See the embryonic expression pattern of p53 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
The expression of Dmp53 transcripts during embryogenesis was examined to assess potential roles for Dmp53 during Drosophila development. Dmp53 RNA is maternally loaded into oocytes and is abundant until cellularization of the blastoderm. Zygotic expression of Dmp53 begins at cellularization and is initially ubiquitous. At midembryogenesis, Dmp53 RNA levels are highest in the mesoderm and gut, with only low levels of RNA detectable in the epidermal and neural cell layers. As development proceeds, the expression of Dmp53 becomes progressively more restricted and falls dramatically in all tissues except for the primordial germ cells and a small patch of hindgut cells. Although one must use caution when inferring function from expression data, the high levels of Dmp53 RNA in germ cells is likely to be significant because germline p53 expression is a common feature in species ranging from clam to human. This conservation of expression suggests an important function for p53 in germline development (Ollmann, 2000).
A developmental profile of Drosophila p53 RNA levels shows that p53 is present throughout development. RNA levels seem to be highest during early embryogenesis and in females, suggesting a maternal contribution. Consistent with this notion, p53 mRNA is found in cells of the egg chamber that provide the maternal contribution, the nurse cells, but p53 mRNA is undetectable in the somatic follicle cells of the egg chamber. Additionally, p53 RNA is expressed ubiquitously in early embryogenesis. The staining inside the blastoderm embryo probably stems from the maternal contribution (Jin, 2000).
Insects and mammals diverged ~150 million years ago in evolution. The striking conservation of p53 in the two systems suggests that p53 is an early-evolved gene and its functions are under strong selection pressure. p53 expression in Drosophila exactly mirrors that of Xenopus and is also very similar to that of mice in early embryonic development. The expression pattern in mice may reflect the function of p53 as teratogenesis suppressor, as shown by the observation that p53-null mice had a higher teratogenesis rate and lower abortion rate upon gamma-irradiation than wild-type mice. This conserved expression pattern in all species examined to date suggests that one major function of p53 might be protecting the genomic integrity of early embryos and that of the germ-line cells. This would of course be critical to ensure proper development of an individual organism, eliminating embryos with DNA damage and genetic defects. Therefore this function is strongly selected and maintained during evolution. The tumor suppressor function of p53 in differentiated somatic cells might be a more recently evolved adaptation. As organisms appeared with long lifespans, activating dividing cells in the adult, the selection pressure to eliminate somatic mutation concomitantly increased. Although p53 minus mice appear to develop normally, it would be interesting to see if p53 minus flies have an elevated degree of developmental defect and germ-line instability (Jin, 2000).
The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. This paper provides evidence that Drosophila p53 has a previously unidentified role in coordinating the compensatory growth response to tissue damage. The sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. These processes are regulated by p53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. These results indicate that once induced, p53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. These data suggest that p53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage (Wells, 2006; full text of article).
The repair of tissue after cellular damage can be critical to the survival of the animal. Previous studies demonstrated that undead cells stimulate the proliferation of neighboring cells, providing a model for how damaged and dying cells contribute to the replacement of lost tissue. With this model, it was found that the wing imaginal disc responds to this damage as a whole by deploying a multi-step process that ends with compensatory growth. p53 functions in a dronc-dependent manner at each step of the tissue-replacement process. Furthermore, p53 and the initiator caspase dronc may be generally required for tissue recovery in imaginal discs, because it was found that blastema formation was significantly impaired during regeneration induced in either p53 or dronc mutant leg discs (Wells, 2006).
The data suggest that p53 is induced and becomes functional in undead cells by a mechanism that does not require DNA-damage sensing or activation of the stress kinase AMPK. Rather, Dronc, an initiator caspase homologous to caspase-9, is necessary and sufficient to induce all aspects of the growth regulation by p53. It is not known how Dronc activity results in p53 expression and activity in these cells, but many caspase substrates are not directly involved in apoptosis. As an example, one of the first caspase substrates identified was the cytokine IL-1β, which regulates many aspects of the inflammatory response. Induction of p53 mRNA in undead cells is prevented in dronc mutant discs, and thus it is possible that a regulator of p53 is cleaved by Dronc, leading to its expression and ultimately to its ability to regulate the compensatory growth response in the imaginal discs. Regardless of the molecular mechanism, the data argue for direct communication between Dronc and p53 in response to tissue damage (Wells, 2006).
