that Dmp53 may be part of the CR pathway of lifespan extension. To understand more about the mechanisms by which neuronal reduction of Dmp53 extends lifespan, the expression of DN-Dmp53 was examined in subsets of neuronal cells. General expression of DN-Dmp53 in the nervous system, using two different, broadly expressing neuronal specific promoters, ELAV (pan-neuronal) or Cha (cholinergic neurons), both lead to significant lifespan extension. When expression of DN-Dmp53 was restricted to smaller subsets of neurons, including dopaminergic neurons, serotoninergic neurons, neurons of the mushroom body, or to IPCs, only expression in the 14 IPCs cells led to lifespan extension. Thus, expression of DN-Dmp53 in only the 14 IPCs is sufficient to extend Drosophila lifespan. When combined with CR, IPC-specific DN-Dmp53 expression did not further extend lifespan, suggesting that reduction of Dmp53 activity in the IPCs may be a component of CR-dependent lifespan extension. Interestingly, IPCs are the functional equivalent of mammalian pancreatic β-cells, but reside in the fly brain. This data suggests that Dmp53 controls insulin secretion from the IPCs. A consequence of inhibition of Dmp53 activity is reduced dILP2 mRNA and subsequent down regulation of IIS in the fat body, Drosophila's major insulin responsive metabolic organ. In this relevant target tissue, two different assays, tGPH and dFoxO subcellular localization, show that IIS is diminished. These data suggest that DN-Dmp53 expression might extend lifespan through modulation of IIS. Interestingly, p53 has been linked to insulin regulation in mammals. Mice over-expressing the shorter p44 isoform of p53, that is thought to resemble Dmp53 more than p53 itself, have elevated levels of IGF-1and IGF-1R. Furthermore, p53 null mice have 75% reduced levels of IGF-1 (Bauer, 2007).

Loss or destruction of IPCs has been postulated to extend lifespan in flies. A loss of the IPCs does not explain the lifespan extension seen with the long-lived flies expressing DN-Dmp53 specifically in IPCs; it was possible to visualize the presence of the IPCs, at least up until day 44, as measured by simultaneous expression of GFP. Evidence was presented suggesting that the IPCs remain functionally active. Of the three dILPs produced in the IPCs, only dILP2 mRNA levels are lowered, whereas dILP3 and dILP5 levels remain unchanged. Furthermore, the fat body cells exhibit a nearly normal insulin response to a short period of starvation and sucrose re-feeding in the long-lived DN-Dmp53- expressing flies. Thus, the lifespan extension induced by expression of DN-Dmp53 in IPCs is not due to loss or damage of the IPCs (Bauer, 2007).

IIS related lifespan extension in the fly is thought to be mediated through alterations in the InR, CHICO (InR substrate), dPTEN and perhaps dFoxO. Interestingly, increasing levels of dPTEN or dFoxO in the fat body, but not the nervous system, extends lifespan. Experiments on another nutrient sensing system, the TOR signaling pathway, also supports the importance of the fat body in lifespan extension; down-regulation of the TOR pathway in the fat body, but not the nervous system leads, to lifespan extension. IIS can modulate TOR signaling via phosphorylation of Tsc2 by protein kinase B/Akt. Thus, down-regulated IIS may lead to suppression of TOR signaling (Bauer, 2007).

The fly thus appears to have at least two different tissues that can influence longevity: one, neuronal in nature, of which Dmp53 is part; the other as part of a larger nutrient-sensing signaling network active in the fat body. Are these two separate systems or is there cross-talk between them? One possible means of connecting these two systems could be through the neuroendocrine system, where alterations in the nervous system, through control of hormonal secretion, could affect the physiology of the fat body. It is thus of considerable interest that the site of production and secretion for three of the major insulin-like peptides, dILP2, dILP3, and dILP5, is the 14 ICPs located in the brain of the fly. The finding that expression of DN-Dmp53 specifically in these IPCs affects IIS in fat body cells and extends lifespan is intriguing. It remains to be determined whether this is one of the cellular sites linking the longevity determining effects of the brain with those associated with the fat body (Bauer, 2007).

It is tempting to speculate even further: The data suggest that the CR and DN-Dmp53 lifespan-extending pathways are related. It may therefore be that the lifespan-extending effects of CR are also accompanied by a down regulation of IIS. Dmp53 might thus be a point of convergence for these two lifespan-extending pathways, but further experiments are needed to clarify this point (Bauer, 2007).

E2F1 and E2F2 have opposite effects on radiation-induced p53-independent apoptosis in Drosophila

The ability of ionizing radiation (IR) to induce apoptosis independent of p53 is crucial for successful therapy of cancers bearing p53 mutations. p53-independent apoptosis, however, remains poorly understood relative to p53-dependent apoptosis. IR induces both p53-dependent and p53-independent apoptoses in Drosophila, making studies of both modes of cell death possible in a genetically tractable model. Previous studies have found that Drosophila E2F proteins are generally pro-death or neutral with regard to p53-dependent apoptosis. This study reports that dE2F1 promotes IR-induced p53-independent apoptosis in larval imaginal discs. Using transcriptional reporters, evidence is provided that, when p53 is mutated, dE2F1 becomes necessary for the transcriptional induction of the pro-apoptotic gene hid after irradiation. In contrast, the second E2F homolog, dE2F2, as well as the net E2F activity, which can be depleted by mutating the common cofactor, dDp, is inhibitory for p53-independent apoptosis. It is concluded that p53-dependent and p53-independent apoptoses show differential reliance on E2F activity in Drosophila (Wichmann, 2010).

This study has taken advantage of the relative simplicity of Drosophila E2F and p53 families to study the role of E2Fs in p53-independent apoptosis. The results indicate that Drosophila E2F homologs play opposing roles in regulating p53-independent apoptosis in response to IR. dE2F1, a homolog of the mammalian 'activator' E2Fs, is required for Chk2-/p53-independent apoptosis, while dE2F2, a homolog of the mammalian 'repressor' E2Fs, limits p53-independent apoptosis. The net E2F activity in the cell, reduced by mutations in dDP, is inhibitory towards p53-independent apoptosis (Wichmann, 2010).

One surprising finding from these studies is that 2 kb of hid promoter confers IR-induced transcriptional activation in a p53-dependent manner. This is surprising because in embryos, transcriptional activation of hid by IR in a p53-dependent manner requires the IRER (irradiation responsive enhancer region) that lies next to rpr, ~ 200 kb away from hid, and is regulated epigenetically by histone modification (Zhang, 2008). Yet, as shown previously, 2 kb of hid promoter is enough to allow IR-induced GFP expression in eye and wing imaginal discs (Tanaka-Matakatsu, 2009). This study shows that this induction is p53-dependent. Clearly, regulation of hid by IR is different between embryos and larval discs (Wichmann, 2010).

Mammalian p53 consensus is a tandem repeat of 10 nucleotides with the sequence RRRCWWGYYY where R = G/A, W = A/T and Y = T/C and invariant C and G are shown in bold. Drosophila p53 binds to a DNA damage response element at the rpr locus that differs from the mammalian consensus at one position shown in lower case; tGACATGTTT GAACAAGTCg. Manual examination of 2 kb of hid promoter fragment that responds to p53 status shows a potential binding sequence at −2006 from the start of hid transcription that deviates from the mammalian consensus at two positions, and another at −1667 that deviates at three positions. These are ttGCATGCTC GctCATGTTC and GtGCAAGagT GtGCTTGaat respectively. Since the consensus for Drosophila p53 has not been determined, it is possible that either or both of these are responsible for the effect of p53 on hid-driven GFP reporter (Wichmann, 2010).

The 2 kb hid enhancer includes E2F consensus sequences. Rb has been shown to repress the expression of hid-driven GFP reporter when E2F binding sequences are intact but not when these are mutated (Tanaka-Matakatsu, 2009). That is, E2F binding sites allow for repression of hid by Rb although which E2F mediates this repression is not known. In any case, the finding that net E2F activity is inhibitory towards apoptosis would be consistent with the published result that Rb inhibits hid expression via E2F consensus sites. It is not known if dE2F1 plays a permissive role (e.g. by allowing elevated basal expression of pro-apoptotic genes) or an instructive role (e.g. by allowing for induction of pro-apoptotic genes by IR), or both. The results with hid-driven GFP reporter are consistent with an instructive role but do not rule out a permissive role (Wichmann, 2010).

In the absence of p53, dE2F1 is needed for the transcriptional induction of hid> GFP reporter by IR. This can explain two published results: that hid is necessary for IR-induced p53-independent apoptosis (McNamee, 2009), and that hid is transcriptionally induced in p53 mutants after a time delay. Human E2F1 can bind to the promoter of a hid ortholog, Smac/DIABLO, and can, when ectopically expressed, activate the transcription of the latter in vivo. The role of p53 status in this process or the significance of this mode of regulation was not investigated. It is speculated that the role of E2F1 in IR-induced, p53-independent transcriptional activation of Smac/DIABLO genes may be conserved in mammals (Wichmann, 2010).

Previous work has shown that dE2F1 and dE2F2 exhibit antagonistic functions, with dE2F1 activating and dE2F2 repressing the transcription of a reporter containing canonical E2F sites from the PCNA promoter. dE2F1 and dE2F2 occupy the PCNA promoter and the ratio of the two E2F proteins influenced the degree of transcriptional activation or repression. In wild type, PCNA expression is tightly coupled to the pattern of S phase, in eye imaginal discs for example. In de2f2 mutants, PCNA is no longer down-regulated outside pattern of the S phase. The loss of all E2F activities, either in de2f1, de2f2 double mutants or in dDP single mutants, results in de-repression of PCNA such that a low but significant level is expressed throughout the cell cycle. Thus the net result of opposing E2F activities is the cyclical expression of PCNA in concert with DNA replication (Wichmann, 2010).

The paradigm of E2F-dependent regulation of PCNA aids in understanding the role of E2F proteins in p53-independent apoptosis. dE2F1 and dE2F2 might similarly influence p53-independent apoptosis by regulating pro-apoptotic gene(s) such as hid. According to this model, dE2F2 (with dDp) provides a net repressive activity that inhibits IR-induced apoptosis. This activity must be operative only in the absence of p53;dE2F2 mutations that have no effect on apoptosis when p53 is present. In p53 mutants, dE2F1 counteracts dE2F2 after irradiation and thus promotes apoptosis. Disabling transcriptional activation by dE2F1, which is what the allelic combination de2f1i2/de2f17172 is predicted to cause, would result in the failure to overcome dE2F2/Dp. Removal of dE2F2 with null alleles, would result in increased gene expression and more apoptosis. Reducing the ability of dDP to interact with dE2Fs, which is what the allelic combination dDPa1/dDPa2 is predicted to cause, would reduce dE2F1 and dE2F2 activities simultaneously. Since this results in more apoptosis, the net E2F activity is inhibitory on apoptosis when p53 is absent (Wichmann, 2010).

This study and published studies in wing imaginal discs reveal significant differences in the effect of E2F/DP mutations on p53-dependent apoptosis (typically assayed at 4–6 h post irradiation) and p53-independent apoptosis (18–24 h after irradiation in p53 mutants). The clearest difference is that dE2F2 null mutations have little or no effect on p53-dependent apoptosis, but increase the level of p53-independent apoptosis. dDP loss-of-function mutations decrease p53-dependent apoptosis throughout eye imaginal discs and in most cells of the wing pouch, whereas they increase the level of p53-independent apoptosis. In contrast, loss-of-function mutations in dE2F1 reduced both p53-dependent apoptosis in most cells of the wing imaginal disc and p53-independent apoptosis in the wing imaginal disc. These differences raise the question ‘how does p53 status alter the role of dE2F2 and dDP in IR-induced apoptosis?’ In the presence of p53, dE2F2 has little effect and dDP is stimulatory. In the absence of p53, dE2F2 and dDP play inhibitory roles. Perhaps the occupancy of transcriptional factors at the target loci such as the hid promoter is sensitive to p53 status (Wichmann, 2010).

In the eye imaginal disc, mutations in ago, a ubiquitin ligase, result in elevated apoptosis. This mode of cell death occurs via elevated E2F1 activity, increased expression of hid and rpr and is independent of apoptosis. Thus, the role of dE2F1 in promoting p53-independent apoptosis is conserved in another tissue of the larvae (Wichmann, 2010).

Previous studies found that the role of Drosophila E2F transcription factors in apoptosis is context-dependent and is influenced by, for example, whether the cells are in the wing pouch or at the dorsal/ventral margin of the wing disc and whether apoptosis is induced by radiation or by the loss of a tumor suppressor homolog, Rb. The current study addresses the role of E2F family members in IR-induced p53-independent apoptosis. The most significant finding in this study is that reducing E2F activity, as in the case of dDP mutants, allows p53-null cells to die following IR exposure. This is in clear contrast to the finding that a similar reduction of E2F activity prevents p53 wild type cells from dying following IR exposure. Several E2F antagonists are being considered in cancer therapy. The current results from Drosophila studies would caution that p53 status must be considered when using such therapies in conjunction with radiation treatment. If the findings in Drosophila apply to human cancers, an E2F antagonist would help kill p53-deficient cancer cells following radiation treatment, but would help p53-wild type cancer cells survive. In addition, an E2F antagonist may be particularly suitable for combination therapy with radiation to eradicate p53-deficient tumors because it may sensitize p53-deficient cancer cells to radiation while protecting p53-wild type somatic cells from the cell-killing effects of IR (Wichmann, 2010).

Maintenance of imaginal disc plasticity and regenerative potential in Drosophila by p53

Following irradiation (IR), the DNA damage response (DDR) activates p53, which triggers death of cells in which repair cannot be completed. Lost tissue is then replaced and re-patterned through regeneration. The role of p53 in co-regulation of the DDR and tissue regeneration was examined following IR damage in Drosophila. After IR, p53 was found to be required for imaginal disc cells to repair DNA, and in its absence the damage marker, γ-H2AX is persistently expressed. p53 is also required for the compensatory proliferation and re-patterning of the damaged discs, and the results indicate that cell death is not required to trigger these processes. An IR-induced delay in developmental patterning in wing discs was identified that accompanies an animal-wide delay of the juvenile-adult transition; both of these delays require p53. In p53 mutants, the lack of developmental delays and of damage resolution leads to anueploidy and tissue defects, and ultimately to morphological abnormalities and adult inviability. It is proposed that p53 maintains plasticity of imaginal discs by co-regulating the maintenance of genome integrity and disc regeneration, and coordinating these processes with the physiology of the animal. These findings place p53 in a role as master coordinator of DNA and tissue repair following IR (Wells, 2011).

The results show that tissue regeneration subsequent to the DDR also requires p53. They add to previous work indicating that a continuum of events follows IR that culminates in regeneration of damaged tissue and survival of the animal. The process initiates with a stereotypical DDR in damaged imaginal disk cells within minutes of IR: damage is sensed and H2AX is phosphorylated, caspases are activated, and cell division in the disk transiently arrests. After approximately 5 h disk cells re-enter the cell cycle and continue to divide at apparently normal rates. Repair of DNA damage leads to loss of γ-H2AX, while ongoing apoptosis eliminates unrepaired cells. The results indicate that the high level of cell death significantly slows the net growth of wing disks, compelling continuous cell division. This is facilitated by a delay of pupariation; in parallel, the expression of late patterning genes is delayed in wing disks. Interestingly, it was found that after 40 Gy of IR, tissue damage was severe enough to require disk cell proliferation to continue not only during the extension of the larval developmental timer, but also into the pupal stage. Thus in contrast to what has been generally believed, disk regeneration is not restricted to the larval “growth phase” of development, but can continue in the early stages of pupal development. The ability of disks to continue regenerative growth after the hormonal cues that stimulate pupariation suggests that disk cell proliferation is only loosely regulated at the juvenile-adult transition (Wells, 2011).

In p53 mutants the events following IR are initially identical to wild-type, but subsequently show several differences. Cells lacking p53 recognize DNA damage, H2AX is phosphorylated, and the cell cycle checkpoint transiently arrests p53 mutant disk cells. p53 mutant cells also reenter the cell cycle with the same kinetics as controls. However, γ-H2AX persists at high levels in mutant disks, indicating that DNA damage is lingering, but the cells are unable to undergo apoptosis. Moreover, the mutant larvae do not significantly delay development, suggesting that p53 is required to regulate the developmental timer. Despite these differences, it was found that disk cells divide at a normal rate and thus the size of the wing disk initially increases after IR. Later, the persistence of damaged DNA coupled with cell division creates aneuploidy, which may contribute to a late wave of apoptosis that continues late into the pupal stage (Wells, 2011).

Cell division continues beyond the normal cessation time in both wild-type and p53 mutants. In wild-type these additional pupal cell divisions are productive. In contrast, the late pupal divisions of p53 mutant disk cells appear to be largely futile since cell death is still prevalent; surviving cells appear to be aneuploid. Perhaps as a result, wing morphogenesis is delayed in irradiated p53 mutants relative to controls, although the mechanisms that pack wing disk cells and re-shape the wing disk ultimately do occur. The lack of DNA repair impairs cell differentiation and/or function throughout the pupa and leads to defects that prevent most animals from eclosing. This is interesting in light of the finding that the persistent DNA damage in p53 mutants did not appear to interfere with cell division during the larval phase of regeneration (Wells, 2011).

p53 functions cell autonomously during disk regeneration, and conditional expression of p53 in the wing disk is sufficient to induce ectopic expression of Wg, compensatory tissue growth, and a systemic developmental delay, all common aspects of regeneration. These results suggest that p53 is activated and operates cell-autonomously in damaged cells to promote regeneration. However, p53 also regulates the larval developmental clock, with the result that it coordinates control of disk regeneration with the physiology of the whole animal. Collectively, the results indicate that p53 functions to ensure repair of damaged DNA, to regulate the developmental timing of the animal, and to coordinate disk and animal maturation via a patterning checkpoint that delays cell fate acquisition in the disk. This linkage provides a mechanism that coordinates the two processes in time and thus facilitates the survival of the animal after DNA and tissue damage (Wells, 2011).

In the absence of p53, DNA damage remains unrepaired, rendering cells incapable of completing the differentiation process. This is exacerbated by the absence of apoptosis immediately after IR in the p53 mutants, allowing cells with DNA damage to persist. Later, some of the persisting damaged cells in the mutants are eliminated by a late surge of disk cell death that continues into the pupal stage. However, although this rids the disk of many damaged cells, it is not induced within a time frame that allows replacement of lost tissue, leading to small pupal wing disk size and small adult wings. Shortly after the onset of the late wave of apoptosis in p53 mutant disks the larval–pupal transition is crossed and metamorphosis is initiated. Although p53 mutant pupal wing disk cells continue to proliferate long after their wildtype counterparts have exited the cell cycle, the results suggest that the juvenile-to-adult transition – the commitment to produce adult traits – prevents critical developmental and patterning cues or render cells incapable of responding to them. However, damaged, aneuploid cells can differentiate trichomes, and this study observed that some damaged cells did carry out aspects of trichome differentiation, including prehair formation. In addition, it is possible that most of the severely damaged cells in p53 mutants were ultimately eliminated (Wells, 2011).

The results are strikingly similar to observations made in mouse and human cells. Loss of the murine DNA damage checkpoint protein Hus1 in a p53-deficient background results in accumulation of damaged cells after IR and prevents the compensatory responses in mammary epithelium. In serial transplantation experiments, self-renewal of irradiated human hematopoietic stem cells (HSCs) is compromised when they are deficient for p53, and, like experiments with wing disk cells, γ-H2AX persisted in the HSCs. Collectively the data indicate that p53's role in Drosophila disk regeneration is analogous to its role in tissue remodeling and stem cell renewal in vertebrates, and suggest that these functions of p53 are conserved (Wells, 2011).

The argument can be made that Drosophila imaginal disks merely take advantage of and extend developmental programs to repair and re-pattern lost tissue. This requires that the appropriate hormonal milieu be maintained by prolonging the juvenile, larval stage. Animals were irradiated late during larval development but still within the disk growth period, and it was found that p53 function is required for the delay of the developmental timer that controls the juvenile-adult transition. Likewise, delay of the timer after RH-damage requires p53. There is a strong correlation between delay of the timer and continued proliferation of disks. Although this relationship remains mysterious it is generally thought that negative feedback from proliferating disks inhibits a neural or humoral target. In contrast to imaginal disks, the polyploid larval cells are relatively insensitive to IR. No induction of p53 activity was detected after either RH damage or IR in tissues known to play key roles in developmental timing, such as the fat body, Dilp-2 expressing neurons, the prothoracic gland, and the corpora allata. The p53 activity reporter contains 2 consensus p53 binding sites and is thus expected to report accurately in many tissues. Although more trivial possibilities cannot be excluded, the absence of induction in these tissues suggests that p53 function in imaginal disks is sufficient for the developmental delay induced upon IR as well as for the disk-autonomous responses. Thus, the data support the view that imaginal disks “signal” to the developmental clock to delay pupariation, and indicates that the putative signal requires p53 for its production (Wells, 2011).

Two independent lines of evidence argue that, in contrast to previous reports, cell proliferation occurs at the same rate during regeneration as it does under normal developmental conditions. First, it was found that the number and distribution of mitotic cells is similar in yw and in p53 mutant disks following IR at every examination from the cell cycle reentry at + 6 h until pupariation, despite the significant differences in the length of this period between the two genotypes. Second, RH-damage in clonal experiments showed that undamaged cells in the vicinity of RH-damaged cells proliferate at the same rate as cells in control disks without damage. These results agree with others, in which an IR dose-dependent lengthening of the developmental timer was observed that correlated with an increase in clone size in adult wings; it was concluded that the remaining cells “undergo additional divisions to compensate for this loss”. As a whole the data indicate that cell divisions occur at the normal rate, with additional divisions that occur during a p53-dependent slowing of the larval timer (Wells, 2011).

In addition, the results indicate that some aspects of disk patterning are delayed while the disk regenerates. This delay is also p53-dependent. One interpretation of these results is that the early, p53-dependent cell death program, by eliminating massive numbers of cells, directly delays ongoing the patterning process. However, the finding that dronc mutant animals, which are unable to induce cell death, exhibit the same regeneration responses as wild-type after IR argues against this idea. An alternative possibility arises from the observation that the disk patterning delay and the animal-wide delay are correlated in time, and thus could be inter-dependent. A third possibility is that p53 induces a disk-wide developmental checkpoint, directly dependent upon its role in the DDR but independent of the disk-produced “signal” that delays the larval timer, which couples regenerative growth to stage-appropriate cell fate specification. Further experiments are required to distinguish between these alternatives. Regardless of the mechanism, however, the finding that cell division proceeds at a similar rate during the delay of patterning regardless of p53 status implies that cell division and late patterning gene expression are independently regulated under these conditions (Wells, 2011).

