Literature reviews of p53 freely available online:
Bourdon, J.-C., et al. (2005). p53 isoforms can regulate p53 transcriptional activity. Genes Dev. 19(18): 2122–2137
Articles on p53 biology available online:
Harms, K. L. and Chen X. (2005). The C terminus of p53 family proteins is a cell fate determinant. Mol. Cell. Biol. 25(5): 2014-30. 15713654
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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