Collectively, these experiments imply that p53 serves as a master coordinator of tissue repair in imaginal discs, regulating both cell-autonomous and non-cell-autonomous cell-cycle arrests, the expression of the pattern-regulating genes wg and dpp, and compensatory cell proliferation and growth. Based on these results, it is suggested that cellular damage activates Dronc, which in a nonapoptotic role causes the induction of p53 mRNA and leads to p53 activity. It is proposed that p53 then acts as an overall damage monitor, in a role that includes its conserved functions in apoptosis (here, induction of hid and rpr expression) and growth arrest (by repression of stg/cdc25), but also allows for induction of signals that promote compensatory growth of the disc. The results suggest that p53 monitors tissue damage through a feed-forward loop with Dronc and the pro-apoptotic genes hid and rpr, which both amplifies and sustains the growth-regulating signal (Wells, 2006).
An intriguing puzzle left unanswered by these results is why the growth response to undead cells occurs only several days after they are generated: both HhGal4 and EnGal4 drive expression of Hid or Rpr from early embryonic stages, yet even with careful observation no growth phenotype was detected until the middle part of the third instar. Caspases are active in cells expressing Hid or Rpr + P35 at early time points, indicating that these cells are not immune to the apoptotic response early in development. The genes involved in the apoptotic response are subject to many levels of control, including that by micro-RNAs (miRNAs). Hid protein expression, for example, is suppressed by Bantam, a miRNA highly expressed early in imaginal disc development, but declining as development progresses. It is likely that rpr is also regulated by miRNA gene silencing. Hence, the delay of the growth response in discs with undead cells may reflect a requirement for threshold levels of these factors to fully activate the feedback loop. At the very least it emphasizes that the regulation of growth and cell death during wing disc development is complex and has multiple inputs, many of which are poorly understood (Wells, 2006).
Activity thresholds appear to play an important role in the processes induced by undead cells. Dronc, for instance, is haploinsufficient for its effect in compensatory proliferation. It is possible that the apoptotic functions of Dronc require a relatively low activity level, but that high Dronc activity allows activation of the p53-dependent tissue-damage response. Regulation of Dronc by critical activity thresholds could provide the animal some regenerative capacity and increase its chances for survival when conditions are appropriate for tissue repair (Wells, 2006).
As expected given its role in coordinating many cellular behaviors, p53 modulates the activity or expression of myriad effectors. Regulatory effectors of Drosophila p53 are only beginning to be identified, and these data add stg/cdc25 to the list. One of the first detectable disc responses to undead cells is G2 arrest, mediated by loss of stg mRNA. Cdc25 is also regulated by vertebrate p53 but is inhibited post-transcriptionally by p53-dependent 14-3-3 activity (Levine, 2006). Experiments with irradiated p53 mutant animals have not revealed a cell-cycle arrest role. However, recent work indicates that dp53 also regulates a G1 checkpoint under conditions of metabolic stress; thus, like vertebrate p53, Drosophila p53 can activate both a G1 and a G2 checkpoint in response to tissue stress. Other effectors and targets involved in the compensatory proliferation process remain unknown, although expression profiling experiments from irradiated p53 mutants identified several potential targets, several of which do not have obvious roles in cell death or DNA repair (Wells, 2006).
How does Drosophila p53 control the signaling that leads to compensatory proliferation? The events observed — G2 arrests in two different cell populations, ectopic expression of wg, and compensatory growth — are all regulated by p53. It is possible that p53 directly and coordinately controls each of these processes by regulating the expression of specific effectors. However, because the response is both cell autonomous and non-cell autonomous, the idea is favored that these processes are interdependent, but sequentially activated. It is envisioned that as a result of Dronc activation in undead cells, p53 induces loss of stg, leading to G2 arrest, and hid and rpr expression, initiating the feedback loop. It is postulate that cells then synthesize factors that stimulate their survival and proliferation. The non-cell-autonomous arrest in the anterior compartment may be a secondary effect of undead cells in the posterior. High levels of TUNEL activity was observed in the anterior cells of these discs, which could feasibly activate p53 in those cells. However, no p53 mRNA was detected in anterior cells. One possibility is that the DNA fragmentation resulting from dying anterior cells could activate ATM and Chk2 in those cells. Consistent with this, although loss of either of these kinases did not affect undead cell induction of Wg expression or compensatory growth, the cell-cycle arrest in anterior cells was reduced in a fraction of atm and chk2 mutants (Wells, 2006).
What is the growth-stimulating signal induced by undead cells? While its identity is still unclear, both Wg and Dpp have been implicated in this role. This makes sense, because Wg and Dpp are the major pattern organizers of all imaginal discs and are also involved in regulating their growth, and furthermore they are known to be induced in disc regeneration. However, although wg and dpp are ectopically expressed in undead cells, it was found that targets of both are sharply downregulated, specifically in the undead cells. These data also show that undead cells are able to proliferate and contribute to the compensatory growth. Thus, although the nonautonomous stimulation of growth (anterior cells near the A/P boundary) could be due to increased Dpp signaling, it is suspected that the autonomous growth stimulation is due to other, unidentified factors (Wells, 2006).