It has been hypothesized that dying cells emit information that stimulates proliferation of surviving cells to regenerate the damaged tissue. The regulation of expression of pro-apoptotic genes and pathways such as Rpr, Hid, Eiger/TNF and JNK by p53, is consistent with the idea that p53 induces apoptosis, which in turn stimulates regeneration. However, the results indicate that this is not the case: the regeneration response is induced after IR even in cells rendered incapable of inducing apoptosis because of a null mutation in dronc. Since these dronc mutant cells remain wildtype for p53, these data support an apoptosis-independent role of p53 in provoking regeneration. Indeed, p53 is required for the tissue repair response even when RH genes are expressed. Thus, at a minimum, the data indicate that expression of pro-death genes and caspase activation are not sufficient to trigger regeneration (Wells, 2011).

Since regeneration does not occur in its absence, p53 appears to be upstream of the signal that triggers regeneration. Three scenarios are suggested for the regeneration trigger downstream of p53 activation. First, it was find that p53 functions cell-autonomously to promote the ectopic induction of Wg, an early event in regeneration induced by a variety of methods in numerous animals. Moreover, expression of p53 under conditional Gal4 control induces ectopic Wg in the wing disk. Thus, the re-organization of disk patterning of the damaged tissue by ectopic expression of Wg, which necessitates a developmental delay for the completion of patterning and growth, may serve to trigger regeneration. However, in contrast to previous models invoking Wg as a mitogen, the current findings indicate that cell proliferation continues at its developmentally programmed pace during this extended period of time (Wells, 2011).

Since JNK activation after tissue damage is p53-dependent, a second candidate for the regeneration trigger is the JNK signaling pathway. JNK is activated early after tissue damage and is important for wound healing. JNK signaling is also activated upon disruptions of Wg and Dpp in the wing disk, and can itself lead to activation of Wg and Dpp expression. JNK activity appears to be upstream of Wg expression, since hep null mutations, which eliminate JNK activity, prevent ectopic expression of Wg after RH damage. A third possibility is that regeneration is triggered via a distinct program of gene expression directed by p53, which is independent of JNK or Wg (Wells, 2011).

Overall, this work suggests that p53 acts as a master regulator of tissue plasticity through its roles in the DDR, in tissue repair, and in coordinating these events with the animal's physiology. In addition to its role in the initiation of regeneration, these results argue that p53 is responsible for regulating the expression of a signal(s) from disks that prolongs larval development to allow regeneration after either RH or IR damage. Studies that identify this signal, that determine from which tissue it arises, and that delineate the mechanism by which p53 controls each aspect of the regeneration process are important goals for the future (Wells, 2011).

Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson's disease model

Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson's disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, it is reported that p53, basket and ICE gene knockdown in dopaminergic neurons prolong life span and locomotor activity in D. melanogaster lines chronically exposed to (1 microM) paraquat [PQ, oxidative stress (OS) generator] compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. It is proposed that a therapy with antioxidants and selectively 'switching off' death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition (Ortega-Arellano, 2013).

p53- and ERK7-dependent ribosome surveillance response regulates Drosophila Insulin-Like peptide secretion

Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. This study uncovered a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. Impairment of ribosome biogenesis specifically in the IPCs was shown to strongly inhibits dILP secretion, consequently leading to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, evidence is provided that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, it is concluded that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions (Hasygar, 2014).

This study reports a novel cell-autonomous control mechanism for dILP secretion. Specifically, it is concluded that 1) inhibition of ribosome biogenesis in the IPCs at any level tested, including ribosomal gene expression (Myc), ribosome maturation (Rio1, Rio2, Tsr1) or by introducing imbalance of ribosomal components (Rpl35A), triggers a response to inhibit dILP secretion, 2) this inhibitory response is dependent on p53, a known surveillance factor for ribosome biogenesis, 3) a downstream effector of this ribosome surveillance pathway is protein kinase ERK7, as erk7 mRNA levels are elevated upon inhibited ribosome biogenesis and p53 activation and erk7 is essential to inhibit dILP secretion in both conditions, 4) the ribosome surveillance mechanism discovered in this study likely contributes to starvation-induced inhibition of dILP secretion. These findings significantly broaden the view about the regulatory functions of the ribosome surveillance pathways, which have been mainly explored at the level of proliferating cells. Ribosome biogenesis serves as the key determinant of cell autonomous growth control and it is finely tuned to match with the cellular nutrient and energy status. Coupling dILP secretion to the ribosome biogenesis pathway is an elegant mechanism for multicellular animals to synchronize the hormonal growth control with the local cell autonomous regulation of growth. Comparable to the finding in the IPCs, inhibition of ribosome biogenesis in the fat body leads to a block of dILP secretion in the IPCs through an unknown humoral mechanism. Linking ribosome biogenesis to growth control through parallel mechanisms likely provides a robust regulatory network to tune down systemic growth signals whenever any region of the body experiences nutrient deprivation. This synchronization is likely important to maintain balanced growth throughout the spectrum of dietary conditions. Future studies should be aimed to explore the possible interrelationship between the fat body-derived signals and the cell-autonomous mechanism discovered here (Hasygar, 2014).

These findings highlight that the p53-mediated ribosome surveillance pathway can serve highly cell type-specific functions in vivo. This is interesting when considering human ribosomopathies, genetic diseases caused by impaired ribosome biogenesis manifesting with a wide spectrum of tissue-specific defects. One of the ribosomopathies, Shwachman-Diamond syndrome (SDS), is manifested with a failure in pancreatic function. A mouse model of SDS displays general preservation of ductal and endocrine compartments, but reduced amount of zymogen granules. Moreover, SDS mutant mice have reduced glucose tolerance, suggesting compromised endocrine function. It will be interesting to learn whether p53 and ERK7 act as mediators of the secretion-related defects observed in SDS (Hasygar, 2014).

Compared to other members of the MAP kinase family, ERK7 has remained relatively poorly characterized. Earlier studies in mammalian and Drosophila cells have shown that ERK7 protein levels are actively regulated at the level of protein degradation. In mammals, an increase in ERK7 levels leads to autophosphorylation and consequent activation. Consistent with the idea that ERK7 is mainly regulated through abundance, it was observed that elevated ERK7 expression had a prominent impact in the function of IPCs. Interestingly, earlier studies have linked ERK7 function to growth regulation by showing that ERK7 protein is stabilized by serum and amino acid starvation. The data provides evidence that impaired ribosome biogenesis as well as starvation increases the expression of erk7 mRNA revealing a novel regulatory level for ERK7 function. In addition to the conditions explored here, ERK7 expression levels increase towards the end of larval development when growth is ceased. It will be interesting to learn further how ERK7 expression is regulated and whether ERK7 has a function in tissue growth control beyond its role in the IPCs. Earlier studies in cell culture have shown evidence that ERK7 regulates cancer cell proliferation and autophagy, suggesting that ERK7 may have a broader role in the regulation of tissue growth (Hasygar, 2014).


EVOLUTIONARY HOMOLOGS

C. elegans p53 homolog

A C. elegans homolog of mammalian p53 has been identified. Using RNAi and DNA cosuppression technology, it has been shown that C. elegans p53 (cep-1) is required for DNA damage-induced apoptosis in the C. elegans germline. However, cep-1 RNAi does not affect programmed cell death occurring during worm development and physiological (radiation-independent) germ cell death. The DNA binding domain of CEP-1 is related to vertebrate p53 members and possesses the conserved residues most frequently mutated in human tumors. Consistent with this, CEP-1 acts as a transcription factor and is able to activate a transcriptional reporter containing consensus human p53 binding sites. The data support the notion that p53-mediated transcriptional regulation is part of an ancestral pathway mediating DNA damage-induced apoptosis and reveals C. elegans as a genetically tractable model organism for studying the p53 apoptotic pathway (Schumacher, 2001).

In order to search the C. elegans genome for distant p53 family members, a series of profiles was constructed from a multiple alignment of accepted members of the p53 family (p53 of human, mouse, hamster, chicken, Xenopus, trout, squid, and mussel, as well as human p63 and p73). Profiles constructed from the whole sequences, as well as those constructed from the DNA binding region, identified a highly significant relationship to the C. elegans predicted ORF F52B5.5 that possesses a region distinctly related to the DNA binding domain of p53. No other p53 family-related sequences were found in the C. elegans genome (Schumacher, 2001).

The putative 645 aa protein termed CEP-1 was aligned with known members of the p53 family. The detectable evolutionary conservation of C. elegans p53 is mostly limited to the regions involved in DNA binding (conserved regions II-V). Comparison of CEP-1 with human p53 indicates that residues critical for DNA binding, as revealed in the three-dimensional structure of p53 bound to DNA, are conserved in C. elegans. Five out of eight amino acids implicated in DNA binding are also conserved, and an additional one is similar. Moreover, out of the six amino acid residues that are most frequently mutated in cancer, two are conserved and two are substituted by similar amino acids. The two conserved amino acids R248 and R273 are by far the most frequently mutated residues and account for more than 20% of tumor-associated p53 mutations. Finally, all four residues implicated in Zn binding are conserved. Although they are weakly conserved, two potential phosphorylation sites were found corresponding to serine 15 and serine 37 of human p53. These sites are implicated in DNA damage-dependent activation of p53 at the N terminus. The one related to serine 15 (CEP-1 S20) lies in a conserved region (PDSQ[D/E]). At the C terminus of CEP-1, a small but distinct amino acid conservation was found at the tetramerization domain. However, there appears to be no obvious acidic domain characteristic for the human transactivation domain (Schumacher, 2001).

Surprisingly, RNAi feeding of cep-1 does not affect cell cycle arrest after irradiation, as demonstrated by the fact that mitotic germ cells from cep-1 RNAi worms respond to irradiation comparably to the wild-type. It is therefore conceivable that Cep-1 is dispensable for DNA damage-induced cell cycle arrest. However, since no cytological markers for various cell cycle phases are currently available, the possibility that Cep-1 might only be required for a G1/S checkpoint cannot be excluded. In the type of experiment performed here, cells defective for cep-1 might still arrest the cell cycle at a G2/M checkpoint and appear to respond to DNA damage in a wild-type manner (Schumacher, 2001).

To confirm that the inactivation of cep-1 leads to an inhibition of radiation-induced cell death, an independent method was used to inactivate cep-1 in the germline. DNA cosuppression is based on the observation that high copy number expression of a gene leads to specific inactivation of this gene in the germline. This effect, which genetically partially overlaps with the RNAi phenomenon, is presumably due to the formation of double-stranded RNAi due to transcription from copies of the transgene oriented in opposite directions. Transgenic worm lines were generated that contained the pRF-4 roller marker as well as a high concentration of cep-1 (promoter and first three exons). Upon the irradiation of cep-1 cosuppression lines, the results of RNAi experiments were confirmed; radiation-induced cell cycle arrest is maintained, whereas radiation-induced pachytene cell apoptosis is completely abrogated (Schumacher, 2001).

While CEP-1 is most closely related to Drosophila p53, the sequence similarity is subtle (<20% identity) and is not revealed by conventional sequence comparison methods such as BLAST. Functional conservation at such a low level of sequence similarity underscores the potential of the generalized-profile method for the detection of homologs in distantly related model organisms. The experimental findings support the notion of an ancient function for p53 in DNA damage-induced apoptosis. As is the case in Drosophila, cep-1 function impinges on radiation-induced programmed cell death but not on radiation-induced cell cycle arrest. It is likely that this ancient p53-dependent pro-apoptotic function depends on the transcriptional activation of target genes that act on the core apoptotic pathway. It will be interesting to determine those targets in C. elegans. It is noteworthy that p53 is highly expressed in the germlines of flies, clams, and mammals. cep-1 function is required for DNA damage-induced germ cell death in the C. elegans germline. It is thus worth speculating about the selective advantage conferred by p53 expression in the germline. In adult C. elegans hermaphrodites, the germline is the only proliferative tissue, and approximately two thirds of embryonic cell division occurs within the very first hours after fertilization, apparently without any DNA damage checkpoints. To guard its progeny from acquiring deleterious mutations, it would seem advantageous to install sensitive DNA damage checkpoints in the germline. In C. elegans this is achieved by making only meiotic pachytene cells competent to die by DNA damage-induced apoptosis. Given that meiotic recombination is being completed in the pachytene stage, this checkpoint also guards from mistakes that may arise when SPO-11-induced double-strand breaks required to initiate the meiotic recombination process are left unprocessed. In light of this, it is interesting that the absence of mouse p53 leads to a reduced amount of germ cell apoptosis, which results in a high frequency of abnormal sperm. Thus, p53 may have an important and conserved role in maintaining the fidelity of germ cells by the elimination of compromised cells (Schumacher, 2001).

The cellular response to genotoxic stress involves the integration of multiple prosurvival and proapoptotic signals that dictate whether a cell lives or dies. In mammals, AKT/PKB regulates cell survival by modulating the activity of several apoptotic proteins, including p53. In Caenorhabditis elegans, akt-1 and akt-2 regulate development in response to environmental cues by controlling the FOXO transcription factor daf-16, but the role of these genes in regulating p53-dependent apoptosis is not known. In this study, it was shown that akt-1 and akt-2 negatively regulate DNA-damage-induced apoptosis in the C. elegans germline. The antiapoptotic activity of akt-1 is independent of its target gene daf-16 but dependent on cep-1/p53. Although only akt-1 regulates the apoptotic activity of cep-1, both akt-1 and akt-2 modulate the intensity of the apoptotic response independently of the transcriptional activity of CEP-1. Finally, it was shown that AKT-1 regulates apoptosis but not cell-cycle progression downstream of the HUS-1/MRT-2 branch of the DNA damage checkpoint (Quevedo, 2007).

In C. elegans, mrt-2, hus-1, and clk-2 encode checkpoint proteins that transmit DNA-damage signals to the core apoptotic pathway through CEP-1/p53. HUS-1 and MRT-2 form part of the 9:1:1 complex, whereas CLK-2 functions in parallel to the 9:1:1 complex. In addition to activating apoptosis, these checkpoint genes also promote cell-cycle arrest in the mitotic region of the germline in response to DNA damage independently of cep-1. Checkpoint mutants also produce inviable embryos after treatment with IR because they are unable to repair damaged DNA. Because AKT-1 appears to act upstream of CEP-1/p53, it was asked whether akt-1 also has a role in the checkpoint response. It was found that germline cell-cycle arrest was not altered in either akt-1 gain-of-function or loss-of-function mutants, and the survival of progeny from akt-1(mg144) and akt-1(ok525) worms were no more sensitive to IR than wild-type worms. Therefore, these results indicate that AKT-1 does not act as a checkpoint protein but likely lies downstream of the DNA damage checkpoint to regulate the apoptotic activity of CEP-1/p53. To test this, double mutants were generated between akt-1(ok525) and loss-of-function alleles in the clk-2, mrt-2, and hus-1 checkpoint genes. It was found that clk-2(qm37);akt-1(ok525) double mutants were as resistant to damage-induced apoptosis as clk-2(qm37) single mutants, indicating that akt-1 does not act downstream of clk-2. However, irradiated mrt-2(e2663);akt-1(ok525) or hus-1(op244);akt-1(ok525) double mutants exhibited similar levels of apoptosis as irradiated wild-type controls, indicating that AKT-1 acts downstream of, or in parallel to, the 9:1:1 checkpoint. This suggests that inhibition of AKT-1 is part of the mechanism by which the HUS-1/MRT-2 complex signals to activate CEP-1/p53-dependent apoptosis in response to DNA damage. To assess this, CEP-1/p53 transcriptional activity was measured in hus-1(op244);akt-1(ok525) and mrt-2(e2663);akt-1(ok525) double-mutant animals. Although CEP-1/p53 is modestly activated in hus-1(op244) and mrt-2(e2663) single mutants treated with IR, presumably because the CLK-2 checkpoint is active, this activation was not enhanced by the akt-1(ok525) allele. Therefore, the increased germ-cell apoptosis observed in mrt-2(e2663);akt-1(ok525) and hus-1(op244);akt-1(ok525) double mutants treated with IR was not due to an increase in CEP-1/p53 transcriptional activity. Because AKT-2 is able to regulate apoptosis without affecting CEP-1/p53 transcriptional activity, hus-1(op244);akt-2(ok393) double mutants were created and similar levels of apoptosis were observed in these mutants as with the hus-1(op244);akt-1(ok525) strain. Because the increased IR-induced apoptosis observed in akt-1(ok525) mutants requires functional cep-1, these results suggest that CEP-1 may also regulate apoptosis independently of its transcriptional activity, as described in mammalian cells. The possibility that CEP-1 regulates the transcription of genes, other than egl-1, that also regulate germline apoptosis cannot be ruled out. A third possibility is that AKT-1/2 can modulate the magnitude of the apoptotic response independently of CEP-1, perhaps by regulating components of the core apoptotic pathway (Quevedo, 2007).

Other invertebrate p53 homologs

The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. This study focused on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. These results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal (Pearson, 2010).

p53 in zebrafish

p53 and its main negative regulator, Mdm2, are key players in mammalian cancer development. Activation of the transcription factor p53 through DNA damage or other stresses can result in cell cycle arrest, apoptosis, or both. Because of the absence of characterized p53 signaling in zebrafish (Danio rerio), the roles of Mdm2 and p53 were studied in zebrafish by generating early embryonic knockdowns, and the involvement of p53 in DNA damage-induced apoptosis was examined. p53-deficient embryos, induced by injection of antisense morpholinos, were morphologically indistinguishable from control embryos, when unperturbed, whereas Mdm2 knockdown embryos were severely apoptotic and arrested very early in development. Double knockdowns showed that p53 deficiency rescues Mdm2-deficient embryos completely, similar to observations in mice. p53 deficiency also markedly decreases DNA damage-induced apoptosis, elicited by ultraviolet irradiation or by the anti-cancer compound camptothecin. p21/Waf/Cip-1 appears to be a downstream target of zebrafish p53, as revealed by relative p21 mRNA levels. In contrast to mammals, zebrafish may regulate p53 activity by using an internal polyA signal site. It is concluded that zebrafish represents a promising model organism for future compound-based and genetic screens and it will be helpful for the identification and characterization of new anticancer drugs and new targets for cancer treatment (Langheinrich, 2003).

p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Delta133p53 and its zebrafish counterpart Delta113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. This study shows here that zebrafish Delta113p53 is directly transactivated by full-length p53 in response to developmental and DNA-damaging signals. More importantly, Delta113p53 functions to antagonize p53-induced apoptosis via activating bcl2L [closest to human Bcl-x(L)], and knockdown of Delta113p53 enhances p53-mediated apoptosis under stress conditions. Thus, it was demonstrated that the p53 genetic locus contains a new p53 response gene and that Delta113p53 does not act in a dominant-negative manner toward p53 but differentially modulates p53 target gene expression to antagonize p53 apoptotic activity at the physiological level in zebrafish. These results establish a novel feedback pathway that modulates the p53 response and suggest that modulation of the p53 pathway by p53 isoforms might have an impact on p53 tumor suppressor activity (Chen, 2009).

Regulation of p53 is critical for vertebrate limb regeneration

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. This study analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, it was shown that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, a potential mechanism was uncovered for the regulation of p53 during limb regeneration, based on its competitive inhibition by DeltaNp73. These results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration (Yun, 2013).

Dimeric combinations of MafB, cFos and cJun control the apoptosis-survival balance in limb morphogenesis

Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. This study showed that bZip factor MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. The data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73 (Suda, 2014).

p53 binding to DNA

Chromatin architectural protein HMGB1 can bind with extremely high affinity (K(d) < 1 pM) to a novel DNA structure that forms a DNA loop maintained at its base by a hemicatenane (hcDNA). The loop of hcDNA contains a track of repetitive sequences derived from CA-microsatellites. Using a gel-retardation assay it is demonstrated that tumor-suppressor protein p53 can also bind to hcDNA. p53 is a crucial molecule protecting cells from malignant transformation by regulating cell-cycle progression, apoptosis, and DNA repair by activation or repression of transcription of its target genes by binding to specific p53 DNA-binding sites and/or certain types of DNA lesions or alternative DNA structures. The affinity of p53 for hcDNA (containing sequences with no resemblance to the p53 DNA consensus sequence) is >40-fold higher (K(d) approximately 0.5 nM) than that for its natural specific binding sites within its target genes (Mdm2 promoter). Binding of p53 to hcDNA remains detectable in the presence of up to approximately 4 orders of magnitude of mass excess of competitor linear DNA, suggesting a high specificity of the interaction. p53 displays a higher affinity for hcDNA than for DNA minicircles (lacking functional p53-specific binding sequence) with a size similar to that of the loop within the hcDNA, indicating that the extreme affinity of p53 for hcDNA is likely due to the binding of the protein to the hemicatenane. Although binding of p53 to hcDNA occurs in the absence of the nonspecific DNA-binding extreme carboxy-terminal regulatory domain (30-C, residues 363-393), the isolated 30-C domain (but not the sequence-specific p53 'core domain', residues 94-312) can also bind hcDNA. Only the full-length p53 can form stable ternary complexes with hcDNA and HMGB1. The possible biological relevance of p53 and HMGB1 binding to hemicatenanes is discussed (Stros, 2004).

Signaling upstream of p53 and p73

A fuller understanding of the function of cyclin G, a commonly induced p53 target, has remained elusive. Cyclin G forms a quaternary complex in vivo and in vitro with enzymatically active phosphatase 2A (PP2A) holoenzymes containing B' subunits. Interestingly, cyclin G also binds in vivo and in vitro to Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate Mdm2 at T216. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at T216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Hdm2 at S166. Thus, these data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to regulate both Mdm2 and p53 (Okamoto, 2002).

Eukaryotic proteins are frequently regulated through their state of phosphorylation. Although protein kinases frequently recognize sequence motifs to target them to their sites in substrates, there is often less specificity in the sequence requirements of the major cellular phosphatases. Therefore, other mechanisms are needed for direction of phosphatases to their substrates, and these results suggest that cylin G serves such a role. In fact, PP2A is likely to be extensively regulated. Individual PP2A complexes have been shown to differ in some cases in their roles, localization, and substrate specificity. Thus, the apparently exclusive association of cyclin G with the B' subfamily is tantalizing. Cyclin G is of course not the only protein that has been shown to be able to interact with PP2A. Among the proteins shown to associate with PP2A and regulate its activity are the small t antigens encoded by SV40 and polyomavirus, the adenovirus E4orf4 protein, casein kinase II, Hox II, PKR, and several others. In some cases, the interaction results in negative regulation of PP2A activity. Although the effect of recruitment of cyclin G on the specific activity of PP2A is not known, cyclin G clearly does not block PP2A enzymatic activity, supporting the possibility that cyclin G serves to recruit PP2A to specific substrates (Okamoto, 2002).

The discovery that cyclin G binds to Mdm2 provided the impetus for testing whether Mdm2 might serve as such a substrate. The data strongly support the conclusion that at least two phosphorylation sites (Mdm2 T216 and Hdm2 S166) are substrates of cyclin G-directed PP2A. Since Mdm2 can associate with a host of cellular proteins, a future challenge will be to determine whether such interactions are regulated by phosphorylation, and if so, which of these are regulated by the cyclin G-PP2A complex. It is, of course, also possible that the cyclin G-PP2A interaction is relevant to other potential substrates, and therefore, the identification of cellular proteins that can interact with cyclin G may prove to be very interesting (Okamoto, 2002).

Mouse cells lacking cyclin G contain both Mdm2 that is hyperphosphorylated at T216 and higher p53 levels when compared to wild-type cells. These two observations are very likely interrelated. Although it is still not fully understood how modification of p53 affects its functions in vivo, phosphorylation of p53 at some N-terminal residues (that are modified in cells in response to DNA damage) decreases the ability of p53 to bind to Mdm2 in vitro. Modification of Mdm2 also impacts on its interactions with p53; phosphorylation of human Hdm2 by DNA PK (at S17 within the N terminus) and phosphorylation of murine Mdm2 by cyclin A/CDK2 (at T216 within the acidic domain) block and attenuate, respectively, the ability of either protein to bind to p53. Moreover, phosphorylation of human Mdm2 (Hdm2) at S395, a process that can be accomplished by ATM kinase in vitro, counteracts Mdm2's ability to target p53 for degradation in vivo. It can thus be speculated that in general, activated p53 and deactivated Mdm2 are the more phosphorylated forms of each protein. The data imply that the function of cyclin G is to serve as a negative regulator of p53 by activating Mdm2 through dephosphorylation. When seen in this context, it becomes less surprising that many previous studies have indicated that cyclin G expression is associated with growth promotion rather than arrest. Most exciting is the evidence that cyclin G null mice have fewer and smaller carcinogen-induced liver tumors, consistent with the hypothesis that cyclin G serves to negatively regulate the tumor suppressor function of p53 (Okamoto, 2002).

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Pirh2 is a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. It is proposed that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function (Leng, 2003).

The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Akt activity has been shown to increase along with cellular senescence; inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology (Miyauchi, 2004).

The ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases respond to DNA damage by phosphorylating cellular target proteins that activate DNA repair pathways and cell cycle checkpoints in order to maintain genomic integrity. The oncogenic p53-induced serine/threonine phosphatase PPM1D (or Wip1: Drosophila homolog Protein phosphatase 2C) dephosphorylates two ATM/ATR targets, Chk1 and p53. PPM1D binds Chk1 and dephosphorylates the ATR-targeted phospho-Ser 345, leading to decreased Chk1 kinase activity. PPM1D also dephosphorylates p53 at phospho-Ser 15. PPM1D dephosphorylations are correlated with reduced cellular intra-S and G2/M checkpoint activity in response to DNA damage induced by ultraviolet and ionizing radiation. Thus, a primary function of PPM1D may be to reverse the p53 and Chk1-induced DNA damage and cell cycle checkpoint responses and return the cell to a homeostatic state following completion of DNA repair. These homeostatic functions may be partially responsible for the oncogenic effects of PPM1D when it is amplified and overexpressed in human tumors (Lu, 2005).

The ARF tumour suppressor (p14ARF in humans, p19ARF in mice) is a central component of the cellular defence against oncogene activation. The expression of ARF, which shares a genetic locus with the p16INK4a tumour suppressor, is regulated by the action of transcription factors such as members of the E2F family. ARF can bind to and inhibit the Hdm2 protein (Mdm2 in mice), which functions as an inhibitor and E3 ubiquitin ligase for the p53 transcription factor. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor-alpha induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor (Rocha, 2005).

Biochemical mechanisms that control the levels and function of key tumor suppressor proteins are of great interest as their alterations can lead to oncogenic transformation. This study identified the human orthologue of Drosophila Ecdysoneless (hEcd) as a novel p53-interacting protein. Overexpression of hEcd increases the levels of p53 and enhances p53 target gene transcription whereas hEcd knockdown has the opposite effects on p53 levels and target gene expression. Furthermore, hEcd interacts with Mdm2 and stabilizes p53 by inhibiting Mdm2-mediated degradation of p53. Thus, hEcd protein represents a novel regulator of p53 stability and function. These studies also represent the first demonstration of a biochemical function for hEcd protein and raise the possibility that altered hEcd levels and/or function may contribute to oncogenesis (Zhang, 2006).

The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. This study has identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. This study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response (Tang, 2008).

MdmX, also known as Mdm4, is a critical negative regulator of p53, and its overexpression serves to block p53 tumor suppressor function in many cancers. Consequently, inhibiting MdmX has emerged as an attractive approach to restoring p53 function in those cancers that retain functional p53. However, the consequences of acute systemic MdmX inhibition in normal adult tissues remain unknown. To determine directly the effects of systemic MdmX inhibition in normal tissues and in tumors, mdmX-/- mice were crossed into the p53ERTAM knockin background. In place of wild-type p53, p53ERTAM knockin mice express a variant of p53, p53ERTAM, that is completely dependent on 4-hydroxy-tamoxifen for its activity. MdmX inhibition was then modeled by restoring p53 function in these MdmX-deficient mice. It was shown that MdmX is continuously required to buffer p53 activity in adult normal tissues and their stem cells. Importantly, the effects of transient p53 restoration in the absence of MdmX are nonlethal and reversible, unlike transient p53 restoration in the absence of Mdm2, which is ineluctably lethal. The therapeutic impact of restoring p53 in a tumor model is enhanced in the absence of MdmX, affording a significant extension of life span over p53 restoration in the presence of MdmX. Hence, systemic inhibition of MdmX is both a feasible therapeutic strategy for restoring p53 function in tumors that retain wild-type p53 and likely to be significantly safer than inhibition of Mdm2 (Garcia, 2011).

PML induces a permanent cell cycle exit and activates p53

The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. This study shows that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors (Vernier, 2011).

Transcriptional regulation by p53 homologs

Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. The possibility was investigated that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. This study shows that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, increased specific activity of p53 protein was detected in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. The possibility was investigated that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. It is proposed that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging (Vaziri, 1997).

The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. Unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites (Espinosa, 2001).

Surprisingly, p300 does not function by facilitating p53 binding to its DNA recognition sites within chromatin. Instead, p300 acts at a later step in the transcription process by acetylating nucleosomes within the proximal and distal p21 promoter when targeted by bound p53. This presumably renders the nucleosomes sufficiently fluid to allow interaction with other components of the transcription machinery. p300-mediated transcriptional activation has been described for other chromatin-assembled genes. These experiments demonstrate that a mechanism by which p300 can regulate the activity of natural promoters operates by acetylating chromatin over a long-range when recruited by a distal transcription factor. In the absence of p53, p300 cannot acetylate nucleosomes due to lack of template targeting, and the p21 promoter remains inactive. p53 proteins containing mutations in lysine residues acetylated by p300 are as active as wild-type p53 in regulating p21 transcription in vitro. This indicates that acetylation of p53 does not contribute to its transactivation potential, and that p300 does not mediate transcription by this mechanism in biochemical assays. This conclusion is in agreement with previous in vivo analyses in which p53 mutants lacking these lysine residues does not show a significant decrease in transcriptional activity. However, p53 acetylation may play a role in protein stabilization or subnuclear localization (Espinosa, 2001).

It is intriguing that p53 binds to the p21 promoter with higher affinity and with different kinetics when assembled into chromatin than it does to DNA. This is particularly interesting because this occurs in the absence of chromatin remodeling or modifying complexes and is not observed with other transcription factors that can also bind to nucleosomes. This could be explained if bending of the DNA, when wrapped around a nucleosome, generates a secondary structure that is more stable for p53 binding. Indeed, previous studies determined the importance of DNA bending for p53 high-affinity binding and predicted that some p53 binding sites would be exposed and accessible when incorporated into a nucleosome. Importantly, the structure recognized by p53 in p21 promoter DNA is preserved and improved or stabilized in chromatin. The physiological significance of the distinct kinetics of p53 occupancy observed on the p21 promoter as chromatin or DNA is unclear. The linear rate of association of p53 with chromatin may indicate that lower concentrations of p53 are required to fully occupy binding sites in vivo than the cooperative binding to DNA would indicate. This could be significant if the cell has to respond efficiently to activate p53-responsive pathways without waiting for a critical threshold of p53 concentration to be reached. It should be emphasized, however, that the nature of p53 binding to chromatin and the requirements for remodeling/modifying activities may vary with individual target promoters (Espinosa, 2001).

Activation of the tumor suppressor p53 by DNA damage induces either cell cycle arrest or apoptotic cell death. The cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21Cip1, whereas the apoptotic effect is mediated by transcriptional activation of mediators including PUMA and PIG3. What determines the choice between cytostasis and apoptosis is not clear. The transcription factor Myc is shown to be a principal determinant of this choice. Myc is directly recruited to the p21Cip1 promoter by the DNA-binding protein Miz-1. This interaction blocks p21Cip1 induction by p53 and other activators. As a result Myc switches, from cytostatic to apoptotic, the p53-dependent response of colon cancer cells to DNA damage. Myc does not modify the ability of p53 to bind to the p21Cip1 or PUMA promoters, but selectively inhibits bound p53 from activating p21Cip1 transcription. By inhibiting p21Cip1 expression Myc favors the initiation of apoptosis, thereby influencing the outcome of a p53 response in favor of cell death (Seoane, 2002).

Several conclusions can be drawn from these results. Myc selectively targets p21Cip1 in the p53 transcriptional program, sparing the ability of p53 to induce the expression of PUMA or PIG3. Myc does not alter the ability of p53 to bind to the p21Cip1 promoter but inhibits p21Cip1 transcriptional activation by promoter-bound p53. In the presence of p21, p53 can still bind to the PUMA promoter and induce the accumulation of its product, but apoptosis is not achieved. Thus, the p21-dependent block in apoptosis maps to a step downstream of the DNA damage-p53-PUMA pathway. The mechanism for this provocative observation is not obvious. These results suggest a model in which Myc selectively prevents p53-dependent transcriptional activation of p21Cip1, enabling pro-apoptotic factors such as PUMA to execute a cell death program. Thus, these results define, in mechanistic terms, how one element of the cellular context, that is, the level of Myc activity, can determine the outcome of the p53 response. Although it remains to be seen whether repression of p21Cip1 would be beneficial in cancer treatment, the mechanism proposed here suggests ways to influence the cell's response to stresses that result in activation of p53 (Seoane, 2002).

REDD1 has been identified as a novel transcriptional target of p53 induced following DNA damage. During embryogenesis, REDD1 expression mirrors the tissue-specific pattern of the p53 family member p63, the most ancient family member most closely related to the single gene present in Drosophila, and TP63 null embryos show virtually no expression of REDD1, which is restored in mouse embryo fibroblasts following p63 expression. In differentiating primary keratinocytes, TP63 and REDD1 expression are coordinately downregulated, and ectopic expression of either gene inhibits in vitro differentiation. REDD1 appears to function in the regulation of reactive oxygen species (ROS): TP63 null fibroblasts have decreased ROS levels and reduced sensitivity to oxidative stress, which are both increased following ectopic expression of either TP63 or REDD1. Thus, REDD1 encodes a shared transcriptional target that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation (Ellisen, 2002).

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress (Mirnezami, 2003).

The recruitment of hCtBP1 by proteins containing a PXDLS motif is regulated by changes in cellular redox potential. The central dehydrogenase domain of hCtBP1 contains a high-affinity binding site for NADH (GXGXXG), occupation of which induces a conformational change in the hCtBP1 molecule and an increase in binding to proteins such as E1A and ZEB. A mutation in hCtBP1 in the GXGXXG motif (G183A) abolishes NADH responsiveness. This site in hCtBP2 is conserved (amino acids 187-192): it was asked whether NADH could regulate the Hdm2:hCtBP2 interaction. NADH concentrations (0.01 to 1 mM) known to promote the interaction of hCtBP1 with PXDLS motif proteins inhibit binding of full-length GST-hCtBP2 to Hdm2. This inhibition did not occur when either GST-hCtBP2(1-110), lacking the dehydrogenase domain, or hCtBP2(G189A), containing a mutation in the NADH binding site, were used in the assays. Therefore, in contrast to interactions with PXDLS motif proteins, the conformational changes induced by NADH binding to the CtBP dehydrogenase domain result in a reduced affinity of hCtBP2 for Hdm2. Exposure of cells in culture to CoCl2 can be used as a model for the induction of a hypoxia-like stress response. CoCl2 treatment (200 μM) induces an increase in the cellular NADH/NAD+ ratio sufficient to promote binding of CtBP proteins to PXDLS motif proteins in the cell. 200 μM CoCl2 reduces the formation of Hdm2:hCtBP2 complexes in MCF-7 cells. Hypoxia, which has a greater effect on the cellular NADH/NAD+ ratio than CoCl2, is more effective than CoCl2 in reducing the Hdm2:hCtBP2 interaction. These data demonstrate, therefore, that the NADH-induced regulation of the Hdm2:hCtBP2 interaction also occurs in vivo (Mirnezami, 2003).

Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. The consequences of deregulated cyclin E expression have been studied in primary cells; cyclin E was found to initiate a p53-dependent response that prevents excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity is not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E leads to increased p53 serine15 phosphorylation that is sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 is rendered nonfunctional, then the excess cyclin E becomes catalytically active and causes defects in S phase progression, increased ploidy, and genetic instability. It is concluded that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity (Minella, 2002).

How might deregulated cyclin E cause S phase abnormalities that activate an S phase checkpoint? In yeast, S phase-promoting cyclins inhibit the transition of replication origins to the prereplicative state. Furthermore, when early-firing origins are inhibited by hydroxyurea, then the stalled early origins inhibit late origins through a checkpoint that requires the Mec1 protein (the budding yeast ATM/ATR homolog. Similarly, inhibition of ATR function in a human cell line by a kinase-inactive ATR mutant renders these cells hypersensitive to treatments that prolong DNA synthesis, and cyclin E overexpression is synthetically lethal with ATR inhibition. Thus, perhaps cyclin E deregulation leads to aberrant licensing of replication origins, and the resultant S phase progression defect may be sensed by a protein such as ATR, which then enforces an S phase checkpoint. Furthermore, the stalled replication origins associated with this prolonged S phase may be fragile and constitute the precursors to genetic instability. Another mechanism through which enforced cyclin E expression might impair normal cell cycle progression is by cyclin A-cdk2 inhibition, since cyclin A-cdk2 activity (and cyclin A expression) drops substantially in cells with ectopic cyclin E expression. However, cyclin E-induced cell cycle anomalies persist in E6-expressing cells with high levels of cyclin A-cdk2 kinase activity, so cyclin A-cdk2 activity cannot be the principle cause of the cyclin E-associated S phase phenotype (Minella, 2002).

The tumor suppressor protein p53 regulates transcriptional programs that control the response to cellular stress. Distinct mechanisms exist to activate p53 target genes as revealed by marked differences in affinities and damage-specific recruitment of transcription initiation components. p53 functions in a temporal manner to regulate promoter activity both before and after stress. Before DNA damage, basal levels of p53 are required to assemble a poised RNA polymerase II initiation complex on the p21 promoter. RNA pol II is converted into an elongating form shortly after stress but before p53 stabilization. Proapoptotic promoters, such as Fas/APO1, have low levels of bound RNA pol II but undergo damage-induced activation through efficient reinitiation. Surprisingly, in a p53-dependent process key basal factors TAFII250 and TFIIB assemble into the transcription machinery in a stress- and promoter-specific manner, behaving as differential cofactors for p53 action after distinct types of DNA damage (Espinosa, 2003).

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters, and endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. It is also shown, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-kappaB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity (Bates, 2005).

The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton. The majority of the Rho family proteins function as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho-family proteins, the Rnd subfamily members, including Rnd1, Rnd2, RhoE (also known as Rnd3), and RhoH, are GTPase deficient and are thus expected to be constitutively active. An unexpected role has been identified for RhoE/Rnd3 in the regulation of the p53-mediated stress response. This study demonstrates that RhoE is a transcriptional p53 target gene and that genotoxic stress triggers actin depolymerization, resulting in actin-stress-fiber disassembly through p53-dependent RhoE induction. Silencing of RhoE induction in response to genotoxic stress maintains stress fiber formation and strikingly increases apoptosis, implying an antagonistic role for RhoE in p53-dependent apoptosis. It was found that RhoE inhibits ROCK I (Rho-associated kinase I) activity during genotoxic stress and thereby suppresses apoptosis. The p53-mediated induction of RhoE in response to DNA damage favors cell survival partly through inhibition of ROCK I-mediated apoptosis. Thus, RhoE is thought to function by regulating ROCK I signaling to control the balance between cell survival and cell death in response to genotoxic stress (Ongusaha, 2006).

Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the functional relationship between both proteins is unknown. This study demonstrates that Dlx3 is a downstream target of p63. Moreover, transcription of Dlx3 is abrogated by mutations in the sterile alpha-motif (SAM) domain of p63 that are associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactylyectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias. These results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis associated with p63 mutations (Radoja, 2007).

Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, evidence is presented that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Genes were defined that are likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, functional evidence is provided that the BCL2 protein is regulated directly by miRNA34. The expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and the restoration of miRNA34 expression inhibits growth of NSCLC cells (Bommer, 2007).

A global decrease in microRNA (miRNA) levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, miRNA expression profiles of wild-type and p53-deficient cells were compared. A family of miRNAs, miR-34a-c, is described whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, consistent with the observed ability of miR-34 to downregulate a program of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation (He, 2007).

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of differentiation into any cell type of the organism (pluripotency) and indefinite self-renewal. H Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss- and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. Rem2 mediates the fibroblastic growth factor 2 (FGF2) signaling pathway to maintain proliferation of hESCs. Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D(1) to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, it was also shown to be as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D(1) expression/localization and suppression of p53 transcription. The effects of Rem2 on cyclin D(1) are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. These studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use (Edel, 2010).

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, the nuclear factor-kappaB (NF-kapaB) subunit p65 was identified as a major transcription factor that accumulates on chromatin of senescent cells. NF-kappaB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-kappaB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-kappaB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. These results demonstrate that NF-kappaB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-kappaB that contributes to the outcome of cancer therapy (Chien, 2011).

The tumor suppressor p53 can induce apoptosis by activating gene expression in the nucleus, or by directly permeabilizing mitochondria in the cytoplasm. It has been shown that PUMA, a downstream target of p53 and a BH3-only Bcl-2 family member, plays an essential role in apoptosis induced by both nuclear and cytoplasmic p53. To understand how PUMA does so, homologous recombination was used to delete the binding sites of p53 in the promoter of PUMA in human colorectal cancer cells. As a result, the induction of PUMA and apoptosis in response to p53 and DNA-damaging agents were abrogated. Transcription coactivator recruitment and histone modifications in the PUMA promoter were suppressed. However, induction of PUMA and apoptosis in response to non-DNA-damaging stimuli were unaffected. These results indicate that the binding of nuclear p53 to the specific sites within the PUMA promoter is essential for its ability to induce apoptosis and is likely to be required for its tumor suppressive capacity (Wang, 2007).

Regulation of the p53 transcriptional response by structurally diverse core promoters

p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, the role of core promoter structures in this process was examined. It was found that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. A downstream element was defined within the Fas/APO1 core promoter that is essential for its activation, and nuclear transcription factor Y (NF-Y) was identified as the downstream element binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress (Morachis, 2010).

In unstressed cells, certain p53 target promoters, like p21, are 'preloaded' with paused RNAP II, whereas proapoptotic promoters, among others, have negligible RNAP II association. Such striking variation in levels of promoter-bound RNAP II may have direct bearing on the differential activation kinetics observed after stress induction of p53-responsive genes. The existence of such regulatory mechanisms acting before DNA damage to establish a default programmatic transcriptional response to stress is very intriguing. These data suggest that the intrinsic properties of diverse p53 core promoters play a key role in regulating RNAP II affinity and dynamics to coordinate appropriate responses to different stress conditions. An unexpected level was found of transcriptional regulation governing RNAP II dynamics that is encoded within the DNA sequence of diverse core promoters that drives expression of p53-responsive genes. The TATA box within the p21 promoter has a critical role in recruiting the transcriptional machinery by promoting rapid formation of a functional PIC that is poised for initiation. However, the p21 core promoter is intrinsically inefficient for reinitiation, which may be enhanced by signal-dependent components acting at other levels of regulation to facilitate PIC reformation and prolonged RNA synthesis. In contrast to p21, the Fas/APO1 promoter does not contain a TATA box or other well-characterized core motifs, and the rate of PIC formation is very slow. A Fas downstream element that binds to NF-Y is essential for core promoter activity in vitro, and may nucleate PIC assembly by direct interaction with the general transcription machinery. Surprisingly, once transcription is engaged, the Fas/APO1 promoter is capable of efficient RNAP II reinitiation events. Published reports have demonstrated that initiation and reinitiation can be experimentally uncoupled and, in one example, reinitiation is faster than initiation, which was also observe with Fas/APO1 (Morachis, 2010).

Thus, two critical parameters of p53-dependent gene activation -- the kinetics of induction and duration of expression through frequent reinitiation -- are intrinsic, DNA-encoded features of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes. The default mode, as seen at the p21 promoter, is to rapidly form a PIC but undergo few rounds of reinitiation, whereas that of Fas/APO1 is the opposite. Of course, sustained p21 expression requiring multiple rounds of RNAP II reinitiation and reduced Fas/APO1 expression by infrequent reinitiation can be achieved by overriding the default programming through sophisticated epigenetic processes that are tailored to specific stress environments. Considering the advantage of preserving flexibility to fine-tune cell fate decisions, having a default, genetic program embedded in core promoter DNA would safeguard against misregulation, particularly of apoptotic genes. This may reflect an evolutionary need to balance cell growth while limiting the ability to self-destruct. It is difficult to envision a cellular system that would evolve to activate cell cycle arrest and apoptotic genes identically. If this were true, apoptosis would likely override the cell cycle arrest program without allowing the cell to recover from stress or DNA damage. Further investigation into the default mechanisms used by structurally diverse p53 target genes may provide insight into how the programmatic response to stress is regulated and how it can be manipulated for targeted therapies (Morachis, 2010).

5'-3'-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage

Optimal induction of p53 protein after DNA damage requires Ribosomal protein L26 (RPL26) mediated increases in p53 mRNA translation. This study reports the existence of a dsRNA region containing complementary sequences of the 5'- and 3'-untranslated regions (UTRs) of human p53 mRNA that is critical for its translational regulation by RPL26. Mutating as few as 3 bases in either of the two complementary UTR sequences abrogates the ability of RPL26 to bind to p53 mRNA and stimulate p53 translation, while compensatory mutations restore this binding and regulation. Short, single-strand oligonucleotides that target this 5'-3'-UTR base-pairing region blunt the binding of RPL26 to p53 mRNA in cells and reduce p53 induction and p53-mediated cell death after several different types of DNA damage and cellular stress. The ability to reduce stress induction of p53 with oligonucleotides or other small molecules has numerous potential therapeutic uses (Chen, 2010).