This study identified a growth-regulatory role for p53 that seems counter to its role as a tumor suppressor in vertebrates. However, it is speculated that the ability of p53 to sense and respond to tissue damage and promote compensatory proliferation and regeneration in Drosophila reflects an ancestral function, aspects of which have been appropriated for developmental processes and distributed among p53, p63, and p73 during vertebrate evolution. Although p63 and p73 initially were proposed to have evolved as duplications of p53, reanalysis of the phylogenetic relationship between the three family members has suggested that p63 may be the ancestral gene. p63 and p73 are structurally similar to p53 but contain an additional SAM domain. p53 is the sole member of the family encoded in the Drosophila genome, and although dp53 does not contain a SAM domain, based on the sequence of the DNA binding domain, the most highly conserved region of p53, it is more related to vertebrate p63 than to p53. After irradiation, cell-cycle arrest is not p53 dependent in either Drosophila or the nematode C. elegans, and therefore it has been proposed that the ancestral p53 function is apoptosis, rather than a “repair, then death” response when damage cannot be repaired. The experiments argue that as in vertebrates, p53 plays a role in cell-cycle arrest after tissue damage. The additional functions of p53 in promoting cell proliferation may have been conserved in p63, which regulates progenitor cell renewal in the epidermis. Other processes that require cell renewal may also be regulated by p53. For example, p53 mutants are reported to have fertility defects, so it is tempting to speculate that stem cell renewal in the gonad requires this previously unappreciated role of Drosophila p53 (Wells, 2006).
Mutant alleles of Dmp53 analogous to the R175H (R155H in Dmp53) and H179N (H159N in Dmp53) tumor-derived mutations in human p53. These mutations in human p53 produce proteins with dominant-negative activity, presumably because they cannot bind DNA but retain a functional tetramerization domain. Thus, DNA binding by any tetramer that incorporates the mutant protein is disrupted. Both Dmp53R155H and H159N proteins inhibit binding of wild-type Dmp53 to a p53 binding site, although they do not bind to DNA themselves. These mutant forms of Dmp53 are useful tools to test the function of wild-type Dmp53 in vivo (Ollmann, 2000).
The similarity between Drosophila and human proteins prompted an exploration of the role Drosophila p53 plays in vivo. It is known that p53 exerts its role as a tumor suppressor partially through initiation of apoptosis. Consistently, expression of human p53 in the fly eye initiates apoptosis. It was reasoned that overexpression of wild-type Drosophila p53 might trigger an ectopic cellular response, thereby revealing some of its in vivo function. Using the UAS/GAL4 binary expression system, Drosophila p53 was expressed under the control of a photoreceptor specific promoter, gmr-GAL4. One of five transgenic fly lines tested shows a rough eye phenotype. At least 2-fold higher p53 RNA levels were observed in the line that shows the phenotype than in any of the other lines. Because the eyes of flies overexpressing Drosophila p53 are smaller than those of wild-type controls, it seemed likely that the observed phenotype was partially caused by ectopic apoptosis. To test this possibility, third-instar eye imaginal discs from animals overexpressing p53 were subjected to an acridine orange staining to visualize cell death. Overexpression of Drosophila p53 in the developing retina causes an increase in cell death. This observation is in accordance with an apoptosis-inducing function of p53. Moreover, ubiquitous expression in transgenic Drosophila results in a high percentage of lethality. This observation is consistent with an induction of extensive apoptosis in essential tissues of the fly, thereby reducing viability (Jin, 2000).
Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).
Wee1 kinases may play a role in regulating genome stability as evidenced by a genetic interaction with Drosophila p53. In humans, the p53 tumor suppressor promotes apoptosis in cells that have suffered DNA damage. Overexpression of Drosophila p53 in the eye promotes extensive cell death by apoptosis, resulting in extremely defective eyes. There is significant suppression of the p53 overexpression eye phenotype by coexpression of either GMR-Dwee1 or GMR-Dmyt1, suggesting that these Cdk1 inhibitory kinases can negatively regulate p53-induced apoptosis. Since Cdk1 activity has been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition. Other reports relevant to this issue are somewhat contradictory, however. In human cell culture, Wee1 can inhibit granzyme B-induced apoptosis; furthermore, Wee1 appears to be downregulated through a p53-dependent mechanism, suggesting that p53 regulation of Wee1 might normally occur during this process. In contrast, Wee1 activity can actually promote apoptosis in a Xenopus oocyte extract system. Further studies are clearly needed to establish the physiological significance of any purported roles for Wee1 or Myt1 in regulating apoptosis, p53-dependent or otherwise (Price, 2002).
Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. The UV response by cells of the Drosophila retina have been explored. The retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells include morphology changes and apoptotic cell death; these defects can be completely accounted for by DNA damage. Cell death, but not morphological changes, is blocked by the caspase inhibitor p53. Utilizing genetic and microarray data, evidence is provided for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, Reaper has no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV-mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue (Jassim, 2003).
Previous work demonstrates that dominant-negative versions of p53 suppress ionizing radiation-induced cell death in larvae, and p53 is able to bind to an upstream P53 consensus binding site within the reaper locus. Surprisingly, flies lacking both functional copies of p53, due to an introduced stop codon, consistently display an enhanced retinal sensitivity to UV light and an increase in apoptotic cell death. It was not possilbe to assess the effect of overexpressing wild-type P53 on UV-mediated damage, since overexpression alone leads to extensive cell death in the retina. The protective effect of p53 is likely mediated by its transcriptional activity: retina-targeted overexpression of two different dominant-negative forms of p53 that lack DNA binding activity (p53259 and p53CT) leads to a similar increase in retinal sensitivity (Jassim, 2003).
Most previous studies have found that P53 acts to promote cell death, in contrast to the current observation. Potential explanations for the protective effect of p53 following irradiation include: (1) p53 is required for cell cycle arrest to provide the time required by cells to repair; or (2) p53 helps direct DNA damage repair. The first possibility is unlikely in the current experimental paradigm, as nearly all of the cells in a 24 h APF pupal retina have been post-mitotic for >14 h. Consistent with this view, BrdU staining of untreated or irradiated retina at 24 h APF indicates no cell divisions (other than the normal, few cell divisions that complete the interommatidial bristle organules). Therefore the photoreactivation repair system was utilized to determine whether loss of p53 compromises DNA repair (Jassim, 2003).
Light-mediated photorepair of genotypically p53-null mutants for 2 h results in a complete reversal of the retinal phenotype. Reducing the dose of photorepair to 30 min still fully restores wild-type retinae, but is less efficient at restoring p53 mutants. This suggests that p53 mutants are more sensitive to UV damage because DNA repair is impaired. Because full rescue of the retina requires intact photorepair and nucleotide excision repair, this result suggests that one of these pathways is deficient in a p53 mutant; alternatively, p53 could function at a downstream step, for example preventing Diap1 degradation. To explore further the potential connection between p53 and DNA repair, a single mutant copy of p53 and of the nucleotide excision repair mutant xpg/mus201D1 were placed in trans to determine whether they demonstrate a dominant genetic interaction. Irradiation of flies containing a single mutant copy of either of these genes by themselves yielded a phenotype similar to irradiated wild-type retinae. Combining a single copy of p53 and xpg/mus201D1 in trans results in a strong enhancement of the UV phenotype. A similar genetic interaction was also observed between p53 and mei-9. Taken together with the repair data, this suggests that p53 promotes DNA repair and cell viability, primarily by acting to enhance nucleotide excision repair (Jassim, 2003).
UV irradiation directs both morphology changes and cell death in the developing retina. Both classes of defects are fully rescued by reversal of DNA damage and exacerbated by removing DNA repair genes, indicating that DNA damage is the primary or sole source of the cells' response. It is presumed the widespread apoptosis upon irradiation is a direct response to DNA damage, although it cannot be rule out that death as a secondary response to cells' release from the apical surface ('anoikis'). In either case, cell death -- but not morphology defects -- is fully blocked by p53, indicating that apoptotic death is caspase dependent. Based on genetic evidence, Dronc, which is not fully inhibited by p53, may play a role in these morphology changes. If so, this activity must diverge before it reaches downstream p53-sensitive caspases (Jassim, 2003).
The major inhibitor of caspase activity in Drosophila is Diap1. Stability of Diap1 is the central point of cell death regulation in the developing retina and this is also true during UV irradiation in the retina. Genetic and microarray results further suggest that the retina requires Hid as a primary regulator of Diap1 stability during UV irradiation. Hid may represent the primary regulator of Diap1 during UV (versus ionizing) irradiation response by the fly. Alternatively, the retina utilizes Hid as its major RHG factor during its development, and this preference may simply extend to its response to UV; other tissues may exploit different Diap1 regulators that reflect their use during development (Jassim, 2003).