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells

Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. This study shows that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. It was also shown that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development (Mardaryev, 2014).

p53 pathway is involved in cell competition during mouse embryogenesis

The function of tumor suppressor p53 (see Drosophila p53) has been under intense investigation. Murine double minute (MDM)2 and MDM4 are two major negative regulators of p53. This study used the strategy of haploinsufficiency of Mdm2 and Mdm4 to induce mild p53 activation in vivo and found that Mdm2+/-Mdm4+/- double-heterozygous mice exhibited normal embryogenesis. However, closer examination demonstrated that the Mdm2+/-Mdm4+/- cells exhibited a growth disadvantage and were outcompeted during development in genetic mosaic embryos that contained wild-type cells. Further study indicated the out-competition phenotype was dependent on the levels of p53. These observations revealed that cells with mild p53 activation were less fit and exhibited altered fates in a heterotypic environment, resembling the cell competition phenomenon first uncovered in Drosophila. By marking unfit cells for elimination, p53 may exert its physiological role to ensure organ and animal fitness (Zhang, 2017).

Translational repression of p53 by RNPC1, a p53 target overexpressed in lymphomas

The p53 pathway is critical for tumor suppression, as the majority of human cancer has a faulty p53. This study identified RNPC1, a p53 target and a RNA-binding protein, as a critical regulator of p53 translation. Ectopic expression of RNPC1 inhibits, whereas knockdown of RNPC1 increases, p53 translation under normal and stress conditions. RNPC1 prevents cap-binding protein eIF4E from binding p53 mRNA via its C-terminal domain for physical interaction with eIF4E, and its N-terminal domain for binding p53 mRNA. Consistent with this, it was found that RNPC1 directly binds to p53 5' and 3'untranslated regions (UTRs). Importantly, it was shown that RNPC1 inhibits ectopic expression of p53 in a dose-dependent manner via p53 5' or 3' UTR. Moreover, loss of RNPC1 in mouse embryonic fibroblasts increases the level of p53 protein, leading to enhanced premature senescence in a p53-dependent manner. Finally, to explore the clinical relevance of this finding, it was shown that RNPC1 was frequently overexpressed in dog lymphomas, most of which were accompanied by decreased expression of wild-type p53. Together, this study identified a novel p53-RNPC1 autoregulatory loop, and these findings suggest that RNPC1 plays a role in tumorigenesis by repressing p53 translation (Zhang, 2011).

Gene-specific repression of the p53 target gene PUMA via intragenic CTCF-Cohesin binding

The p53 transcriptional program orchestrates alternative responses to stress, including cell cycle arrest and apoptosis, but the mechanism of cell fate choice upon p53 activation is not fully understood. PUMA (p53 up-regulated modulator of apoptosis), a key mediator of p53-dependent cell death, is regulated by a noncanonical, gene-specific mechanism. Using chromatin immunoprecipitation assays, it was found that the first half of the PUMA locus (approximately 6 kb) is constitutively occupied by RNA polymerase II and general transcription factors regardless of p53 activity. Using various RNA analyses, it was found that this region is constitutively transcribed to generate a long unprocessed RNA with no known coding capacity. This permissive intragenic domain is constrained by sharp chromatin boundaries, as illustrated by histone marks of active transcription (histone H3 Lys9 trimethylation [H3K4me3] and H3K9 acetylation [H3K9Ac]) that precipitously transition into repressive marks (H3K9me3). Interestingly, the insulator protein CTCF (CCCTC-binding factor) and the Cohesin complex occupy these intragenic chromatin boundaries. CTCF knockdown leads to increased basal expression of PUMA concomitant with a reduction in chromatin boundary signatures. Importantly, derepression of PUMA upon CTCF depletion occurs without p53 activation or activation of other p53 target genes. Therefore, CTCF plays a pivotal role in dampening the p53 apoptotic response by acting as a gene-specific repressor.

Protein interactions of p53 family members

Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. The nuclear phosphoprotein p73 can engage in a physical association with the Yes-associated protein (YAP). This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine (Y) of the PPPPY motif of p73 is essential for the association with YAP. Unlike p73alpha, p73beta, and p63alpha, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73gamma isoform do not interact with YAP. Indeed, YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73alpha transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling (Strano, 2001).

A family of proteins termed ASPP has been identified. ASPP1 is a protein homologous to 53BP2, the C-terminal half of ASPP2. ASPP proteins interact with p53 and specifically enhance p53-induced apoptosis but not cell cycle arrest. Inhibition of endogenous ASPP function suppresses the apoptotic function of endogenous p53 in response to apoptotic stimuli. ASPP proteins enhance the DNA binding and transactivation function of p53 on the promoters of proapoptotic genes in vivo. Two tumor-derived p53 mutants with reduced apoptotic function were defective in cooperating with ASPP in apoptosis induction. The expression of ASPP is frequently downregulated in human breast carcinomas expressing wild-type p53 but not mutant p53. Therefore, ASPP regulates the tumor suppression function of p53 in vivo (Samuels-Lev, 2001).

Nuclear localization of p53 is essential for its tumor suppressor function. Parc, a Parkin-like ubiquitin ligase, has been identified as a cytoplasmic anchor protein in p53-associated protein complexes. Parc directly interacts and forms a ~1 MDa complex with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of Parc induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Overexpression of Parc promotes cytoplasmic sequestration of ectopic p53. Furthermore, abnormal cytoplasmic localization of p53 was observed in a number of neuroblastoma cell lines; RNAi-mediated reduction of endogenous Parc significantly sensitizes these neuroblastoma cells in the DNA damage response. These results reveal that Parc is a critical regulator in controlling p53 subcellular localization and subsequent function (Nikolaev, 2003).

YY1 is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. Direct physical interactions of YY1 with Hdm2 (the human ortholog of Mdm2) and p53 have been identified and the basis for YY1-regulating p53 ubiquitination has been shown to be its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor and cyclin-dependent kinase inhibitor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis (Sui, 2004).

The tumor suppressor p53 regulates cell-cycle progression and apoptosis in response to genotoxic stress, and inactivation of p53 is a common feature of cancer cells. The levels and activity of p53 are tightly regulated by posttranslational modifications, including phosphorylation, ubiquitination, and acetylation. The transcription factor YY1 interacts with p53 and inhibits its transcriptional activity. YY1 disrupts the interaction between p53 and the coactivator p300, and expression of YY1 blocks p300-dependent acetylation and stabilization of p53. Furthermore, expression of YY1 inhibits the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. YY1 also interacts with Mdm2 and the expression of YY1 promotes the assembly of the p53-Mdm2 complex. Consequently, YY1 enhances Mdm2-mediated ubiquitination of p53. Inactivation of endogenous YY1 enhances the accumulation of p53 as well as the expression of p53 target genes in response to DNA damage, and it sensitizes cells to DNA damage-induced apoptosis. Hence, these results demonstrate that YY1 regulates the transcriptional activity, acetylation, ubiquitination, and stability of p53 by inhibiting its interaction with the coactivator p300 and by enhancing its interaction with the negative regulator Mdm2. YY1 may, therefore, be an important negative regulator of the p53 tumor suppressor in response to genotoxic stress (Gronroos, 2004).

In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. This study describes an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. These data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53 (Sasaki, 2007).

Numb is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. Numb is also an endocytic protein, and the Notch-Numb counteraction has been linked to this function. There might be, however, additional functions of Numb, as witnessed by its proposed role as a tumour suppressor in breast cancer. This study describes a previously unknown function for human Numb as a regulator of tumour protein p53 (also known as TP53). Numb enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of Numb expression. In primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of Numb expression causes increased activity of the receptor Notch5. Thus, in these cancers, a single event -- loss of Numb expression -- determines activation of an oncogene (Notch) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that Numb-defective breast tumours display poor prognosis. These results uncover a previously unknown tumour suppressor circuitry (Colalucam, 2008).

TGFβ ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. This study shows that TGFβ-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFβ acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, two candidate metastasis suppressor genes associated with metastasis risk were identified in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFβ-dependent inhibition of p63 function (Adorno, 2009)

Fine-tuning p53 activity through C-terminal modification significantly contributes to HSC homeostasis and mouse radiosensitivity

Cell cycle regulation in hematopoietic stem cells (HSCs) is tightly controlled during homeostasis and in response to extrinsic stress. p53, a well-known tumor suppressor and transducer of diverse stress signals, has been implicated in maintaining HSC quiescence and self-renewal. However, the mechanisms that control its activity in HSCs, and how p53 activity contributes to HSC cell cycle control, are poorly understood. This study used a genetically engineered mouse to show that p53 C-terminal modification is critical for controlling HSC abundance during homeostasis and HSC and progenitor proliferation after irradiation. Preventing p53 C-terminal modification renders mice exquisitely radiosensitive due to defects in HSC/progenitor proliferation, a critical determinant for restoring hematopoiesis after irradiation. Fine-tuning the expression levels of the cyclin-dependent kinase inhibitor p21, a p53 target gene, contributes significantly to p53-mediated effects on the hematopoietic system. These results have implications for understanding cell competition in response to stresses involved in stem cell transplantation, recovery from adverse hematologic effects of DNA-damaging cancer therapies, and development of radioprotection strategies (Wang, 2011).

In vitro and in vivo studies show that p53 activity is determined to a significant extent by mechanisms that regulate its abundance and stability. Mdm2 and Mdmx reduce p53 activity by binding to the N-terminal p53 transactivation domain (TAD) and by promoting ubiquitin-dependent p53 degradation. Control of p53 degradation is thought to partially require ubiquitylation of highly conserved C-terminal lysine residues. The same lysines can also be acetylated by coactivators such as p300 and CBP to promote transactivation of target genes. In vitro studies suggest a model in which p53 is activated by damage-mediated kinases that induce phosphorylation in N-terminal serines to produce a conformational change leading to Mdm2/Mdmx dissociation and p300/CBP recruitment. These factors then acetylate the C terminus to stabilize p53 and enhance its transcriptional activity. However, in vivo, these modifications appear to be dispensable during embryogenesis, and do not significantly affect p53 activity in mouse embryonic fibroblasts (MEFs). This does not address tissue and condition-specific effects of these residues in vivo, nor whether such effects would be manifested through regulation of a subset of p53 target genes. Evidence of the importance of lysine modification for p53 in vivo functions comes from recent studies showing that mutation of K120 impairs puma activation, and that a K120R/K164R double mutant reduces induction of puma and p21, but not mdm2. Thus, lysine modifications in the DNA-binding region appear to play a key role in differential gene regulation (Wang, 2011).

These observations led to an examination of the impact of modification of the conserved seven C-terminal lysine residues in a knock-in model in which they were replaced with arginine (7KR) to prevent ubiquitylation or acetylation. This study showa that the p537KR mutation plays a critical role in regulating p53 transcriptional activity on a subset of genes in the hematopoietic system. This, in turn, affects maintenance of HSCs during unchallenged homeostatic growth, in the setting of BM transplantation, and after radiation exposure. Notably, the p537KR mutation causes extraordinary radiosensitivity, which is partially rescued by loss of a single p21 allele, implicating C-terminal modification in controlling p21 transcription in the HSC and progenitor pools that play critical roles in radiation responses (Wang, 2011).

Structures of three distinct activator-TFIID complexes

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) was determined and their structures were compared as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, these results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation (Liu, 2009).

Three D density difference maps generated from reconstructions of the three independent activator/TFIID assemblies (i.e., p53-IID, Sp1-IID, and c-Jun-IID) and free holo-TFIID have served as a method to map the most likely contact sites of these activators within the native TBP-TAF complex. Remarkably, each activator contacts TFIID via select TAF interfaces within TFIID. The unique and localized arrangements of these three activators contacting different surfaces of TFIID could be indicative of the wide diversity of potential activator contact points within TFIID that would be dependent on both the specificity of activation domains as well as core promoter DNA sequences appended to target gene promoters. It is also possible, however, that these distinct activator-TFIID contacts can form a common scaffold when TFIID binds to the core promoter DNA (Liu, 2009).

It is well established that activators including p53, Sp1, and c-Jun frequently work synergistically with each other or other activators to potentiate selective gene expression programs in response to a variety of stimuli in vivo. Therefore, combinatorial mechanisms of promoter activation might favor distinct nonoverlapping activator-binding sites within TFIID, which can be achieved by specific interactions between selective TAF subunits and activators. Indeed, it was established that TAF1 and TAF4 serve as coactivators for Sp1, while TAF1, TAF6, and TAF 9 mediate p53-dependent transactivation and TAF1 and TAF7 subunits are thought to be coactivators for c-Jun. Since activators make sequence-specific contacts with the DNA template at various positions upstream of the core promoter, it is also plausible that activators bound to unique surfaces of TFIID can influence specific structures of a promoter as the DNA traverses along TFIID resulting in distinct activator/promoter DNA structures (Liu, 2009).

Activator mapping results also complement and structurally extend the functional relevance of previous biochemical and immunomapping studies of TFIID. For example, label transfer studies show that the N-terminal activation domain of p53 contacts TAF6, confirming previous biochemical evidence showing that amino acids 1-42 of p53 contact TAF6/9. In support of this observation, the p53-IID 3D structure indicates that p53 contacts TFIID at lobes A and C where TAF6/9 are located as determined by EM immunomapping. In addition, previous studies have shown that both TBP and TAF1 can directly contact p53 in the absence of additional TFIID subunits. Interestingly, body-labeled p53 cross-linked to TAF1, TAF5, and weakly to TBP, thus extending the immunomapping studies that determined the locations of TBP and the N terminus of TAF1 at lobe C. Thus, EM activator mapping studies show a significant interface between p53 and specific TAFs located at lobes A and C of TFIID. Likewise, Sp1 label transfer results confirmed previous biochemical data showing a direct interaction between TAF4 and the N-terminal glutamine-rich domains of Sp1. In addition to TAF4, TAF6 was identified as weakly cross-linked to Sp1, suggesting that TAF6 may also be in the vicinity but perhaps more distal to the N terminus of Sp1. The largest TFIID subunit, TAF1, was cross-linked when body-labeled Sp1 was used. This result was not entirely unexpected, since previous studies found that TAF1 is required for Sp1-dependent transactivation, possibly through a direct interaction between TAF1 and Sp1 (Liu, 2009).

In comparison with p53 and Sp1, body-labeled c-Jun was shown to contact TAF1 and TAF6 in label transfer studies with no subunits contacting the N-terminal activation domain of c-Jun. This N-terminal activation domain of c-Jun may be structurally flexible or predominantly unstructured and is apparently positioned away from TFIID contacts. Indeed, successful structural studies of c-Jun thus far have been limited to the C-terminal leucine zipper DNA-binding region when bound to DNA. Previous biochemical assays have shown that the C-terminal basic leucine zipper DNA-binding region also contacts the N terminus of TAF1 (Liu, 2009).

It is worth noting that the extra density representing c-Jun and the other activator polypeptides in EM studies may not reflect the full-expected size of the activators. This is due to the presence of large unstructured regions in these proteins that are averaged out during structural analysis. As activators contain multiple molten globular domains that likely interact with different partners, one would expect a high degree of structural disorder in the domains that are not in direct contact with TFIID. Thus, the extra density associated with each activator determined from the single-particle reconstructions likely only represents minimally the most stably associated portion of activators bound to TFIID. This common situation would invariably lead to underrepresenting the actual size of the activator in a manner not unlike crystal structures of domains with flexible loops that become 'invisible' in the crystal structure (Liu, 2009).

Based on EM immunomapping, there are two copies of TAF6 within TFIID, wherein one copy resides in lobe A and another in lobe B. Collectively, the current studies suggest that two distinct activators (p53 and c-Jun) strongly contact the two different TAF6 subunits that are each located in different lobes of TFIID. It is unknown how p53 or c-Jun discriminates between TAF6 on lobe A versus B when binding to TFIID. In the future, it will be interesting to investigate if these two activators can bind to a single TFIID molecule simultaneously and decipher 3D structures of TFIID assemblies bound to select endogenous promoter DNA sequences in the presence and absence of distinct activators that are engaged in synergistic transcriptional activation (Liu, 2009).

It is of note that unlike the radical, diverse, and global structural changes observed with CRSP/Mediator complexes upon activator binding, TFIID largely retains its overall architecture when bound by three different activators. Interestingly, this study found that two of the activator/IID structures, p53-IID and Sp1-IID assemblies appear to be more constricted around the central cavity with narrower ChB-D and ChA-B channels, while the third structure, c-Jun-IID, remains most similar to free holo-TFIID. In particular, the p53-IID structure more closely resembles the closed conformational state of the previous cryo-TFIID structure. To test if p53-bound TFIID mimics the most closed conformational form of holo-TFIID, 3D reconstructions were performed using either the most closed or 'open' cryo-TFIID structures as an initial reference volume for refinement. Interestingly, it was found that both newly refined 3D structures generated from either the closed or open reference volume are fairly similar, with possibly a partial occupancy of p53 on lobe A. These findings suggest that the overall p53-TFIID structure tends to move toward the closed conformation with moderate movement at the outer tips of lobes A and B, even though p53-IID is predominantly observed in an intermediate average conformational form between the most closed and open forms. Perhaps factors contacting lobe A or C can induce certain coordinated movements within lobes that lead to a closed conformation of TFIID (Liu, 2009).

Although TFIID largely retains its prototypic global architecture upon activator binding, several common localized structural changes induced upon activator binding were observed in the 3D reconstruction. For example, a prominent and consistent induced extra density protrusion located in lobe D was observed when each of the three different activators binds TFIID. Given that all these activators are represented by distinct densities with unique sizes and shapes within the bound TFIID structure, and the fact that it has been demonstrated that they each can target different subunits within TFIID by a number of independent biochemical assays, it seems reasonable to assign 'unique and significant' extra densities located at distinct sites as representing the different bound activators. In contrast, the common similarly sized extra density seen at lobe D of each activator-IID structure most likely represents a conserved conformational change induced by these three different activators. Interestingly, this protrusion in lobe D resides distal to each of the activator-binding sites, suggesting that these three activators may potentially induce a long-range internal conformational change within TFIID. It would be intriguing to identify which TAF subunits are located at the tip of lobe D and eventually determine the function, if any, of this extended lobe in activator-induced transcription initiation. However, despite the potential significance of these structural changes induced by activators, it is premature to speculate regarding their functional importance (Liu, 2009).

p21 transcription is regulated by differential localization of histone H2A.Z directed by p53

In yeast cells, H2A.Z regulates transcription and is globally associated within a few nucleosomes of the initiator regions of numerous promoters. H2A.Z is deposited at these loci by an ATP-dependent complex, Swr1.com. H2A.Z suppresses the p53 --> p21 transcription and senescence responses. Upon DNA damage, H2A.Z is first evicted from the p21 promoter, followed by the recruitment of the Tip60 histone acetyltransferase to activate p21 transcription. p400, a human Swr1 homolog, is required for the localization of H2A.Z, and largely colocalizes with H2A.Z at multiple promoters investigated. Notably, the presence of sequence-specific transcription factors, such as p53 and Myc, provides positioning cues that direct the location of H2A.Z-containing nucleosomes within these promoters. Collectively, this study strongly suggests that certain sequence-specific transcription factors regulate transcription, in part, by preferentially positioning histone variant H2A.Z within chromatin. This H2A.Z-centered process is part of an epigenetic process for modulating gene expression (Gévry, 2007).

Eukaryotic DNA is condensed many fold (e.g., 10,000) into chromatin, the basic unit of which contains 146 base pairs (bp) of DNA and an octamer of histone proteins (H2A, H2B, H3, and H4). Due to the high level of compaction, chromatin typically represses certain cellular DNA transactions, including transcription. For successful transcription, it is argued that nucleosomes need to be remodeled or evicted from promoter regions for the transcriptional machinery to be efficiently recruited to a target gene (Gévry, 2007).

The incorporation of histone variants into specific nucleosomes within a promoter region constitutes a mechanism by which promoter region chromatin can become more permissive to transcription initiation and elongation following receipt of a proper physiological cue. One such histone variant is H2A.Z. In Saccharomyces cerevisiae, it can elicit positive effects on gene expression. In addition, H2A.Z regulates genes that are proximal to telomeres and acts as a 'buffer' to antagonize the spread of heterochromatin into euchromatic regions (Meneghini, 2003). Furthermore, recent reports (Guillemette, 2005; Li, 2005; Raisner, 2005; Zhang, 2005) have shown that H2A.Z is preferentially localized within a few nucleosomes of the initiator regions of multiple promoters in the yeast genome. Interestingly, these H2A.Z-rich loci are largely devoid of transcriptional activity, which suggests that the variant histone prepares genes for activation (Guillemette, 2005) and/or operates as a transcriptional repressor. Finally, yeast H2A.Z has been shown to regulate nucleosome positioning, which provides mechanistic insight into how its presence can alter promoter transcriptional state (Gévry, 2007).

An ATP-dependent chromatin remodeling complex that specifically loads H2A.Z onto chromatin and exchanges it with H2A exists in yeast (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). This complex, in which the catalytic subunit is Swr1, also shares essential subunits with the NuA4 histone acetyltransferase complex (Krogan, 2003; Kobor, 2004). In addition to their importance in gene regulation, the Swr1 complex, H2A.Z, and NuA4 are all involved in the regulation of yeast chromosome stability (Krogan, 2004). This is noteworthy because, in mammalian cells, depletion of H2A.Z causes major nuclear and chromosomal abnormalities (Rangasamy, 2004) as witnessed by a high incidence of lagging chromosomes and chromatin bridges (Gevry, 2007).

There are two homologs of Swr1 in human cells: p400/Domino (referred to as p400), and SRCAP. There are also three uncharacterized p400-type SWI2-SNF2 molecules, including hIno80. Members of this family of SWI2/SNF2 chromatin remodeling enzymes each contain a spacer region inserted into the SWI2/SNF2 homology region (Gevry, 2007).

p400 was originally isolated as an E1A-associated protein, and it was also shown to interact with p53, Myc, and SV40 large T antigen. It is also required for E1A to induce p53-mediated apoptosis. SRCAP has been isolated as a CREB-binding protein. While one report shows that both p400 and SRCAP constitute part of the same complex, a recent study shows that SRCAP and p400 exist in distinct complexes with H2A.Z (Jin, 2005; Ruhl, 2006). Recently an SRCAP-containing complex was purified, and it was shown to have the ability to exchange H2A-H2B for H2A.Z-H2B in reconstituted mononucleosomes (Ruhl, 2006). It remains to be determined whether mammalian homolog(s) of Swr1, such as p400 and SRCAP, also catalyze H2A.Z deposition in vivo (Gevry, 2007).

Depletion of p400 elevates p21 synthesis to initiate premature senescence in primary human fibroblasts (Chan, 2005). Senescence has been observed in tissue culture cells as a stable form of cell growth arrest provoked by diverse stresses. Recently, oncogene-induced senescence was shown to occur in various precancerous lesions both in humans and mice, further suggesting that senescence acts as a defense mechanism against malignant cell development. Importantly, the action of p400 at p21 depends on the function of p53, a key regulator of p21 transcription (Gevry, 2007).