This close similarity between periods of developmental cell death and sensitivity to DNA damage is also seen in the developing mammalian CNS. The extreme sensitivity of the developing CNS to irradiation has limited the usefulness of radiation therapy as a treatment for pediatric CNS tumors. Although clear links have been made between the status of a cell in the cell cycle and its response to DNA damage, this study, performed on a developing post-mitotic nervous system, suggests a mechanism behind this radiosensitivity. Hid is both necessary and sufficient to confer radiation sensitivity to at least the interommatidial cells, mirroring its requirement during normal retinal development. Recent work in the mammalian nervous system indicates that the functionally related RGH family member Smac is capable of conferring cell death sensitivity to neurons. The factor has yet to be identified that confers a similar sensitivity to, for example, the photoreceptor neurons in irradiated Drosophila retinae (Jassim, 2003).
Reaper, also an inhibitor of Diap1 function, is thought to be of central importance during the larval wing disc's response to ionizing radiation; however, no evidence was found for its use in the retina. This suggests that p53 -- which is active in this experimental paradigm -- can act in a manner independent of any regulation of Reaper. p53 has been shown to be capable of targeting sequences upstream of reaper, and it is not known if p53 is required for the observed upregulation in hid expression in irradiated retinae; the results suggest its primary targets may be DNA repair enzymes (Jassim, 2003).
Remarkably, although all the cells in the pupal retina are sensitive to UV during the 18-25 h APF window, only the interommatidial cells are rescued when Hid activity is removed. Reaper has been ruled out as a regulator of the retina's UV response, leaving open the question regarding what factor(s) acts to destabilize Diap1 within the ommatidial core. An equally intriguing question is why the ommatidial cores demonstrate a window of UV competence identical to the interommatidial cells; no cell death occurs within this cell population at any stage of normal development (Jassim, 2003).
Mammalian P53 can arrest proliferation to permit repair or it can promote cell death, depending on the cellular context. In the fly retina a different result was observed: p53 is required to prevent cell death following UV irradiation, but its role is unrelated to cell cycle regulation as these retinal cells are post-mitotic. Nor is it likely to be linked directly to caspase stability, as in the case of Diap1. Instead, genetic and photorepair evidence is presented that p53 functions to promote DNA repair and viability. There is growing data that supports the idea that P53 can direct repair of DNA damage; the current work provides in situ support for this proposal. Recent work has reported that p53 mutant larvae are more sensitive to ionizing radiation; this effect was ascribed to a block in the death of severely damaged cells. An alternative interpretation is proposed: the cells of irradiated larvae can not repair DNA damage proficiently, leading to an increased likelihood of cell death as well as the observed increase in mutation rates. The connection between DNA repair, p53 and transcription of repair enzymes remains to be elucidated; future experiments comparing upregulated transcripts in wild-type and p53-null tissues should help address this issue (Jassim, 2003).
Together, these results identify two points of regulation during a retinal cell's response to UV irradiation. The early step involves pyrimidine dimers, and requires proper repair from factors that include XPG and p53. The second step involves activation of caspases and requires regulation of Diap1 stability; interommatidial cells utilize Hid at this step, and the remaining cells employ a different (unknown) regulator. One challenge will be to connect these two points of regulation. Multiple signaling pathways are suggested by the microarray data. These include EGFR/Ras1 signaling (a central regulator of Hid), JNK pathway signaling and TGFß pathway signaling. The role of these factors is not known, but understanding them may help to connect early and late events (Jassim, 2003).
Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. The Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene (alternative name: ATM). In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. It is proposed that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus, 2004).
In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence. This analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres (van Steensel, 1998; Takai, 2003; Wong, 2003). Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM (Oikemus, 2004).
Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae
Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. It is concluded that DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. It is proposed that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes (Jaklevic, 2004).
In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell cycle as needed. Central components of this checkpoint in mammals include four kinases: ATM, ATR, Chk1, and Chk2. Homologs of these exist in other eukaryotes and assume similar roles where examined. Human patients with ATM mutations, as well as their cells, show a dramatic sensitivity to killing by ionizing radiation. The importance of checkpoints in cellular survival to DNA damaging agents is presumed to be due to the role of checkpoints in cell cycle regulation. This is because mutants in the budding yeast gene rad9, the first checkpoint gene to be characterized, fail to arrest the cell cycle following damage and show increased radiation sensitivity; the latter phenotype is rescued by experimental induction of cell cycle delay. Consequently, cell cycle delay is thought to allow time for DNA repair and thereby ensure survival (Jaklevic, 2004 and references therein).
Components of the DNA damage checkpoint are found to activate DNA repair and to promote programmed cell death, which would cull cells with damaged DNA. For example, phosphorylation of NBS (a component of the Mre11 repair complex) by human ATM is of functional importance, while ATM knockout mice show a reduction in radiation-induced cell death in the CNS. Therefore, the essential role of checkpoints in conferring survival to genotoxins may be due to DNA repair and cell death responses in addition to or instead of cell cycle regulation. Furthermore, what is important for survival at the cellular level may not be so in a multicellular context. For instance, the failure to arrest the cell cycle by checkpoints may be detrimental to individual cells, but removal of these by cell death and replacement via organ homeostasis may make cell cycle regulation inconsequential for survival of multicellular organs (Jaklevic, 2004).
To address how DNA damage checkpoints operate in the context of multicellular organisms in vivo, the effect of ionizing radiation on Drosophila melanogaster is being studied. In Drosophila, mei-41 (ATR homolog) and grp (Chk1 homolog) are required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. Thus, mei-41 and grp play similar roles to their homologs in other systems. Moreover, mei-41 mutants are deficient in DNA repair. The role of mei-41 and grp in radiation-induced cell death has not been tested, but mei-41 is dispensable for cell death after enzymatic induction of DNA double-strand breaks (Jaklevic, 2004 and references therein).
Mutants in mei-41, grp, p53, and okra, a homolog of budding yeast RAD54 that functions in repair of DNA double-strand breaks (DSB) have been used to address the relative importance of cell cycle regulation, cell death, and DNA repair to the ability of a multicellular organism to survive ionizing radiation. The three responses are affected to different degrees in these mutants: wild-type larvae regulate S and M phases and repair DNA; grp mutants are unable to regulate the cell cycle but are able to repair DNA; okra mutants are able to regulate the cell cycle but are deficient in DNA repair; and mei-41 mutants are unable to regulate the cell cycle and are also deficient in DNA repair. All genotypes with the exception of p53 mutants are proficient in radiation-induced cell death, suggesting that mei-41 and grp do not contribute to this response. Under these conditions, it is found that while mei-41 and okra mutants are highly sensitive to killing by ionizing radiation, p53 mutants show reduced but significant survival and grp mutants resemble wild-type. These results suggest that cell death is neither sufficient nor absolutely necessary, DNA repair is essential, and optimal cell cycle regulation is dispensable for surviving ionizing radiation in Drosophila larvae (Jaklevic, 2004).
The effects of DNA damage by ionizing radiation on the maintenance and survival of Drosophila larvae was studied. Despite an extensive loss of cells to radiation-induced cell death, organ size and morphology are maintained remarkably well, and larvae survive to produce viable adults. Surprisingly, optimal cell cycle regulation by checkpoints is neither necessary (as in grp mutants) nor sufficient (as in okra mutants) to ensure organ homeostasis and organismal survival. p53-dependent cell death is also largely dispensable in this regard. Instead, DNA repair appears to be of paramount importance as might be expected (Jaklevic, 2004).
Genetic and microarray analysis have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member Eiger. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).
Previous studies have established that Drosophila p53 mediates X-irradiation-induced apoptosis and expression of rpr and skl. This study characterized the pathway that transduces the DNA damage signal to the apoptosis and cell cycle machineries. The results indicated that a number of genes in this pathway are largely specific to the cell cycle or apoptotic response. Both cellular assays and transcriptional profiling suggest that Drosophila p53 is required for IR-induced regulation of apoptosis but is not required for G2 arrest. In contrast, mei-41, mus304, and grps were required for cell cycle arrest, but not induction of apoptosis. The biochemical experiments suggested that mnk, which encodes a conserved damage-activated kinase, is required for phosphorylation of p53 following IR. All IR-induced transcription required both mnk and p53. The absence of genes that required mnk or p53 only was consistent with a linear signaling pathway of MNK activating p53, which acts as a global regulator of IR-induced transcription (Brodsky, 2004).
Although mnk and p53 mutant animals have similar defects in IR-induced transcription, mnk also acts in p53-independent pathways. In animals with mutations in double-strand break repair enzymes, unrepaired breaks formed during meiotic recombination activate an mnk-dependent checkpoint signal that disrupts oocyte patterning and nuclear morphology. Induction of the meiotic checkpoint differs from IR-induced transcription in at least two respects: (1) activation of mnk during meiosis requires mei-41, the Drosophila homolog of ATR; (2) p53 is not required for this damage response pathway. In a different damage response pathway, mnk, but not p53, is required for damage-induced inactivation of centrosomes. In this study, IR was found to induced a p53-independent decrease in RNA levels of at least 17 genes, including many developmental regulators. Although this observation could indicate a transcriptional repressor that is regulated by mnk, a model is favored in which an mnk-dependent cell cycle delay following IR has a secondary effect on the developmental induction of these genes. Together, these results and previous studies indicate that mnk regulates multiple signaling pathways in addition to p53-dependent induction of gene expression (Brodsky, 2004).