Given the possibility of a link between p400 and H2A.Z, it was asked whether H2A.Z is also an important regulator of p21 expression. The results of this effort show that H2A.Z depletion induces p21 expression in a p53-dependent fashion, as well as the premature senescence of primary diploid fibroblasts. Similar to senescence induced by p400 depletion, inactivating p53 or p21 blocked the emergence of certain senescent phenotypes following H2A.Z depletion. In a normal setting, H2A.Z is highly enriched at discrete p53-binding sites that lie within the p21 promoter. This distinctive localization pattern depends on the presence of p53, and was detected at other p53 target gene promoters as well. The presence of p400 is required to localize H2A.Z at those loci, and purified recombinant p400 from insect cells can carry out in vitro exchange of H2A.Z-H2B dimers into chromatin. H2A.Z and p400 localization at the p53-binding sites in p21 is severely diminished following p21 induction, and this process is not dependent on active p21 transcription per se. After H2A.Z and p400 eviction from the p53-binding sites in p21, it was observed that the Tip60 histone acetyltransferase isrecruited to the distal p53-binding site in the promoter to positively regulate p21 expression. Finally, overexpression of Myc, a known suppressor of p21 synthesis, significantly increases H2A.Z localization at the Myc-binding site in the TATA initiator region of the p21 promoter. This observation is consistent with the view that Myc represses p21 expression by preferentially recruiting H2A.Z-containing nucleosome(s) to this element (Gevry, 2007).

SKIP counteracts p53-mediated apoptosis via selective regulation of p21Cip1 mRNA splicing

The Ski-interacting protein SKIP/SNW1 functions as both a splicing factor and a transcriptional coactivator for induced genes. Transcription elongation factors such as SKIP have been shown to be dispensable in cells subjected to DNA damage stress. However, this study reporta that SKIP is critical for both basal and stress-induced expression of the cell cycle arrest factor p21Cip1. RNAi chromatin immunoprecipitation (RNAi-ChIP) and RNA immunoprecipitation (RNA-IP) experiments indicate that SKIP is not required for transcription elongation of the gene under stress, but instead is critical for splicing and p21Cip1 protein expression. SKIP interacts with the 3' splice site recognition factor U2AF65 and recruits it to the p21Cip1 gene and mRNA. Remarkably, SKIP is not required for splicing or loading of U2AF65 at other investigated p53-induced targets, including the proapoptotic gene PUMA. Consequently, depletion of SKIP induces a rapid down-regulation of p21Cip1 and predisposes cells to undergo p53-mediated apoptosis, which is greatly enhanced by chemotherapeutic DNA damage agents. ChIP experiments reveal that SKIP is recruited to the p21Cip1, and not PUMA, gene promoters, indicating that p21Cip1 gene-specific splicing is predominantly cotranscriptional. The SKIP-associated factors DHX8 and Prp19 are also selectively required for p21Cip1 expression under stress. Together, these studies define a new step that controls cancer cell apoptosis (Chen, 2011).

Degradation of p53 family members

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. mdm2 gene is shown here to be regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2 (Ries, 2000).

Thus Mdm2 expression is modulated by the Ras/Raf/MEK/MAP kinase pathway through activation of Ets and AP-1 sites in the P2 promoter, upstream from the p53 responsive element and independent of its activity. Furthermore, Mdm2 induced by the Ras/Raf/MEK/MAP kinase pathway is functionally active and leads to degradation of p53. This signaling pathway is intact in tumor cells expressing activated Ras because Mdm2 protein levels decrease dramatically after inhibiting MEK activity in these cells. Importantly, the effects of induced Mdm2 on p53 are regulated by p19ARF. Ras therefore acts on p53 through two competing pathways. Activation of the Ras/Raf/MEK/MAP kinase cascade results in elevated levels of Mdm2 protein. However, in normal cells, this pathway also induces the expression of p19ARF, which inhibits Mdm2 activity. Thus, in normal cells, levels of p53 are determined by a balance between opposing effects of the Ras/Raf/MEK/MAP kinase pathway. In mouse embryonic fibroblasts (MEFs), these opposing effects are equivalent, and Raf is ineffective at inducing p53, despite its effects in p19ARF. In different cell types, or even in MEFs growing under slightly different conditions, the balance of these opposing pathways is likely to be different. For example, in IMR90 human diploid fibroblasts, activated MEK leads to accumulation of p53, presumably because p14ARF exceeds Mdm2 induction (Ries, 2000 and references therein).

The stability of p53 tumor suppressor is regulated by Mdm2 via the ubiquitination and proteasome-mediated proteolysis pathway. The c-Abl and PTEN tumor suppressors are known to stabilize p53 by blocking the Mdm2-mediated p53 degradation. This study investigated the correlation between p53 and merlin, a neurofibromatosis 2 (NF2)-related tumor suppressor, in association with the Mdm2 function. The results showed that merlin increases p53 stability by inhibiting the Mdm2-mediated degradation of p53, which accompanies the increase in the p53-dependent transcriptional activity. The stabilization of p53 by merlin appears to be accomplished through Mdm2 degradation, and the N-terminal region of merlin is responsible for this novel activity. This study also showed that overexpression of merlin induces apoptosis of cells depending preferentially on p53 in response to the serum starvation or a chemotherapeutic agent. These results suggest that merlin may be a positive regulator of p53 in terms of tumor suppressor activity, and provide the promising therapeutic means for treating tumors with non-functional merlin or Mdm2 overexpression (Kim, 2004).

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. This study shows that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), Mdm2-dependent NEDD8 modification of p53 was demonstrated to inhibits p53 transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins (Xirodimas, 2004).

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. This ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1), a flavoenzyme that catalyzes two-electron reductive metabolism and detoxification of quinones and their derivatives leading to protection of cells against redox cycling and oxidative stress. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. This pathway plays a role in p53 accumulation in response to ionizing radiation. These findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level (Asher, 2005).

The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, the link between TAF1 and p53 was investigated. TAF1 was found to induce G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAi-mediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduces Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation is reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage (Li, 2004).

NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Curcumin, a natural phenolic compound found in the spice turmeric, induces ubiquitin-independent degradation of WT p53 and inhibits p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibits the activity of recombinant NQO1 in vitro, inhibits the activity of endogenous cellular NQO1 in vivo, and dissociates NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociates the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore does not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induces degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway (Tsvetkov, 2005).

A p53 family and checkpoint pathways

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. How rereplication is prevented in normal mammalian cells has been investigated; these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 (Drosophila homolog: Double parked) and Cdc6 (Drosophila homolog: Origin recognition complex subunit 1) along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability (Vaziri, 2003).

To test whether geminin inhibits rereplication induced by Cdt1, geminin was overexpressed along with Cdt1 and Cdc6. Overexpression of geminin partially inhibits the rereplication mediated by Cdt1+Cdc6. Overexpression of Cdt1 (by itself) leads to a paradoxical increase in geminin levels in the rereplicating cells. In order to confirm that there was free Cdt1 (uncomplexed with geminin) in the cell lines, all the geminin was precleared from these cell extracts before immunoblotting for residual Cdt1 in the supernatant. The results show that despite the induction of geminin, not enough of the protein is produced to associate with and inhibit all the overexpressed Cdt1. The increase in geminin was attributed to a 10-fold induction of geminin mRNA seen upon overexpression of Cdt1. The mechanism of this induction is currently unclear but suggests the existence of a feedback loop between Cdt1 and its antagonist geminin (Vaziri, 2003).

The activation of the DNA damage checkpoint pathway and the tumor suppressor protein p53 provides a pathway by which mammalian cells prevent rereplication. Rereplication appears to lead to DNA damage. The data suggest that activation of ATM/ATR kinases caused by overexpression of Cdt1 and Cdc6 leads to direct phosphorylation of p53 and indirect phosphorylation of p53 through Chk2 kinase. Phosphorylation of p53 stabilizes the protein and leads to increased transcription and expression of p21. The latter is a potent inhibitor of cyclin A-cdk2 kinase and could therefore prevent any rereplication. Consistent with this hypothesis, overexpression of wild-type p53 or of p21 effectively inhibits rereplication in the p53-negative H1299 cells, while inactivation of p53 in A549 cells by overexpressing Mdm2 prevents p21 induction and permits rereplication. Because of the concurrent induction of proapoptotic genes like PIG3, p53 could also promote apoptosis of cells that have already undergone significant rereplication. Since mutations in p53 have been widely documented to promote genomic instability and gene amplification, these results provide a partial explanation of this observation by proposing a mechanism by which p53 stabilizes the genome. Genes other than p53, however, also prevent gene amplification, so it is unlikely that p53 is the only barrier to rereplication upon overexpression of Cdt1 and Cdc6 in all cell lines (Vaziri, 2003).

The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. In several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance (Urist, 2004).

Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, this study generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. Single-cell microscopic analyses were performed to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, knockouts were generated across cell lines of diverse origins. p53 (see Drosophila p53) was shown to modulate the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression (McKinley, 2017).

p53 and inherited predisposition to cancer

Germ-line mutations in the p53 gene predispose individuals to Li-Fraumeni syndrome (LFS). The cell cycle checkpoint kinases CHK1 and CHK2 act upstream of p53 in DNA damage responses, and rare germ-line mutations in CHK2 have been reported in LFS families. CHK1, CHK2, and p53 genes were analyzed for mutations in 44 Finnish families with LFS, Li-Fraumeni-like syndrome, or families phenotypically suggestive of LFS with conformation-sensitive gel electrophoresis. Five different disease-causing mutations were observed in 7 families: 4 in the p53 gene and 1 in the CHK2 gene (2 of 44 families). Interestingly, the other CHK2-mutation carrier also has a mutation in the MSH6 gene. The cancer phenotype in the CHK2-families is not characteristic of LFS, and may indicate variable phenotypic expression in the rare families with CHK2 mutations. No mutations in the CHK1 gene were identified (Vahteristo, 2001).

Inactivation of p53 and Pten promotes invasive bladder cancer

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. This study shows that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. These integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease (Puzio-Kuter, 2009).

The combined status of ATM and p53 link tumor development with therapeutic response

While the contribution of specific tumor suppressor networks to cancer development has been the subject of considerable recent study, it remains unclear how alterations in these networks are integrated to influence the response of tumors to anti-cancer treatments. This study shows that mechanisms commonly used by tumors to bypass early neoplastic checkpoints ultimately determine chemotherapeutic response and generate tumor-specific vulnerabilities that can be exploited with targeted therapies. Specifically, evaluation of the combined status of ATM and p53, two commonly mutated tumor suppressor genes, can help to predict the clinical response to genotoxic chemotherapies. This study shows that in p53-deficient settings, suppression of ATM dramatically sensitizes tumors to DNA-damaging chemotherapy, whereas, conversely, in the presence of functional p53, suppression of ATM or its downstream target Chk2 actually protects tumors from being killed by genotoxic agents. Furthermore, ATM-deficient cancer cells display strong nononcogene addiction to DNA-PKcs for survival after DNA damage, such that suppression of DNA-PKcs in vivo resensitizes inherently chemoresistant ATM-deficient tumors to genotoxic chemotherapy. Thus, the specific set of alterations induced during tumor development plays a dominant role in determining both the tumor response to conventional chemotherapy and specific susceptibilities to targeted therapies in a given malignancy (Jiang, 2009).

Mutant p53 Drives Pancreatic Cancer Metastasis through Cell-Autonomous PDGF Receptor beta Signaling

Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. This study shows that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors (Weissmueller, 2014).

p53 suppresses the self-renewal of adult neural stem cells

There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. Ghe prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal (Meletis, 2005; full text of article).

Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of an ongoing program to distinguish the biological functions of these isoforms, mice were generated that were selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, a completely new role was discovered for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. It was found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy (Wilhelm, 2010).

p53, p73 and apoptosis

p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, homologous recombination and LoxP/Cre-mediated deletion were used to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53Gln25Ser26 is stable but does not accumulate after DNA damage; the expression of p21/Waf1 and PERP is not induced, and p53-dependent repression of MAP4 expression is abolished. Therefore, p53Gln25Ser26 is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53Gln25Ser26 is phosphorylated at Ser18 but is not acetylated at C-terminal sites, and its DNA binding activity does not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53Gln25Ser26 mouse thymocytes and ES cells, like p53-/- cells, did not undergo DNA damage-induced apoptosis. It is concluded that the transcriptional activities of p53 are required for p53-dependent apoptosis (Chao, 2000).

Analyses employing constructed mutants have suggested that transcriptional activation by p53 is critical for the induction of apoptosis. Furthermore, several p53 target genes have been identified that are known to play a role in apoptosis. Bax, a pro-apoptotic member of the Bcl-2 family, has p53 binding sites in its promoter; thus, direct activation by p53 could provide a link with the apoptotic machinery. Nevertheless, the requirement for Bax in p53-dependent cell death is only partial, and Bax is fully dispensable for the p53-dependent cell death of thymocytes in response to gamma-irradiation. These results suggest that Bax induction may be relevant to p53-induced apoptosis only in certain cellular contexts. Other potential apoptosis target genes such as KILLER/DR5 and other PIGs (p53 inducible genes) have been described, but it remains to be seen whether these play critical roles in p53-dependent apoptosis. Nevertheless, a recently identified p53 target gene, PERP, which is specifically induced upon DNA damage during apoptosis, provides a potentially compelling demonstration of a candidate effector in the p53 transcriptionally dependent apoptotic pathway. The transcriptional activation of PERP by p53 appears crucial for PERP’s ability to induce cell death, and PERP apparently functions only to induce apoptosis and not cell cycle arrest. PERP is a new member of the PMP-22/gas3 family of tetraspan transmembrane proteins that have been implicated in cell growth regulation and apoptosis. A second gene, Pw1yPeg3, is also specifically induced during apoptosis. Interestingly, Pw1yPeg3 cooperates with Siah1a, another p53-inducible gene, to induce apoptosis. Furthermore, the induction of Pw1yPeg3 during apoptosis requires activation of both p53 and c-myc expression. These data strongly suggest that Pw1yPeg3, like PERP, may be a critical downstream effector of the p53-mediated cell death pathway (Chao, 2000 and references therein).

Although a growing number of p53-induced genes are implicated in the DNA damage-induced apoptotic pathway, it remains unclear whether any of them is directly involved in p53-dependent apoptosis and whether p53 also induces apoptosis through mechanisms that are independent of transcriptional activation. One formal hypothesis is that p53 may repress the transcription of certain genes required for cell survival. In support of this notion, it was shown that p53-mediated repression of MAP4 expression might be involved in p53-dependent apoptosis. Importantly changing Leu25 and Trp26 of murine p53 to Gln and Ser, respectively, simultaneously disrupts the transcriptional activation and repression activity of p53 in vivo as well as its apoptotic function. Therefore, in murine ES cells and thymocytes, the induction of apoptosis in response to DNA damage requires the p53-dependent transcriptional activation and/or repression of certain gene products. However, the relative contributions to apoptosis of p53 transcriptional activation activity and repression activity remain to be determined. In addition, it remains possible that in certain cells or conditions, apoptosis can be induced through the accumulation of p53 by mechanisms that do not require transcriptional activity (Chao, 2000).

Studies from several laboratories have begun to elucidate the steps leading to p53 activation. It is now clear that several sites in p53, including Ser15, become phosphorylated in response to DNA damage-inducing agents. In vitro, Ser15 can be phosphorylated by DNA-PK and the related protein kinase ATM, and in vivo, efficient phosphorylation of Ser15 after cells have been exposed to IR requires a functional ATM gene. These results, coupled with the observation that p53 accumulation is delayed in ATM-deficient cells after exposure to IR, suggest that phosphorylation of Ser15 may be important for stabilizing p53. Phosphorylation of the N-terminal serines 15, 33 and 37 has also been proposed to permit subsequent modification of the C-terminal lysine residues through the recruitment of p300/CBP/PCAF. The finding that p53Gln25Ser26 is not acetylated in response to UV light is consistent with the notion that the N-terminus of p53 is involved in recruitment of the histone acetylases and that acetylation of p53 at the C-terminus activates the specific DNA binding activity of p53. It is suggested that interaction of p53 with components of the transcriptional apparatus may be a further requirement for C-terminal acetylation. Phosphorylation of Ser15 alone, in response to DNA damage, which still occurs on the transcriptionally inactivated mutant p53, is not sufficient to promote acetylation of the C-terminal residues (Chao, 2000).

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point, independent of the 'tumor suppressor' protein p53. Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. It is concluded that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli (Lissy, 2000).

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumor-suppressor protein p14ARF, which neutralizes HDM2 (human homolog of MDM2) and thereby stabilizes the p53 protein. E2F-1 induces the transcription of the p53 homolog p73. Disruption of p73 function inhibits E2F-1-induced apoptosis in p53-defective tumor cells and in p53-/- mouse embryo fibroblasts. It is conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53 (Irwin, 2000).

The tumor-suppressor gene p53 is frequently mutated in human cancers and is important in the cellular response to DNA damage. Although the p53 family members p63 and p73 are structurally related to p53, they have not been directly linked to tumor suppression, although they have been implicated in apoptosis. Given the similarity between this family of genes and the ability of p63 and p73 to transactivate p53 target genes, their role in DNA damage-induced apoptosis has been explored. Mouse embryo fibroblasts deficient for one or a combination of p53 family members were sensitized to undergo apoptosis through the expression of the adenovirus E1A oncogene. Using the E1A system facilitates the ability to perform biochemical analyses. The functions of p63 and p73 were also examined using an in vivo system in which apoptosis has been shown to be dependent on p53. Using both systems, it has been shown that the combined loss of p63 and p73 results in the failure of cells containing functional p53 to undergo apoptosis in response to DNA damage (Flores, 2002).

An affinity purification method has been used to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage (Basu, 2003).

YAP is a 65 kDa protein (sometimes termed YAP65 or YAP1) that was originally identified due to its interaction with the Src family tyrosine kinase Yes. YAP contains either one or two WW domains depending on alternative splicing and also a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. YAP has been reported to interact with p53 binding protein-2, an important regulator of the apoptotic activity of p53. Through its carboxyl terminus, YAP binds to the PDZ-containing protein EBP50, a submembranous scaffolding protein. YAP is a transcriptional coactivator that binds and activates Runx transcription factors and the four TEAD/TEF transcription factors. YAP is homologous to TAZ (45% identity), a transcriptional coactivator that is regulated by interaction with 14-3-3 and PDZ domain-containing proteins. YAP also interacts with the p53 family member p73, resulting in an enhancement of p73's transcriptional activity. YAP phosphorylation by Akt suppresses its ability to promote p73-mediated transcription of proapoptotic genes in response to DNA damaging agents and the resulting cell death. This extends the range of mechanisms whereby Akt can promote cellular survival in the face of apoptotic stimuli (Basu, 2003).

p53 induces apoptosis by target gene regulation and transcription-independent signaling. A fraction of induced p53 translocates to the mitochondria of apoptosing tumor cells. Targeting p53 to mitochondria is sufficient to launch apoptosis. Evidence has been found that p53 translocation to the mitochondria occurs in vivo in irradiated thymocytes. Further, the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming complexes with the protective BclXL and Bcl2 proteins, resulting in cytochrome c release. p53 binds to BclXL via its DNA binding domain. The significance of mitochondrial p53 has been probed; tumor-derived transactivation-deficient mutants of p53 concomitantly lose the ability to interact with BclXL and promote cytochrome c release. This opens the possibility that mutations might represent 'double-hits' by abrogating the transcriptional and mitochondrial apoptotic activity of p53 (Mihara, 2003).

The transcription factor c-Jun mediates several cellular processes, including proliferation and survival, and is upregulated in many carcinomas. Liver-specific inactivation of c-Jun at different stages of tumor development was used to study its role in chemically induced hepatocellular carcinomas (HCCs) in mice. The requirement for c-jun is restricted to early stages of tumor development, and the number and size of hepatic tumors is dramatically reduced when c-jun is inactivated after the tumor has initiated. The impaired tumor development correlates with increased levels of p53 and BH3-only protein Noxa, which is a known target gene of p53; this response results in the induction of apoptosis without affecting cell proliferation. Primary hepatocytes lacking c-Jun shows increased sensitivity to TNF-alpha-induced apoptosis -- this sensitivity is abrogated in the absence of p53. These data indicate that c-Jun prevents apoptosis by antagonizing p53 activity, illustrating a mechanism that might contribute to the early stages of human HCC development (Efer, 2003).

The tumor suppressor p53 exerts its versatile function to maintain the genomic integrity of a cell, and the life of cancerous cells with DNA damage is often terminated by induction of apoptosis. The role of Noxa, one of the transcriptional targets of p53 that encodes a proapoptotic protein of the Bcl-2 family, was studied by the gene-targeting approach. Mouse embryonic fibroblasts deficient in Noxa [Noxa-/- mouse embryonic fibroblasts (MEFs)] show notable resistance to oncogene-dependent apoptosis in response to DNA damage, which is further increased by introducing an additional null zygosity for Bax. These MEFs also show increased sensitivity to oncogene-induced cell transformation in vitro. Furthermore, Noxa is also involved in the oncogene-independent gradual apoptosis induced by severe genotoxic stresses, under which p53 activates both survival and apoptotic pathways through induction of p21WAF1/Cip1 and Noxa, respectively. Noxa-/- mice show resistance to X-ray irradiation-induced gastrointestinal death, accompanied with impaired apoptosis of the epithelial cells of small intestinal crypts, indicating the contribution of Noxa to the p53 response in vivo (Shibue, 2003).

A Drosophila p53 protein has been identified that mediates apoptosis via a novel pathway involving the activation of the Reaper gene and subsequent inhibition of the inhibitors of apoptosis (IAPs). The present study found that CIAP1, a major mammalian homolog of Drosophila IAPs, is irreversibly inhibited (cleaved) during p53-dependent apoptosis and this cleavage is mediated by a serine protease. Serine protease inhibitors that block CIAP1 cleavage inhibit p53-dependent apoptosis. Furthermore, activation of the p53 protein increases the transcription of the HTRA2 gene, which encodes a serine protease that interacts with CIAP1 and potentiates apoptosis. These results demonstrate that the mammalian p53 protein may activate apoptosis through a novel pathway functionally similar to that in Drosophila, which involves HTRA2 and subsequent inhibition of CIAP1 by cleavage (Jin, 2003).

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. This study analyzed the consequences of inactivating E2f7 and E2f8 in mice, and showed that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis (Li, 2008).