In mammals, Chk2 and other checkpoint kinases block Mdm2-mediated turnover and inhibition of p53. Several lines of evidence suggest that this regulatory mechanism is not conserved in Drosophila. (1) Simple sequence searches have not revealed an obvious Mdm2 homolog in the Drosophila genome. (2) The Drosophila p53 protein sequence does not contain a conserved binding site for Mdm2. (3) p53 protein levels were not dramatically altered following IR. p53 did exhibit an IR-induced change in gel mobility due to mnk-dependent phosphorylation. Thus, these results provide a clear example of damage-induced activation of p53 without changes in p53 protein levels (Brodsky, 2004).
Phosphorylation of p53 by Chk2 may represent an important step in the evolution of DNA damage responses in multicellular animals. Checkpoint pathways regulating cell cycle control and DNA repair have been highly conserved in eukaryotes, including unicellular organisms such as yeast. In contrast, induction of apoptosis during development or in response to cellular stress is confined to multicellular organisms. p53 phosphorylation by Chk2/MNK was found to be a conserved molecular link between DNA damage detection and the core apoptotic machinery in metazoans. Mdm2 adds an additional layer of complexity to the regulation of mammalian p53 compared to Drosophila p53. Regulation of p53 turnover by Mdm2 may provide mammalian cells with greater control of the levels or timing of p53-dependent transcription (Brodsky, 2004).
Microarray analysis was used to perform a comprehensive analysis of p53 targets following exposure to IR. The number of genes identified in these experiments was substantially smaller than the number of p53 targets identified in mammals. In part, this observation may reflect underlying differences in the damage response pathway in flies and mammals. For example, induction of p21 by mammalian p53 mediates G1 arrest following damage. IR-induced G1 arrest has not been described in Drosophila, consistent with the observation that the Drosophila p21/p27 homolog dacapo is not induced by IR. However, the smaller number of targets identified in Drosophila also reflects experimental differences. Expression changes induced by IR were examined during a defined window of embryonic development. In contrast, targets of mammalian p53 have been identified in many different cell types following different types of DNA damage or simply overexpression of p53. It is likely that additional targets of Drosophila p53 will be identified using other types of cellular stresses in different cell types or developmental stages. For example, UV irradiation of Drosophila embryos has been shown to induce Apaf1 through either E2F or mei-41, depending on the developmental stage (Brodsky, 2004).
The most prominent group of p53 targets identified in this study regulates two apoptotic pathways that are also targeted by mammalian p53. hid, rpr, and skl are part of a group of genes that induce apoptosis by blocking the caspase-inhibiting activity of IAP proteins. Recent experiments have confirmed that HTRA2, a functional homolog of these genes, is a target of mammalian p53. The Drosophila p53 target Eiger is a member of the TNF ligand family and can induce apoptosis when overexpressed. In mammals, FAS and DR5/Killer are p53 targets that can regulate apoptosis by acting as receptors for TNF ligand family members. Thus, two examples of mammalian and Drosophila p53 regulating common signaling pathways have been identified. Combined with the many other proapoptotic targets of mammalian p53, these results support the general hypothesis that multiple components of proapoptotic signaling pathways can be targets for transcriptional regulation following stresses such as DNA damage (Brodsky, 2004).
Although FAS and DR5/Killer are targets of mammalian p53 and act in the extrinsic apoptosis pathway, it is unclear what role they play in DNA damage-induced apoptosis. Analysis of deletion mutations in the Drosophila p53 target Eiger indicates that this gene is not required to initiate IR-induced apoptosis. This negative result is not due to redundancy with a related molecule, since Eiger is the only TNF-related gene in the Drosophila genome sequence. It is possible that the conserved activation of the TNF pathway by p53 is required for the induction of apoptosis under specific conditions not tested in these experiments. Alternatively, induction of Eiger may activate other cellular responses to DNA damage. Further characterization of Eiger function should reveal how cell-cell signaling contributes to survival or genomic stability following DNA damage in multicellular organisms (Brodsky, 2004).