The genetic mechanisms that regulate neurodegeneration are only poorly understood. This study shows that loss of one allele of the p53 family member, p73, makes mice susceptible to neurodegeneration as a consequence of aging or Alzheimer's disease (AD). Behavioral analyses demonstrated that old, but not young, p73+/- mice displayed reduced motor and cognitive function, CNS atrophy, and neuronal degeneration. Unexpectedly, brains of aged p73+/- mice demonstrated dramatic accumulations of phospho-tau (P-tau)-positive filaments. Moreover, when crossed to a mouse model of AD expressing a mutant amyloid precursor protein, brains of these mice showed neuronal degeneration and early and robust formation of tangle-like structures containing P-tau. The increase in P-tau was likely mediated by JNK; in p73+/- neurons, the activity of the p73 target JNK was enhanced, and JNK regulated P-tau levels. Thus, p73 is essential for preventing neurodegeneration, and haploinsufficiency for p73 may be a susceptibility factor for AD and other neurodegenerative disorders (Wetzel, 2008).

p53 family members and development

Epidermal stem cells play a critical role in producing the multilayered vertebrate skin. Products of the p63 gene not only mark the epidermal stem cells, but also are absolutely required for the formation of mammalian epidermis. Early zebrafish embryos express a dominant-negative form of p63 (DeltaNp63), which accumulates in the nucleus just as epidermal growth begins. Using antisense morpholino oligonucleotides, it has been shown that DeltaNp63 is needed for epidermal growth and limb development and is specifically required for the proliferation of epidermal cells by inhibiting p53 activity. While the structure of fish epidermis is very different from that of higher vertebrates, this study shows that DeltaNp63 has an essential and ancient role in the development of skin (Lee, 2002).

Bone morphogenetic proteins (Bmps) promote ventral specification in both the mesoderm and the ectoderm of vertebrate embryos. Zebrafish DeltaNp63, encoding an isoform of the p53-related protein p63, is identified as an ectoderm-specific direct transcriptional target of Bmp signaling. DeltaNp63 itself acts as a transcriptional repressor required for ventral specification in the ectoderm of gastrulating embryos. Loss of DeltaNp63 function leads to reduced nonneural ectoderm followed by defects in epidermal development during skin and fin bud formation. In contrast, forced DeltaNp63 expression blocks neural development and promotes nonneural development, even in the absence of Bmp signaling. Together, DeltaNp63 fulfills the criteria to be the neural repressor postulated by the 'neural default model' (Bakkers, 2002).

p63, initially isolated from mammals and also known as p51 or KET, is a homolog of the tumor suppressor and transcription factor p53. The p63 gene is transcribed from two different promoters, which in combination with alternative splicing gives rise to at least six isoforms. Use of the distal promoter generates TAp63 isoforms with the three domains also present in p53: an amino-terminal acidic transactivating domain (TAD), a central DNA binding domain (DBD), and an oligomerization domain (OD). However, use of the second transcriptional start site in intron 3 leads to the generation of N-terminally truncated DeltaNp63 isoforms, which lack the TA domain. Both the TAp63 and the DeltaNp63 transcripts can undergo differential splicing, resulting in proteins with different C-terminal regions. The longest isoforms (alphas) contain a fourth domain, the sterile alpha motif (SAM), also found in numerous other developmental regulators, while ß and gamma forms lack most or all of their SAM domains, respectively. All six proteins act as transcription factors, which can either activate or repress the expression of genes under the control of p53-responsive elements (Bakkers, 2002).

In contrast to p53 mutant mice, mice lacking the p63 gene have severe developmental defects. They lack all squamous epithelia and their derivatives, including skin, hair, whiskers, teeth, as well as mammary, lacrimal, and salivary glands, and they die shortly after birth due to dehydration. In addition, they fail to form limbs, probably as a result of the incapability to maintain the apical ectodermal ridge (AER), a structure required for limb outgrowth. Two human disorders have recently been shown to result from mutations in p63. Patients suffering from the ectodermal dysplasia, ectrodactyly, and cleft plate syndrome (EEC, OMIM 604292) have skin defects and severe limb and craniofacial abnormalities, while the ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC or Hay-Wells, OMIM 106260) is characterized by fused eyelids and severe scalp dermatitis, but normal limb formation. These phenotypes, together with the high expression rates of p63 in proliferating basal cells of the epidermis, have led to the proposal that p63 is involved in the regulation of proliferation and differentiation programs in epithelial tissues. Since differentiated cells can be detected in the epidermis of knockout mice, it has been further proposed that p63 might be required to maintain the regenerative character of epithelial stem cells, rather than for keratinocyte differentation. It has been, however, impossible to specify which of the different p63 isoforms is essential for these processes. Also, little is known about the regulation of p63 expression (Bakkers, 2002).

The isolation of three different DeltaNp63 isoforms from the zebrafish is described. By using antisense morpholino oligonucleotides directed against DeltaNp63, it has been shown that p63 lacking the transactivation domain is required for skin formation and AER maintenance in zebrafish pectoral fin buds. Analyses of earlier stages of morphant embryos and overexpression studies further reveal that DeltaNp63 acts as a transcriptional repressor with a much earlier role during DV patterning of the zebrafish ectoderm. The early expression of DeltaNp63 in the ventral ectoderm is directly activated by Smad4/5-mediated Bmp signaling and is sufficient to block anterior neural specification while promoting early steps of epidermal specification, even in embryos lacking Bmp signaling (Bakkers, 2002).

The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. In the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, the expression was examined of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. In dnp53-expressing C2C12 cells, as well as in p53 minus primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous p53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions (Porrello, 2000).

In physiological proliferating conditions, p53-impaired myoblasts did not show any modification of the Rb gene expression. These observations are consistent with the notion that p53 is not involved in cell cycle control in normal proliferating conditions. In contrast, it is well known that different types of stressing stimuli promote p53 activation. In this type of situation, p53 is known to promote pRb hypophosphorylation and inhibition of DNA synthesis through the transcriptional induction of p21Waf1/Cip1. Indeed, compared with the parental cells, C2-dnp53 cells do not arrest in the G1 phase of the cell cycle in response to doxorubicin-induced DNA damage. Together with the findings obtained in differentiating conditions, these results indicate the presence of two different types of p53-dependent regulation of pRb. One operates through p21Waf1/Cip1 transcription, and the other through direct Rb transcription. These observations are consistent with the emerging idea that p53 regulates transcription of different genes, depending on the type of stimuli that provoked its activation. Interestingly, the existence of a positively regulated p53-binding site on the Rb promoter has been known for several years, but no transcriptional induction of the Rb gene was found in apoptotic or growth-arresting situations, so far. These results reveal the existence of a physiological condition in which p53 directly transactivates the Rb gene (Porrello, 2000)

Pax-3 is a transcription factor that is expressed in the neural tube, neural crest, and dermomyotome. Apoptosis is associated with neural tube defects (NTDs) in Pax-3-deficient Splotch (Sp/Sp) embryos. p53 deficiency, caused by germ-line mutation or by pifithrin-alpha, an inhibitor of p53-dependent apoptosis, rescues not only apoptosis, but also NTDs, in Sp/Sp embryos. Pifithrin-alpha inhibits p53-dependent transcription and apoptosis. The precise mechanisms are not known, but given that nuclear accumulation of p53 is reduced, this suggests that pifithrin-alpha stimulates nuclear export, inhibits nuclear import, or decreases p53 stability. Pax-3 deficiency has no effect on p53 mRNA, but increases p53 protein levels. These results suggest that Pax-3 regulates neural tube closure by inhibiting p53-dependent apoptosis, rather than by inducing neural tube-specific gene expression (Pani, 2002).

Both thyroid hormone (TH) and retinoic acid (RA) induce purified rat oligodendrocyte precursor cells in culture to stop division and differentiate. These responses are blocked by the expression of a dominant-negative form of p53. Moreover, both TH and RA cause a transient, immediate early increase in the same 8 out of 13 mRNAs encoding intracellular cell cycle regulators and gene regulatory proteins, but only if protein synthesis is inhibited. Platelet-derived growth factor (PDGF) withdrawal also induces these cells to differentiate, but the intracellular mechanisms involved are different from those involved in the hormone responses: the changes in cell cycle regulators differ, and the differentiation induced by PDGF withdrawal (or that which occurs spontaneously in the presence of PDGF) is not blocked by the dominant-negative p53. These results suggest that TH and RA activate the same intracellular pathway leading to oligodendrocyte differentiation, and that this pathway depends on a p53 family protein. Differentiation that occurs independently of TH and RA apparently involves a different pathway. It is likely that both pathways operate in vivo (Tokumoto, 2001).

The p53 tumor suppressor belongs to a family of proteins that sense multiple cellular inputs to regulate cell proliferation, apoptosis, and differentiation. Whether and how these functions of p53 intersect with the activity of extracellular growth factors is not understood. Key cellular responses to TGF-ß signals rely on p53 family members. During Xenopus embryonic development, p53 promotes the activation of multiple TGF-ß target genes. Moreover, mesoderm differentiation is inhibited in p53-depleted embryos. In mammalian cells, the full transcriptional activation of the CDK inhibitor p21WAF1 by TGF-ß requires p53. p53-deficient cells display an impaired cytostatic response to TGF-ß signals. Smad and p53 protein complexes converge on separate cis binding elements on a target promoter and synergistically activate TGF-ß induced transcription. p53 can physically interact in vivo with Smad2 in a TGF-ß-dependent fashion. The results unveil a previously unrecognized link between two primary tumor suppressor pathways in vertebrates (Cordenonsi, 2003).

To identify molecules that modulate TGF-β/Activin/Nodal signaling during development, an unbiased functional screen was performed for genes whose expression promotes the differentiation of embryonic cells into endoderm and mesoderm, as this is the hallmark of TGF-β signaling in early vertebrate embryos. A mouse gastrula (embryonic day [E]6.5) cDNA library was generated, constructed in an RNA expression plasmid. Synthetic mRNA was prepared from pools of 100 bacterial colonies and injected into the animal hemisphere of 2-cell Xenopus embryos. At the blastula stage, the ectoderm was explanted and cultivated until siblings reached the gastrula stage. The injected animal caps were then assayed by RT-PCR to identify pools able to activate the expression of Mixer (endoderm) and Xbra (mesoderm). Of five positive pools, two of the active cDNAs isolated after sib selection corresponded to Smad2 and, unexpectedly, three corresponded to p53AS, a natural variant of p53 generated by alternative splicing at the C terminus. p53AS shares with commonly spliced p53 (p53R) the N-terminal transactivation domain, the central DNA binding and oligomerization domains, but lacks the most C-terminal 26 amino acids of p53R (Cordenonsi, 2003).

A wealth of data indicates that the TGF-β and p53 signaling networks operate independently as powerful tumor suppressors in mammalian cells; yet, the cloning of a p53 isoform in a TGF-β screen unveiled the possibility of a previously unrecognized partnership between these two types of molecules. Evidence is provided that p53 family members are critical determinants for key TGF-β gene responses in different cellular and developmental settings. p53 is shown to associates with Smad2 and Smad3 in vivo in a TGF-β-dependent manner, and p53 family members can strongly cooperate with the activated Smad complex. Several TGF-β target genes in mammalian cells and Xenopus embryos are under such joint control of p53 and Smad (Cordenonsi, 2003).

Using a combination of loss-of-function approaches, evidence of the biological importance of such cooperation is provided. In frog cells, specific depletion of p53 leads to diminished responsiveness to Activin signaling and, in the context of the whole embryo, to severe developmental phenotypes recapitulating aspects of Nodal/Derriere deficiencies. In mammalian cells, the biological relevance of the p53/Smad cooperation was investigated in the context of TGF-β growth arrest program. Transient depletion of p53 or its genetic ablation impairs the antiproliferative response to Activin/TGF-β1 signaling. Finally, in p53 null cancer cells that do not respond to TGF-β signaling, reintroduction of p53 activity leads to the rescue of Smad-dependent growth inhibition (Cordenonsi, 2003).

The combinatorial control of gene expression by p53 and Smad establishes a new tier in the regulation of TGF-β gene responses. These data indicate that p53 neither serves as a DNA binding platform for the Smads, nor can it adjust the general magnitude of gene responses to TGF-β. Depletion of p53 leaves the Smad response fully operational on artificial promoters containing only the Activin/TGF-β responsive element and on some endogenous TGF-β targets, such as goosecoid or TIEG. Instead, p53 appears as an independent input that is integrated on specific target promoters to modulate TGF-β induced transcription. Multiple cellular inputs converge on p53 and it is tempting to speculate that specific posttranslational modifications of p53 may further tune its crosstalk with Smads. A model is proposed in which p53 and the activated Smad complex are recruited at distinct cis-regulatory elements on a common target promoter, leading to synergistic activation of transcription. This model is demonstrated for the Mix.2 promoter, a paradigm of TGF-β-induced transcription. A point mutation in the p53 binding element of the Mix.2 promoter causes a reduced Activin responsiveness in human cells and in the frog embryo, suggesting that p53 activity is required on DNA for full TGF-β transactivation. Of note, a correlation is found between other genes that are under joint control of p53 and Smad, and the presence of a functional p53 binding element in their promoters. This is the case for p21WAF1, PAI-1, and MMP2. In contrast no putative p53 elements were identified in the known regulatory sequences of goosecoid or TIEG, two genes not aided by p53 (Cordenonsi, 2003).

The transcription factor p53 has been shown to mediate cellular responses to diverse stresses such as DNA damage. However, the function of p53 in cellular differentiation in response to growth factor stimulations has remained obscure. Evidence suggests that p53 regulates cellular differentiation by modulating signaling of the TGFß family of growth factors during early Xenopus embryogenesis. p53 functionally and physically interacts with the activin and bone morphogenetic protein pathways to directly induce the expression of the homeobox genes Xhox3 and Mix.1/2. Furthermore, functional knockdown of p53 in embryos by an antisense morpholino oligonucleotide reveals that p53 is required for the development of dorsal and ventral mesoderm. These data illustrate a pivotal role of interplay between the p53 and TGFß pathways in cell fate determination during early vertebrate embryogenesis (Takebayashi-Suzuki, 2003).

Development of stratified epithelia, such as the epidermis, requires p63 expression. The p63 gene encodes isoforms that contain (TA) or lack (DeltaN) a transactivation domain. TAp63 isoforms are the first to be expressed during embryogenesis and are required for initiation of epithelial stratification. In addition, TAp63 isoforms inhibit terminal differentiation, suggesting that TAp63 isoforms must be counterbalanced by DeltaNp63 isoforms to allow cells to respond to signals required for maturation of embryonic epidermis. These data demonstrate that p63 plays a dual role: initiating epithelial stratification during development and maintaining proliferative potential of basal keratinocytes in mature epidermis (Koster, 2004).

Oligodendrocytes make myelin in the vertebrate central nervous system. They develop from oligodendrocyte precursor cells (OPCs), most of which divide a limited number of times before they stop and differentiate. OPCs can be purified from the developing rat optic nerve and stimulated to proliferate in serum-free culture by PDGF. They can be induced to differentiate in vitro by either thyroid hormone (TH) or PDGF withdrawal. A dominant-negative form of p53 can inhibit OPC differentiation induced by TH but not by PDGF withdrawal, suggesting that the p53 family of proteins might play a part in TH-induced differentiation. Since the dominant-negative p53 used inhibited all three known p53 family members (p53, p63 and p73) it was uncertain which family members are important for this process. Evidence is provided that both p53 and p73, but not p63, are involved in TH-induced OPC differentiation and that p73 also plays a crucial part in PDGF-withdrawal-induced differentiation. This is the first evidence of a role for p73 in the differentiation of a normal mammalian cell (Billon, 2004).

The prostate contains two major epithelial cell types -- luminal and basal cells -- both of which develop from urogenital sinus epithelium. The cell linage relationship between these two epithelial types is not clear. Luminal cells can develop independently of basal cells, but basal cells are essential for maintaining ductal integrity and the proper differentiation of luminal cells. Urogenital sinus (UGS) isolated from p63+/+ and p63-/- embryos developed into prostate when grafted into adult male nude mice. Prostatic tissue that developed in p63-/- UGS grafts contained neuroendocrine and luminal cells, but basal cells were absent. Therefore, p63 is essential for differentiation of basal cells, but p63 and thus basal cells are not required for differentiation of prostatic neuroendocrine and luminal epithelial cells. p63-/- prostatic grafts also contain atypical mucinous cells, which appear to differentiate from luminal cells via activation of Src. In the response to castration, regression of p63-/- prostate is inordinately severe with almost complete loss of ducts, resulting in the formation of residual cystic structures devoid of epithelium. Therefore, basal cells play critical roles in maintaining ductal integrity and survival of luminal cells. However, regressed p63-/- prostate regenerates in response to androgen administration, indicating that basal cells are not essential for prostatic regeneration (Kurita, 2004b).

In conclusion, p63 is essential for differentiation of prostatic basal cells, and basal cells are essential in maintaining normal differentiation of luminal cells and integrity of prostatic ducts. However, basal cells (therefore p63) are not required for development and regeneration of prostate. Further experimentation is required to define the role of p63 in basal cell differentiation. p63 isoforms are functionally distinct in regard to cell fate commitment, particularly in epidermal differentiation. The differentiation of epidermis appears to be regulated by the balance between isoforms containing and lacking the transactivation domain. To understand the function of p63 in basal cell differentiation in prostate may require detailed analysis of isoform expression in the developing UGS and the adult prostate (Kurita, 2004b).

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. This study demonstrates that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. These studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation (Qin, 2006).

p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, siRNAs directed against p63 was used to down-regulate p63 expression in regenerating human epidermis. Loss of p63 results in severe tissue hypoplasia and inhibits both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibits hypoproliferation, differentiation defects are not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescues the cell proliferation defect of p63 knockdown alone but fails to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Three p63 isoforms contain N-terminal transcriptional activation (TA) sequences while the other three (DeltaN) do not. p63 TA and DeltaN isoforms are further subjected to alternative splicing at their C termini, resulting in α, β, and γ variants. DeltaNp63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes (Truong, 2007).

The distinguishing feature of adult stem cells is their extraordinary capacity to divide prior to the onset of senescence. While stratified epithelia such as skin, prostate, and breast are highly regenerative and account disproportionately for human cancers, genes essential for the proliferative capacity of their stem cells remain unknown. This study analyzed p63, a gene whose deletion in mice results in the catastrophic loss of all stratified epithelia. p63 is strongly expressed in epithelial cells with high clonogenic and proliferative capacity, and stem cells lacking p63 undergo a premature proliferative rundown. Additionally, p63 is dispensable for both the commitment and differentiation of these stem cells during tissue morphogenesis. Together, these data identify p63 as a key, lineage-specific determinant of the proliferative capacity in stem cells of stratified epithelia (Senoo, 2007).

During gastrulation of the amphibian embryo, specification of the three germ layers, endoderm, ectoderm, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-beta signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. This study reports that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, XFDL156 and its mammalian homologs were found interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53 (Sasai, 2008).

Mdm2 (Murine Double Minute-2) is required to control cellular p53 activity and protein levels. Mdm2 null embryos die of p53-mediated growth arrest and apoptosis at the peri-implantation stage. Thus, the absolute requirement for Mdm2 in organogenesis is unknown. This study examined the role of Mdm2 in kidney development, an organ which develops via epithelial-mesenchymal interactions and branching morphogenesis. Mdm2 mRNA and protein are expressed in the ureteric bud (UB) epithelium and metanephric mesenchyme (MM) lineages. This paper reports the results of conditional deletion of Mdm2 from the UB epithelium. UB(mdm2-/-) mice die soon after birth and uniformly display severe renal hypodysplasia due to defective UB branching and underdeveloped nephrogenic zone. Ex vivo cultured UB(mdm2-/-) explants exhibit arrested development of the UB and its branches and consequently develop few nephron progenitors. UB(mdm2-/-) cells have reduced proliferation rate and enhanced apoptosis. Although markedly reduced in number, the UB tips of UB(mdm2-/-)metanephroi continue to express c-ret and Wnt11; however, there was a notable reduction in Wnt9b, Lhx-1 and Pax-2 expression levels. It is further shown that the UB(mdm2-/-) mutant phenotype is mediated by aberrant p53 activity because it is rescued by UB-specific deletion of the p53 gene. These results demonstrate a critical and cell autonomous role for Mdm2 in the UB lineage. Mdm2-mediated inhibition of p53 activity is a prerequisite for renal organogenesis (Hilliard, 2011).

PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRalpha signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRalpha is unable to bind PI3K (Pdgfra(PI3K/PI3K)) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRalpha signaling, Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates were immunoprecipitated, and the peptides were analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). This analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. It was demonstrated that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra(PI3K/PI3K)-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. Ybox1 binds both the p53 promoter and the p53 protein and expression of p53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, this study demonstrated that introduction of a p53-null allele attenuates the vertebral defects found in Pdgfra(PI3K/PI3K) neonates. These findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRalpha signaling that regulates survival and proliferation during skeletal development in vivo (Fantauzzo, 2014).

ATR maintains chromosomal integrity during postnatal cerebellar neurogenesis and is required for medulloblastoma formation

Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. This study reports that ATR (see Drosophila ATR), which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation (see Drosophila p53), apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. These data show that p53-driven apoptosis and cell cycle arrest - and, in the absence of p53, non-apoptotic cell death - redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition (Lang, 2016).

The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells

Stem-like cells may be integral to the development and maintenance of human cancers. Direct proof is still lacking, mainly because of poor understanding of the biological differences between normal and cancer stem cells (SCs). Using the ErbB2 transgenic model of breast cancer, it was found that self-renewing divisions of cancer SCs are more frequent than their normal counterparts, unlimited and symmetric, thus contributing to increasing numbers of SCs in tumoral tissues. SCs with targeted mutation of the tumor suppressor p53 possess the same self-renewal properties as cancer SCs, and their number increases progressively in the p53 null premalignant mammary gland. Pharmacological reactivation of p53 correlates with restoration of asymmetric divisions in cancer SCs and tumor growth reduction, without significant effects on additional cancer cells. These data demonstrate that p53 regulates polarity of cell division in mammary SCs and suggest that loss of p53 favors symmetric divisions of cancer SCs, contributing to tumor growth (Cicalese, 2009).

The PKH-26/mammosphere assay allows for the isolation to near purity of bona fide mammary SCs. Direct imaging of the initial self-renewing division of WT SCs revealed that, in most cases, it generates two daughter cells with different proliferative fates: one that is quiescent and another that proliferates actively. Evaluation of the correlation between proliferation potential and SC fate during mammosphere growth showed that the less proliferating cell subset (PKHhigh) was highly enriched in SCs (one out of three) and the only subset containing SCs, suggesting that the observed replicative asymmetry of WT SCs generates daughter cells with different developmental fate. This was confirmed by analysis of the intracellular localization of the cell fate determinant Numb after the first mitotic division of WT PKHhigh cells, which showed that cell divisions are intrinsically asymmetric. The behavior of the ErbB2 tumor MICs was remarkably different: each produced two cells with identical proliferation potential and uniform distribution of the cell fate determinant Numb. In the formed mammosphere, SCs were found also within the most proliferating cell subset (PKHneg), indicating that they underwent multiple rounds of cell divisions without losing self-renewal potential. Thus, one relevant feature of cancer SCs is their acquired property to divide symmetrically and, as consequence, to increase their numbers. Consistently, it was found that during clonal expansion in vitro (as occurs during mammosphere formation) cancer SCs increase in number, whereas WT SCs do not, and that primary tumors contain increased numbers of SCs (Cicalese, 2009).

Though increased in numbers, the ErbB2 tumor SCs remain minor cell subpopulations within cultured mammospheres or in the primary tumors, which are mainly composed of SC progenies at different stages of differentiation. These observations imply that ErbB2 tumor SCs can increase in number without, however, losing their developmental potential. In principle, this can be achieved, in a pool of SCs, by the alternate use of symmetric and asymmetric divisions. This is consistent with the observation that cancer SCs could divide either asymmetrically or symmetrically, though with inverted relative frequencies as compared to WT SCs. It was noticed, however, an apparent discrepancy between the numbers of SCs found in the formed ErbB2 tumor mammosphere and the theoretical numberthat is obtained if one assumes that the frequency of symmetric divisions of the mammosphere-initiating cells remains the same during its expansion in the growing mammosphere. Notably, the frequency of SCs in the primary ErbB2 tumors is even lower than in the formed mammosphere. To address this issue, the proliferative history of SC proliferation during mammosphere formation was modeled. The resulting model indicates that the relative frequency of symmetric divisions adopted by ErbB2 tumor SCs decreases progressively during mammosphere growth, suggesting that the mammosphere or the tissue environments, through as yet unidentified mechanisms, influence the binary fate decision of mammary ErbB2 tumor SCs in favor of the asymmetric divisions (Cicalese, 2009).

Although SCs have an enormous self-renewal capacity, there is evidence that the number of times that a SC replicates is restricted, suggesting that self-renewal of SCs is intrinsically limited. In fact, WT mammary SCs rapidly lose self-renewal potential in culture, and the whole mammary tissue can be serially transplanted less than six to seven times. On the contrary, ErbB2 tumor SCs are nearly immortal, and the potential of ErbB2 tumors to be serially transplanted is virtually unlimited. Thus, extended replicative potential, together with increased frequency of symmetric divisions, might be responsible for the continuous and geometric expansion of cancer SCs. ErbB2 tumor SCs, however, can also divide asymmetrically, a property that might account for their ability to originate differentiated progeny, thus maintaining tumor cell heterogeneity and leading to the continuous expansion of the tumor mass (Cicalese, 2009).

Like the ErbB2 tumor SCs, p53 null SCs are near immortal in culture and undergo symmetric self-renewing divisions, two properties that are consistent with their ability to expand geometrically in culture. Most notably, in the mammary gland of p53 null mice, numbers of SCs are increased and expand progressively over time, thus indicating that p53 null SCs divide symmetrically also in vivo. It has been reported that the mammary epithelium of mice with increased WT p53 activity (p53+/m mice) has decreased regenerative capabilities upon serial transplantation, suggesting early stem cell exhaustion. Together, these data suggest that one physiological function of p53 is to maintain a constant number of SCs in the mammary gland by imposing an asymmetric mode of self-renewing divisions. Interestingly, loss of p53 increases self-renewal of neural SCs, suggesting that this might represent a general function of p53 in SCs of different tissues (Cicalese, 2009).

The molecular mechanisms underlying this effect of p53 on self-renewal are unclear. Increased levels of Nanog expression was found in both p53-/- and ErbB2 tumor mammospheres, which were reverted after N3 treatment. RNA interference of Nanog expression in p53-/- or ErbB2 tumor mammospheres, however, did not affect their growth kinetics, suggesting that, in this system, p53 role in self-renewal is independent of Nanog (Cicalese, 2009).

A role for p53 loss in mammary carcinogenesis is suggested by the high proportion of breast cancers with p53 mutations and the prevalence of breast tumors in women with germline mutations of p53. Although the mammary gland of p53 null mice is apparently normal and p53 null mice rarely develop mammary tumors (probably because of the early occurrence of lymphomas), high incidence of mammary tumors develops after somatic inactivation of p53 or transplantation into WT fat pads of the p53 null mammary epithelium. Thus, the p53 null mammary epithelium contains increasing numbers of SCs and is highly susceptible to tumor development, suggesting that increased frequency of symmetric divisions might contribute to mammary tumorigenesis in the p53 null mice by expanding the pool of putative tumor target cells. Notably, the mammary gland mass, which is likely to correlate with the number of mammary SCs, is an important breast cancer risk factor. Since p53 is a potent suppressor of mammary transformation, an intriguing possibility is that inhibition of SC symmetric divisions by p53 is one mechanism of tumor suppression in the mammary epithelium (Cicalese, 2009).

This study found that p53 signaling is attenuated in the ErbB2 tumor mammospheres and that restoration of p53 by N3 provokes the rapid exhaustion of cultured mammospheres and reduces tumor growth in vivo. Analysis of biological mechanisms suggests that the antitumor activity of N3 is due to a selective effect of the drug on the self-renewing divisions of ErbB2 tumor SCs. First, N3 converted the prevailing mode of division of ErbB2 tumor SCs from symmetric to asymmetric. Consistently, the frequency of ErbB2 tumor SCs decreased dramatically in cultured mammospheres and primary tumors after N3 treatment. Furthermore, N3 did not induce apoptosis in either ErbB2 mammospheres or primary tumors, nor did it reduce the fraction of proliferating cells. Together, these data imply that increased frequency of symmetric divisions and extended replicative potential of ErbB2 tumor SCs contribute to tumor growth in vivo (Cicalese, 2009).

Three groups have recently reported that re-expression of p53 causes regression of different p53 null tumors, including lymphomas, sarcomas, and hepatocellular carcinomas. This study shows that this can be achieved also with drugs that target p53 in tumors carrying WT p53 alleles and attenuated p53 signaling, a situation that is common to ~50% of human cancers. The current findings, however, suggest that restoration of p53 by N3 selectively affects the self-renewal of cancer SCs. This is surprising, since re-expression of p53 in p53 null tumors promoted senescence or apoptosis (depending on the tumor cell type) and a rapid reduction of the tumor mass (within days). To resolve this issue, it would be important to determine treshold and targets (cellular or molecular) of p53 restoration in different tumor types (Cicalese, 2009).

Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis

Women exposed to diethylstilbestrol (DES) in utero develop abnormalities, including cervicovaginal adenosis that can lead to cancer. Transient disruption of developmental signals by DES permanently changes expression of p63, thereby altering the developmental fate of Müllerian duct epithelium. The cell fate of Müllerian epithelium to be columnar (uterine) or squamous (cervicovaginal) is determined by mesenchymal induction during the perinatal period. Cervicovaginal mesenchyme induces p63 in Müllerian duct epithelium and subsequent squamous differentiation. In p63-/- mice, cervicovaginal epithelium differentiates into uterine epithelium. Thus, p63 is an identity switch for Müllerian duct epithelium to be cervicovaginal versus uterine. p63 is also essential for uterine squamous metaplasia induced by DES-exposure. DES-exposure from postnatal day 1 to 5 inhibits induction of p63 in cervicovaginal epithelium via epithelial ER{alpha}. The inhibitory effect of DES is transient, and most cervicovaginal epithelial cells recover expression of p63 by 2 days after discontinuation of DES-treatment. However, some cervicovaginal epithelial cells fail to express p63, remain columnar and persisted into adulthood as adenosis (Kurita, 2004a).

Diethylstilbestrol is a synthetic estrogen that was prescribed to prevent miscarriage in pregnant women. Two to four million individuals were exposed to DES during pregnancy from 1946 to 1971. Women exposed to DES in utero (DES daughters) exhibit genital tract abnormalities, including cervicovaginal adenosis, that are characterized as the development of columnar epithelium in the cervix and/or vagina. DES daughters are at risk of developing cervicovaginal clear-cell adenocarcinoma, and cervicovaginal adenosis is thought to be the precursor of adenocarcinoma. Perinatal exposure of mice to DES generates a spectrum of reproductive tract lesions similar to those observed in humans. Using this animal model, many genes have been identified as a potential cause of DES-induced abnormalities in the female reproductive tract. For example, perinatal DES exposure disrupts expression of Wnt7a in the upper Müllerian duct. These genes play important roles in development and/or function of the uterus. However, the mechanism of DES-induced cervicovaginal adenosis is not understood. Estrogen receptor alpha (ERalpha) is essential for development of cervicovaginal adenosis induced by neonatal DES-exposure; however, the target of DES and ERalpha in the cervicovaginal adenosis is still unclear (Kurita, 2004a and references therein).

Columnar and squamous epithelia are dramatically different. The major functions of columnar epithelium are absorption and secretion, while stratified squamous epithelia form barriers. In addition, cytoskeletal and cell-adhesion molecules are different in columnar versus squamous epithelia. For example, cytokeratins 5 and 14 are expressed in squamous epithelial cells, and are essential to maintain the integrity of squamous epithelium. It is not understood how squamous and columnar epithelia differentiate from their embryonic precursors. The female reproductive tract is an excellent model with which to study the program of epithelial differentiation because it is lined with two distinct types of epithelia that differentiate from a common precursor. The Müllerian vagina, cervix, uterus and oviduct develop from the embryonic Müllerian duct, which is composed of a uniform layer of pseudo-stratified columnar epithelial cells. In the mouse, the Müllerian duct epithelium undergoes organ-specific morphogenetic changes during postnatal development induced by uterine and vaginal mesenchyme. In the uterus, the epithelium gives rise to columnar luminal and glandular epithelia. In the Müllerian vagina and cervix, the columnar epithelium transforms into a stratified squamous epithelium. In adulthood, columnar uterine and squamous cervicovaginal epithelia meet at the squamocolumnar junction (SCJ) in the cervix. In the mouse, epithelial cells of the Müllerian duct are fully capable of being induced by heterotypic mesenchyme to undergo uterine or vaginal differentiation prior to 7 days postnatal, after which this developmental plasticity is gradually lost. By adulthood, most uterine and cervicovaginal epithelial cells do not change their phenotype in response to induction by heterotypic mesenchyme (Kurita, 2004a and references therein).

Historically, uterine and vaginal epithelial phenotypes have been judged by histology. However, 17ß-estradiol (E2) and progesterone (P4) modify epithelial morphology in the uterus and vagina, and thus the effects of ovarian steroids must be always considered. For example, uterine epithelium can stratify as a result of hyper-proliferation in response to E2. In this case, the stratification is reversible, and does not involve expression of squamous-epithelial markers. The uterus of progesterone receptor (PR) knockout mice shows a stratified epithelial phenotype due to hyperplasia caused by unopposed estrogen action, which is due to loss of PR in the stromal cells. Thus, epithelial stratification (histology) per se is not the most reliable marker distinguishing uterine versus cervicovaginal epithelia. Unequivocal identification of cervicovaginal epithelial differentiation can be achieved by examination of squamous markers such as K14, which are not modified by steroid hormones. Likewise, uterine epithelial differentiation is best assessed by estrogen-independent expression of PR, which is a unique feature of rodent uterine epithelium. Multiple markers have been used in this study to assess differentiation of uterine and cervicovaginal epithelia (Kurita, 2004a).

p63 has been shown to act as an identity switch in differentiation of Müllerian duct epithelium. P63 (KET, p51A, p51B, p40 or p73L) is a homolog of the p53 tumor suppressor gene. p63–/– mice have skin defects and lack organs arising from epidermis such as mammary and salivary glands. Since development of uterus, cervix and vagina occurs mostly during postnatal stages, the phenotype of p63–/– mice in the mature female reproductive tract is unknown because of newborn lethality. Through rescue of p63–/– cervicovaginal rudiments by grafting, it has been shown that cervicovaginal epithelium of p63–/– mice expresses the full spectrum of uterine epithelial markers. This study describes the ontogeny of p63 in the mouse female reproductive tract and demonstrates a key role for p63 in DES-induced cervicovaginal adenosis (Kurita, 2004a).

p53 and the temporal order of gene activation induced by NO in mammalian cells

Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. This study has determined he temporal order of gene activation induced by NO in mammalian cells and the signaling pathways that mediate the action of NO have been examined. Using microarrays to study the kinetics of gene activation by NO, it was determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. The results were verified using quantitative real time PCR and the conclusions about the effects of NO were further validated by using cytokines to induce endogenous NO production. Pharmacological and genetic approaches were appled to determine the signaling pathways that are used by NO to regulate gene expression. Inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, were used to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. The results demonstrate that NO utilizes several independent signaling pathways to induce gene expression (Hemish, 2004).

One conclusion of this study is that there are distinct waves of gene induction events initiated by NO in mammalian cells. The first wave activates genes that are immediate targets of the NO signals. These genes (group I) include several of the known immediate-early genes, such as c-fos and egr-1. Several group I genes code for transcription factors; this is consistent with the fact that this initial wave of gene activation is followed by a second wave (activation of group II genes). Group II genes may include direct targets of transcription factors activated in the first wave. Finally, a distinct third wave of gene activation can be detected that starts at ~12 h after the addition of the NO donor. These genes may represent the targets of the group II genes; they may also reflect changes inherent to the cell cycle arrest status induced by NO. It will be interesting to determine whether there are any key regulatory genes in these groups required for the transition to the next stage (Hemish, 2004).

Genes in group I are especially interesting because they represent immediate targets of NO, and their activation may reflect changes in the transcription machinery (e.g., S-nitrosylation of some transcription factors). Most of these genes are activated within 30 min after addition of the NO donor; using Q-PCR it was also found that some of them are activated as early as 10-15 min after addition of the donor. The regulatory regions of these genes may be good candidate sites to search for putative NO response elements; they may also lead to identification of transcription factors affected by NO (Hemish, 2004).

The findings were validated by quantitating the NO-induced changes using Q-PCR technique. Furthermore, it was found that the tested genes induced by exogenous NO donor were also induced by the mixture of cytokines, which gives rise to endogenously produced NO. The degree of contribution of the NO signaling pathways varies widely from fully underlying the action of cytokines on gene expression (e.g., in the case of HO-1 and mdm2) to mediating only a part of the signaling cascades that lead to gene activation (e.g., BNIP3 and gly96). The overlap between the sets of genes activated in NIH3T3 cells by NO and by cytokines may reflect an important role for NO in the response of fibroblasts to cytokines in vivo during inflammation and tissue repair. It will be interesting to compare these results with the transcriptional profiles of cells exposed to individual cytokines to estimate the relative contribution of NO in the action of these effectors (Hemish, 2004).

Specific groups of genes were identified that require the activity of PI 3-kinase, PKC, or NF-kappaB to be induced by NO. These data correspond well to reports of the involvement of these proteins in the physiological changes induced by NO or changes in the enzymatic activity of these proteins induced by NO. A distinct group of genes was found whose activation by NO was prevented by the lack of p53. These data show that the p53 protein is up-regulated in response to NO and plays a role in the antiproliferative function of NO. This provides further support for the relevance of the profiling data in explaining the long term biological effect of NO (Hemish, 2004).

p53 and cellular senescence

Self-renewal, proliferation and differentiation properties of stem cells are controlled by key transcription factors. However, their activity is modulated by chromatin remodeling factors that operate at the highest hierarchical level. Studies on these factors can be especially important to dissect molecular pathways governing the biology of stem cells. SWI/SNF complexes are adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes that have been shown to be required for cell cycle control, apoptosis and cell differentiation in several biological systems. This study investigated the role of these complexes in the biology of mesenchymal stem cells (MSCs). To this end, in MSCs a forced expression of the ATPase subunit of SWI/SNF (Brg1 - also known as Smarca4) by adenoviral transduction was induced, forcing a significant cell cycle arrest of MSCs in culture. This was associated with a huge increase in apoptosis that reached a peak 3 days after transduction. In addition, signs of senescence were observed in cells having ectopic Brg1 expression. At the molecular level these phenomena were associated with activation of Rb- and p53-related pathways. Inhibition of either p53 or Rb with E1A mutated proteins suggested that both Rb and p53 are indispensable for Brg1-induced senescence, whereas only p53 seems to play a role in triggering programmed cell death. Effects were examined of forced Brg1 expression on canonical MSC differentiation in adipocytes, chondrocytes and osteocytes. Brg1 did not induce cell differentiation per se; however, this protein contributed, at least in part, to the adipocyte differentiation process. In conclusion, these results suggest that whereas some ATP-dependent chromatin remodeling factors, such as ISWI complexes, promote stem cell self-renewal and conservation of an uncommitted state, others cause an escape from 'stemness' and induction of differentiation along with senescence and cell death phenomena (Napolitano, 2007).

p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling

The tumor suppressor p53 is activated upon genotoxic and oxidative stress and in turn inhibits cell proliferation and growth through induction of specific target genes. Cell growth is positively regulated by mTOR, whose activity is inhibited by the TSC1:TSC2 complex. Although genotoxic stress has been suggested to inhibit mTOR via p53-mediated activation of mTOR inhibitors, the precise mechanism of this link was unknown. This study demonstrates that the products of two p53 target genes, Sestrin1 and Sestrin2 (see Drosophila Sestrin), activate the AMP-responsive protein kinase (AMPK) and target it to phosphorylate TSC2 and stimulate its GAP activity, thereby inhibiting mTOR. Correspondingly, Sestrin2-deficient mice fail to inhibit mTOR signaling upon genotoxic challenge. Sestrin1 and Sestrin2 therefore provide an important link between genotoxic stress, p53 and the mTOR signaling pathway (Budanov, 2008).

The mTOR signaling pathway is a central regulator of cell growth and survival. It is therefore not surprising that adverse environmental conditions negatively regulate cell growth by inhibiting mTOR. In addition to nutrient limitation, mTOR activity is negatively regulated by genotoxic stress and hypoxia, conditions that activate tumor suppressor p53. The ability of p53 to inhibit mTOR signaling is in line with its function as a negative regulator of cell growth and proliferation. The results described above strongly suggest that the ability of p53 to inhibit mTOR signaling depends on two of its target genes: Sesn1 and Sesn2 (Budanov, 2008).

The Sestrins belong to a small and evolutionary conserved family composed of 3 members in mammals, of which Sesn1 and 2 are stress inducible and p53 regulated. The ability of Sesn1/2 to inhibit cell growth and proliferation was attributed to their redox activity. The present work, however, demonstrates that Sesn1/2 are potent inhibitors of mTOR signaling, acting in a manner that does not depend on their redox activity, which only makes a partial contribution to their growth inhibitory activity. Sesn1 and 2 inhibit TORC1 activity towards p70S6K and 4E-BP1 in a variety of human and mouse cell lines, as well as in mouse liver. Notably, the ability of the hepatocarcinogen DEN to inhibit S6 phosphorylation is restricted to zone 3 hepatocytes, which are the main site in which it undergoes metabolic activation to become a potent alkylating agent, and this inhibitory activity is Sesn2-dependent. By inhibiting 4E-BP1 phosphorylation, Sesn2 enhances its interaction with eIF-4E and inhibits expression of growth regulatory proteins, such as cyclin D1 and c-Myc, whose translation is eIF-4E-dependent and sensitive to 4E-BP1 phosphorylation (Budanov, 2008).

The Sestrins impact TORC1 activity through the TSC1:TSC2 complex. Being a GAP for Rheb, the direct activator of TORC1, the TSC1:TSC2 complex is a central regulator of mTOR signaling. Sesn2 expression decreases Rheb GTP loading and the ability of both Sesn1 and Sesn2 to inhibit mTOR signaling is TSC2-dependent. One way to regulate TSC1:TSC2 GAP activity is through TSC2 phosphorylation, but other modes of regulation may also exist. Although the Sestrins have no effect on ERK and its target RSK or GSK3β, which can all serve as TSC2 kinases, they stimulate the activity of AMPK, a major TSC2 kinase. Furthermore, Sestrin expression enhanced TSC2 phosphorylation in live cells and this effect required the N-terminus of Sesn2, which mediates AMPKα binding. Sesn2 did not stimulate TSC1 phosphorylation and Sesn2-activated AMPK did not phosphorylate TSC1 (Budanov, 2008).

Importantly, the mTOR inhibitory activity of Sesn1/2 depends on AMPKα, whose phosphorylation at the activation loop was enhanced upon Sestrin expression. Inhibition of AMPK using compound C as well as shRNA silencing of AMPKα1 attenuated the ability of Sesn2 to inhibit mTOR signaling. Co-immunoprecipitation and gel filtration analyses revealed an interaction between Sesn2 and AMPKα, suggesting that Sestrins are engaged in formation of a large protein complex containing AMPK and TSC1:TSC2. It is proposed that Sesn1/2 induction in response to genotoxic stress results in binding of Sestrins, most likely as dimers, to AMPK and TSC1:TSC2, as well as auto-activation of AMPK through a mechanism based on induced proximity. In addition to activation of AMPK the Sestrins recruit it to phosphorylate TSC2. Phosphorylation of TSC2 correlates with enhancement of its GAP activity that leads to inhibition of Rheb and mTOR (Budanov, 2008).

Importantly, ample and clear evidence was obtained that Sesn1/2 are critical mediators of p53's ability to inhibit mTOR signaling. Using shRNA-mediated silencing it was found that both Sesn1 and Sesn2 participate in mTOR inhibition upon p53 activation in human cancer cells. Furthermore, disruption of the Sesn2 gene in mice attenuated the inhibition of p70S6K activity by the DNA-damaging agents: camptothecin in fibroblasts and DEN in hepatocytes. In both cases inhibition of p70S6K was p53-mediated, but unlike the p53 deficiency, the absence of Sesn2 has no effect on induction of p21Waf1, another p53 target gene. Thus, Sesn2 (and presumably Sesn1) seems to mediate only one aspect of p53 signaling -- inhibition of mTOR. Correspondingly, the growth-inhibitory activity of Sesn2 is not as strong as that of p53, which has additional targets with anti-proliferative activity, such as p21Waf1 (Budanov, 2008).

p53 deficiency and activation of mTOR signaling are hallmarks of human cancer. Several mechanisms account for mTOR activation in cancer, including activation of Ras, PI3K and AKT and inactivation of tumor suppressors that negatively regulate these molecules: PTEN, TSC1, TSC2 and LKB1. Although p53 can induce expression of several negative regulators of mTOR, including PTEN, TSC2, AMPKβ1 and IGF-BP3 in a cell type-dependent manner, the results demonstrate that p53-mediated inhibition of mTOR depends mainly on Sesn1 and 2 in mouse fibroblasts and certain human cancer cell lines and on Sesn2 in mouse liver (Budanov, 2008).

Inhibition of mTOR suppresses cell growth and proliferation. Sesn2 was known to inhibit cell proliferation, but its mechanism of action was heretofore unknown. The results strongly suggest that Sesn1 and Sesn2 exert their growth inhibitory effect via mTOR and may cooperate with other anti-proliferative p53 targets, such as p21Waf1. Interestingly, the SESN1 (6q21) and SESN2 (1p35) loci are frequently deleted in a variety of human cancers, suggesting they harbor one or more tumor-suppressors. Sesn2 deficiency was found to render murine fibroblasts more susceptible to oncogenic transformation and this effect may depend on mTOR inhibition. Hence, SESN1 and SESN2 may indeed be important components of the tumor suppressor network activated by p53 (Budanov, 2008).

In summary, while more remains to be learned about Sestrin biology and mechanism of action, the results establish these proteins as critical links between p53 and mTOR that enable p53 to inhibit cell growth (Budanov, 2008).

p53, life span and senescence

Overexpression of the short isoform of p53 (p44) has unexpectedly uncovered a role for p53 in the regulation of size and life span in the mouse. Hyperactivation of the insulin-like growth factor (IGF) signaling axis by p44 sets in motion a kinase cascade that clamps potentially unimpeded growth through p21Cip1. This suggests that pathways of gene activity known to regulate longevity in lower organisms are linked in mammals via p53 to mechanisms for controlling cell proliferation. Thus, appropriate expression of the short and long p53 isoforms might maintain a balance between tumor suppression and tissue regeneration, a major requisite for long mammalian life span (Maier, 2004).

Although a p53-like protein has been identified in C. elegans (CEP-1) and in Drosophila melanogaster (Dmp53), both of these p53 proteins lack regions with significant homology to the N-terminal domain of mammalian full-length p53. The function of this domain is to alter gene transcription at a number of different targets, some of which are directly involved in cell-cycle control and can cause proliferation arrest. Thus, the p53 homologs in lower organisms more closely resemble a short form of p53 (DeltaN-p53) that was recently identified in mammalian cell lines and in normal cells from several different tissues. In cells in which this short form is the only p53 protein present, the ability to transactivate target genes such as Mdm2 and p21/Cip1/Waf1 is lost and, along with it, the ability of p53 to control cellular proliferation and growth. In the absence of full-length p53, DeltaN-p53 is tumorigenic, whereas in the presence of full-length p53, it is growth-suppressive. The fact that this would have no functional consequences in the postmitotic cells of adult C. elegans and Drosophila suggests that the short form of p53 might represent the primitive form of the p53 protein (Maier, 2004).

In order to determine the mechanism by which the short form of p53 might control growth, in particular mammalian growth, transgenic mice were generated in which a genomic fragment coding for the short form of p53 is expressed in the context of full-length p53. Translation of the short isoform initiates at codon 41 in exon 4 and produces a 44-kD protein. Overexpression of p44 upsets the balance that normally exists between the full-length and short forms of p53 and leads to a phenotype of growth suppression and premature aging in mice. Growth suppression by p44 links small size, proliferation deficits, cellular senescence, and organismal aging to abnormal IGF signaling in the mouse (Maier, 2004).

A role has been uncovered for the short isoform of p53 in the regulation of the IGF axis in the mouse. Overexpression of the short form of p53 and disruption of the normal ratio of p44 to p53 have effects on both size and life span that can be linked to changes in the intracellular signaling cascades initiated by IGF. p53 exerts control over IGF signaling at several key points. First and foremost, p53 controls the level of the IGF-1 receptor. Second, p53 controls both the level and activity of the dual lipid-protein-phosphatase, PTEN. PTEN modulates the IGF signal transduced to Akt, mainly through dephosphorylation of phosphatidyl-inositol triphosphate. The level of PTEN is controlled by direct trans-activation of the PTEN promoter by p53. Although trans-activation of PTEN is enhanced only slightly by the presence of p44, the level of the protein actually goes down in MEFs and in tissues from p44+/+ mice. In addition to this apparent protein instability, phosphorylation at the site modified by casein kinase II (CKII) is dramatically increased. Collectively, these results can be interpreted as interference by p44 with protein-protein interactions between full-length p53 and CKII, which are known to be inhibitory, rather than with protein-DNA interactions during assembly of the trans-activation complex on the PTEN promoter. Increased phosphorylation of PTEN by CKII leads not only to its inactivation, but also to its stabilization, which seems to be responsible for the apparent accumulation of phospho-PTEN and loss of degradation-sensitive PTEN in cells of p44 mice. Because phosphorylation blocks its recruitment into complexes at the cell membrane and inhibits its phosphatase activity, p44 interference effectively results in functional inactivation of PTEN (Maier, 2004).

Control of the IGF axis by p53 is exerted at the earliest steps in the cascade, modulating effectors of both growth and proliferation further downstream. Although the exact mechanism by which the Ras-Raf-MEK-ERK pathway switches from one promoting proliferation to one inducing cell-cycle arrest is unknown, blocking this pathway pharmacologically can prevent replicative senescence and/or restore the ability of presenescent cells to proliferate. Pharmacological reversal of replicative senescence by blocking the expression of p21Cip1 is similar to a result with presenescent human fibroblasts in which senescence can be reversed in cells expressing p21, but not in cells expressing p16. This strengthens the argument that hyperactivation of the IGF signaling pathway plays a causal role in the phenotype of p44 transgenic mice by setting in motion a 'fail-safe' pathway to clamp downstream pathways that would otherwise lead to uninhibited growth. This also helps to resolve the paradox of small size with hyperactivity of an axis that otherwise would be expected to enhance growth. The outcome of affecting a major pathway that controls both growth and proliferation is perhaps best illustrated by contrasting the phenotype of p44 mice in which p53 function is disturbed with mice in which proliferation alone is affected. Hypomorphic alleles of Myc cause comparable reductions in overall size, but have no effect on longevity (Maier, 2004).

The p53 tumor suppressor plays a key role in organismal aging. Cellular senescence is a cellular mechanism postulated to drive the aging process, mediated in part by p53. Although senescent cells accumulate in elderly individuals, most studies have relied on correlating in vitro senescence assays with in vivo phenotypes of aging. Two different mouse models have been used in which the p53-related protein p63 is compromised; cellular senescence and organismal aging are intimately linked, and these processes are mediated by p63 loss. p63+/- mice were found to have a shortened life span and display features of accelerated aging. Both germline and somatically induced p63 deficiency activates widespread cellular senescence with enhanced expression of senescent markers SA-beta-gal, PML, and p16INK4a. Using an inducible tissue-specific p63 conditional model, it was further shown that p63 deficiency induces cellular senescence and causes accelerated aging phenotypes in the adult. These results suggest a causative link between cellular senescence and aging in vivo, and demonstrate that p63 deficiency accelerates this process (Keyes, 2005).

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. This study shows that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3' untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation (Burns, 2008).

The telomere-capping complex shelterin protects functional telomeres and prevents the initiation of unwanted DNA-damage-response pathways. At the end of cellular replicative lifespan, uncapped telomeres lose this protective mechanism and DNA-damage signalling pathways are triggered that activate p53 and thereby induce replicative senescence. This study identified a signalling pathway involving p53, Siah1 (a p53-inducible E3 ubiquitin ligase) and TRF2 (telomere repeat binding factor 2; a component of the shelterin complex). Endogenous Siah1 and TRF2 were upregulated and downregulated, respectively, during replicative senescence with activated p53. Experimental manipulation of p53 expression demonstrated that p53 induces Siah1 and represses TRF2 protein levels. The p53-dependent ubiquitylation and proteasomal degradation of TRF2 are attributed to the E3 ligase activity of Siah1. Knockdown of Siah1 stabilized TRF2 and delayed the onset of cellular replicative senescence, suggesting a role for Siah1 and TRF2 in p53-regulated senescence. This study reveals that p53, a downstream effector of telomere-initiated damage signalling, also functions upstream of the shelterin complex (Fujita, 2010).

p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease

Evidence is presented for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington’s disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific; it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-α, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. These findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD (Bae, 2005).

To explore the role of p53 in mHtt-induced cell death of intact animals, the effect of p53 deletion was evaluated in mHtt-Tg flies. The mHtt-Tg flies, which express mHtt N170-120Q under the control of the eye-specific expression GMR promoter, manifest prominent cell death of photoreceptor neurons. mHtt-Tg flies were crossed with p53 mutant flies in which the p53 gene was deleted by homologous recombination and the effects of deleting p53 was assessed in the compound eyes. Wild-type compound eyes contain ~800 ommatidia, each of which includes seven photoreceptor cells in any given plane of section. The photoreceptor cells contain a microvillar structure referred to as the rhabdomere. Wild-type (wt) and p53 knockout flies (p53) display a normal composition of seven photoreceptor cells in each ommatidium. mHtt-Tg flies (Htt), however, manifest strong age-dependent loss of rhabdomeres and photoreceptor cells. Deletion of two copies of p53 in mHtt-Tg flies (Htt;p53) suppresses this phenotype. Thus, p53 mediates mHtt-induced neurotoxicity in intact organisms (Bae, 2005).

This study presents evidence favoring a specific role for p53 in HD pathology. Mutant Htt binds p53 and elicits increases in the levels of p53 protein in the nucleus and p53 transcriptional activity. These elevations occur in PC12 cells, primary neuronal cultures, mHtt-Tg mice, and postmortem brains of HD patients. Perturbation of p53 by pifithrin-α, RNAi, or genetic deletion prevents mHtt-induced cellular dysfunction and abnormal behavior in vivo. Mitochondrial membrane depolarization and cytotoxicity by mHtt is prevented by inhibition of p53. By contrast, mHtt nuclear and cytoplasmic aggregates are not influenced by p53 deletion. Genetic deletion of p53 suppresses mHtt-induced neurodegeneration in Drosophila. Some of the neurobehavioral defects in mHtt-Tg mice, including dyskinesia of the hindlimbs, rotational activity, prepulse inhibition, and rotarod performance, are prevented by genetic deletion of p53 (Bae, 2005).

In summary, this study establishes a specific role for p53 in HD. Since p53 is a nuclear transcription factor that regulates various mitochondrial genes and insofar as mitochondrial dysfunction appears important in HD, these findings provide a molecular mechanism linking disturbances of nuclei and mitochondria in HD. A lower incidence of cancer has been reported in HD patients. Since p53 is a tumor suppressor, its upregulation in multiple HD tissues might account for diminished carcinogenesis, though dietary and other extrinsic factors in cancer incidence must be ruled out (Bae, 2005).

The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. This study used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53R175H, genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin 'pearl' formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma (Okawa, 2007).

The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. Mice were generated with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway, was examined. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences (Terzian, 2008).

p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. This study tests whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. The approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. It was shown that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. These results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity (Zhao, 2010).

Selective activation of p53-mediated tumour suppression in high-grade tumours

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10%-15%. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras. p53 is also frequently inactivated in NSCLC and, because oncogenic Ras can be a potent trigger of p53, it seems likely that oncogenic Ras signalling has a major and persistent role in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease. This study models the probable therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras. Surprisingly, p53 restoration failed to induce significant regression of established tumours, although it did result in a significant decrease in the relative proportion of high-grade tumours. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked upregulation in Ras signal intensity and induction of the oncogenic signalling sensor p19(ARF). These data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and in the efficacy of therapeutic p53 restoration to eradicate cancers (Junttila, 2010).

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive in p53 mutants

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. This report demonstrates that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, this aspect of pRB function was studied in isolation, and it was shown to be an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice (the murine version of the p53 gene) succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, it was demonstrated that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes (Coschi, 2009).

This study relied on a viable, gene targeted mouse strain in which pRB is mutated to block LXCXE-dependent interactions, such as those with viral oncoproteins and chromatin remodeling enzymes like histone deacetylases. Cells from these mice have limited proliferative control defects, except for the responsiveness to transforming growth factor β (TGF-β) and senescence-inducing stimuli. It was demonstrated that pRB interacts with the Condensin II complex to establish proper chromosome structure. These experiments reveal that condensation defects caused by a deficiency in pRB-LXCXE interactions occur before metaphase, and are unrelated to the ability to regulate G1-to-S-phase progression. Rb1deltaL/deltaL; Trp53-/- mice were used as well as Trp53-/- controls -- both of which are uniformly defective in their response to G1 arrest stimuli such as DNA damage - and oncogene-induced senescence -- to study pRB's role in chromosome condensation in isolation. Rb1deltaL/deltaL; Trp53-/- mice succumb to much more aggressive forms of cancer than p53-deficient controls, and their tumors are characterized by elevated levels of chromosome instability. Furthermore, defective chromosome condensation caused by mutant pRB can accelerate loss of heterozygosity and cancer onset in Trp53+/- mice. This study reveals that participation in mitotic chromosome condensation is an integral aspect of pRB's function as a tumor suppressor protein (Coschi, 2009).

Cyclin B2 and p53 control proper timing of centrosome separation

Cyclins B1 and B2 are frequently elevated in human cancers and are associated with tumour aggressiveness and poor clinical outcome; however, whether and how B-type cyclins drive tumorigenesis is unknown. This study shows that cyclin B1 and B2 transgenic mice are highly prone to tumours, including tumour types where B-type cyclins serve as prognosticators. Cyclins B1 and B2 both induce aneuploidy when overexpressed but through distinct mechanisms, with cyclin B1 inhibiting separase activation, leading to anaphase bridges, and cyclin B2 triggering aurora-A-mediated Plk1 hyperactivation, resulting in accelerated centrosome separation and lagging chromosomes. Complementary experiments revealed that cyclin B2 and p53 act antagonistically to control aurora-A-mediated centrosome splitting and accurate chromosome segregation in normal cells. These data demonstrate a causative link between B-type cyclin overexpression and tumour pathophysiology, and uncover previously unknown functions of cyclin B2 and p53 in centrosome separation that may be perturbed in many human cancers (Nam, 2014).

Stage-specific sensitivity to p53 restoration during lung cancer progression

Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. This study shows that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. Amplification of MAPK signalling is a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. It is proposed that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. These observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations (Feldser, 2010).

A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection

The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. This study identified a polymorphic p53 responsive element and demonstrates its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. A single-nucleotide polymorphism (SNP) was uncovered in a functional p53-binding site, and its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene was establish. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. It was establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but it was then shown that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans (Zeron-Medina, 2013).

This report presents strong evidence supporting the hypothesis that well-placed polymorphisms in functional p53-binding sites can result in differential p53-dependent transcriptional regulation and cancer risk, through the identification and characterization of the KITLG p53-RE SNP. The KITLG p53-RE SNP is strongly linked to three SNPs shown, in three independent GWASs, to associate with differential risk for developing seminomatous and nonseminomatous testicular cancer, with a per allele odds ratio of up to 3.07, one of the highest and most significant findings among all cancer GWASs. Based on linkage data, individuals with the G allele, and therefore the stronger KITLG p53-RE, harbor the greater testicular cancer risk. This finding supports the intriguing hypothesis that p53-regulated paracrine growth factor signaling may be associated with promoting tumor development. Indeed, activating mutations of the KIT receptor have been shown to promote tumor formation and the receptor is currently targeted by many therapeutic agents in cancer treatments. In testicular cancer, the KIT pathway is central to its molecular pathology, and many components of this pathway are somatically mutated to activate KIT signaling. Moreover, the six SNPs identified in testicular cancer GWASs to associate with differential cancer risk reside in three genes in this pathway: KITLG, SPRY4, and BAK1. Although p53 is the most commonly mutated gene in human cancer, whereby 50% of all cancers have mutant p53, less than 3% of testicular cancers do. In many cancers that retain wild-type p53 genes, the cells have frequently attenuated p53 through other mechanisms such as the overexpression of direct inhibitors, like the amplification of the MDM2 oncogene. In dramatic contrast to these observations, p53 signaling is often found to be robust in human testicular cancers. Intriguingly, 80% of testicular germ cell tumors can be cured with DNA-damaging therapies, and the retention of p53 activity has been implicated in this astounding cure rate through chemotherapeutics. Together with the data presented in this report, these observations suggest that the G allele of KITLG p53-RE allows increased p53-dependent upregulation of KITLG expression, which drives male germ cell proliferation rather than arrest in the presence of DNA damage, thus promoting tumorigenesis and thereby offering an explanation for the abnormal retention of p53 activity in testicular tumors (Zeron-Medina, 2013).

Drosophila SCE/dRING E3-ligase inhibits apoptosis in a Dp53 dependent manner

The Polycomb group (PcG) of proteins control developmental gene silencing and are highly conserved between flies and mammals. PcG proteins function by controlling post-translational modification of histones, such as ubiquitylation, which impacts chromatin compaction and thus gene transcription. Changes in PcG cellular levels have drastic effects on organismal development and are involved in the generation of human pathologies such as cancer. However, the mechanisms controlling their levels of expression and their physiological effects are only partially understood. This work describes the effects of modulating levels of SCE/dRING (Sex combs extra), a conserved E3 ubiquitin ligase and member of the PcG known to mono-ubiquitylate histone H2A. Inactivation of Sce induces apoptosis, an effect that is decreased in the absence of Dp53 function. However, over-expression of SCE produce no developmental effects but inhibits DP53-induced apoptosis. Thus, Sce functions as a Dp53-dependent apoptosis inhibitor. The SCE inhibition of DP53-induced apoptosis requires Ring and YY1 Binding Protein (dRYBP), an ubiquitin binding protein and member of the PcG. Moreover, this inhibition of apoptosis involves the reduction of DP53 protein levels. Finally, high levels of SCE inhibit X-ray induced apoptosis as well as the apoptosis associated with tumor growth. It is proposed that SCE, together with dRYBP, inhibits apoptosis either by epigenetically regulating Dp53 transcription or by controlling the stabilization of DP53 protein levels thus promoting its ubiquitylation for proteaosomal degradation. This function may generate a homeostatic balance between apoptosis and proliferation during development that provides cell survival during the initiation and progression of disease processes (Simoes da Silva, 2017).

These results identify a novel apoptotic role for the SCE protein. The results show that Sce functions as a repressor of apoptosis, as inactivation of Sce promotes apoptosis. This novel function of Sce has gone unidentified most likely because the strong effect of Sce inactivation on the de-repression of UBX expression in the wing has masked its apoptotic function. This activity is revealed when Ubx and Sce are simultaneously inactivated (Simoes da Silva, 2017).

Components of the PRC1 complex, such as PH and PSC/SU(Z)2, have been classified as tumor suppressors because, when inactivated, they induce tumor growth in the wing disc. Inactivation of Sce in the wing disc does not induce tumor growth thus adding complexity to the analysis of PcG targets. Interestingly, SCE-mediated repression of apoptosis requires Dp53 indicated by the decrease in apoptosis when both Sce and Dp53 were inactivated and the response in the activation of the P53R-GFP reporter expression construct when Sce function was inactivated (Simoes da Silva, 2017).

The results on the impact of high SCE levels also support its proposed anti-apoptotic function. This analysis showed SCE and dRYBP, an ubiquitin binding protein previously found to interact genetically and physically with SCE (Fereres, 2014), participates in the inhibition of apoptosis. Curiously, over-expression of SCE produces no developmental phenotype in the wings and the SCE-mediated inhibition of apoptosis is revealed only under stress conditions such as high levels of DP53, X-ray treatment or tumorogenesis. Moreover, human RYBP has been shown to induce apoptosis in transformed cells but not in normal cells. It will be very interesting to investigate if human SCE (Ring1b/RNF2) is involved in human RYBP's apoptotic activity, which could make the combination of RNF2 and RYBP a potential cancer therapeutic candidate (Simoes da Silva, 2017).

The results indicate that SCE-mediated inhibition of apoptosis both requires Dp53 and also modulates DP53 levels, as measured by the activation of P53R-GFP, P53-GFP-FLAG and by DP53 immuno-staining. Thus, the results show that SCE inhibits apoptosis through the modulation of DP53 levels by a mechanism that is, as yet, not clear (Simoes da Silva, 2017).

The well described functions of the PcG proteins and E3-ubiquitin ligases suggest two mechanisms through which SCE could modulate DP53 levels: 1) SCE, as part of the PRC1 complex, could epigenetically act to regulate Dp53 transcription; and 2) SCE, as an E3 ubiquitin ligase, could control the stabilization of DP53 protein levels by promoting its ubiquitylation for proteaosomal degradation. Several observations combined with results from the experiments described in this study support a mechanism by which SCE inhibits apoptosis by promoting DP53 protein degradation. There is no evidence for the presence of Polycomb Response Elements (PREs) in the Dp53 gene, as SCE protein was not seen to bind to Dp53 genomic sequences in wing imaginal discs by ChiP experiments. Further, RNF2, the vertebrate SCE counterpart, has been shown to function as a p53 E3-ubiquitin ligase. The current results show that SCE requires Dp53 to inhibit apoptosis and that inactivation of Sce function increases DP53 protein levels as measured by the activation of a P53R-GFP reporter construct. Further, high levels of SCE produce no phenotype in wing cells, implying that it has no effect on transcription in wing disc cells. Importantly, the results indicate that high levels of SCE modulate apoptosis and DP53 protein levels. It would be interesting to know whether, and to which degree, each of the possible mechanisms contribute to the modulation of DP53 cellular levels mediated by high levels of SCE (Simoes da Silva, 2017).

In Drosophila, SCE function has been associated with protein mono-ubiquitylation rather than poly-ubiquitylation. Further studies are needed to determine the SCE function in the mono- or poly-ubiquitylation of DP53 in the fly. In vertebrates, RNF2 appears to have poly-ubiquitylation activity and, also in vertebrates, MDM2, the major E3-ligase for p53 poly-ubiquitylation, is able to mono- and poly-ubiquitylate p53. In the fly, these functions could be provided by either SCE itself or other E3-ligases acting on DP53, such as Bonus, Synoviolyn or CORP, or via through the cooperation of E3-ligases such as PSC, a member of the PRC1 complex known to have transcriptional independent E3-ligase activity on Cyclin B (Simoes da Silva, 2017).

This study has also shown that SCE is capable of controlling both hyperplasic growth induced by high levels of ABRUPT and neoplasic growth induced by inactivation of polyhomeotic function. Each of these is achieved by reducing apoptosis (Simoes da Silva, 2017).

Curiously neither high levels of DIAP1 nor inactivation of Dp53 function were capable of inhibiting apoptosis induced by either over-expression of ABRUPT or by inactivation of polyhomeotic. Thus, in these tumor conditions high levels of SCE efficiently inhibit apoptosis without inducing proliferation. Further studies will be needed to characterize the mechanisms underlying the SCE-dependent inhibition of apoptosis. In vertebrates, as elevated RNF2 levels have been found in many tumors, RNF2 has been proposed to function as an oncogene via its ability to decrease levels of p53. In Drosophila, high levels of SCE do not produce overgrowth during normal development or during tumoral growth. The results show that SCE acts to inhibit apoptosis in the fly, a function that may include oncogenic activity. This oncogenic activity may be based on the regulation of DP53 levels, as proposed for vertebrates. It appears that in Drosophila the mechanisms of DP53 regulation may also involve SCE, highlighting the evolutionarily conservation of these mechanisms and the utility of the fly for their study (Simoes da Silva, 2017).


REFERENCES

Search PubMed for articles about Drosophila p53

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date revised: 25 April 2018

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