Analysis of the remaining proapoptotic targets of p53 indicates that they are part of a dosage-sensitive mechanism that regulates IR-induced apoptosis. In contrast to Eiger, the proapoptotic genes in the genetic region containing hid, rpr, and skl are both sufficient and necessary for apoptosis. Animals with deletions that include genes in this region are defective in IR-induced apoptosis. Because these proapoptotic genes act, at least in part, by inhibiting a common target (IAP1/Thread), it has been proposed that they contribute to a rheostat-like mechanism in which the added activity of all proapoptotic proteins present must pass a threshold before a cell undergoes the irreversible decision to undergo programmed cell death. Following the observation that three of these genes are induced following DNA damage, the effect of lowering the dose of all proapoptotic genes in this region by half was tested. It was found that deletions in this region were haploinsufficient for IR-induced apoptosis. Dose sensitivity may represent an important feature of damage-induced apoptosis. Animals heterozygous for these deletions exhibit apparently normal morphology and fertility, suggesting that they are not haploinsufficient for developmentally regulated apoptosis. One possible interpretation of these results is that the apoptotic signal in many developmental contexts is well past the threshold required to commit to apoptosis, while the apoptotic signal following DNA damage is closer to that threshold. A lower apoptotic signal following DNA damage may allow cells to monitor DNA repair and block apoptosis if repair is successful. Haploinsufficiency of some tumor suppressor genes, including p53, has been proposed to contribute to cancer development. If stress-induced apoptosis in mammals is sensitive to the dose of p53 target genes, haploinsufficiency of these genes may also contribute to suppression of apoptosis, particularly in cells with extensive aneuploidy (Brodsky, 2004).
Analysis of animals heterozygous for deletions that removed a subset of genes has revealed that loss of one copy of hid is sufficient to reduce IR-induced apoptosis. A greater reduction was observed in larger deletions, indicating that additional genes in this region, likely rpr and skl, also contribute to IR-induced apoptosis. Previous analysis of animals heterozygous for two overlapping deletions [Df(3L)H99 and Df(3L)xr38] that remove both copies of rpr demonstrate reduced levels of IR-induced apoptosis. The current results indicate that part of that reduction is due to haploinsufficiency of hid and other genes in this region. Although the induction of hid RNA was lower than that observed for rpr and skl, hid may exhibit a greater absolute difference in RNA and protein levels following IR. Because null mutations in hid are embryonic lethal, the effects of completely removing hid function were not investigated. The dose-sensitive effects of hid suggest that total loss of hid would completely block IR-induced apoptosis. However, even in animals with normal levels of hid, increased levels of rpr and skl may be required to pass the proapoptotic signaling threshold required for a full DNA damage response. The Ras pathway and a micro-RNA in the bantam locus regulate hid expression. These and other pathways regulating hid may help determine which cells in the developing wing are most sensitive to DNA damage (Brodsky, 2004).
The other class of p53 targets identified in these experiments includes components of the Ku and Mre11 DNA repair complexes. Both of these complexes participate in repair of double-strand DNA breaks by nonhomologous end joining (NHEJ). Compared with homologous recombination, NHEJ is a potentially error-prone mechanism for DNA repair. Mutagenic DNA repair mechanisms are a prominent feature of the SOS response in bacteria that apparently promotes cell survival following DNA damage at the expense of genomic integrity. The ability of multicellular animals to eliminate damaged cells by apoptosis might suggest that low-fidelity mechanisms of DNA repair would not be favored following damage. However, the induction of NHEJ components by p53 suggests that mechanisms such as apoptosis or cell cycle arrest that are presumed to prevent mutations following DNA damage may compete with mechanisms that promote cell survival and prevent aneuploidy by error-prone DNA repair. The previous demonstration that an isoform of Ku86 is also a target of mammalian p53 suggests that this is an evolutionarily conserved response to DNA damage in metazoans that may modulate mutagenesis following DNA damage (Brodsky, 2004).
It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. This work explores if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, K9 and K14 H3 acetylation patterns were analyzed in situ after UV irradiation of wild-type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild-type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3-K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3-K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans (Rebollar, 2006).
The results presented here show that there are some similarities and differences between fly and human cells. For instance, in wild type third instar larvae there is an increase in the acetylation of K9 and K14 in the histone H3 in response to UV light irradiation. This observation is similar to previous reports in mammalian cells. Other similarity between both systems is that mutations in p53 affect the increase in the K9 acetylation after DNA damage, but not the acetylation of K14 in H3. In contrast, both human p53 and Dm p53 are required to maintain the basal histone H3 acetylation levels. In the case of human cells, the basal K9 acetylation level seems to be preferentially diminished when human p53 is mutated. In the case of Drosophila, K14 basal acetylation is dramatically reduced by the absence of Dm p53. Several scenarios may explain these differences. The first is that since not all p53 functions are conserved between human p53 and Dm p53 and the only region with significant identity between both proteins is the DNA binding domain, it is possible that the interactions with factors involved in histone modifications are different. Another possibility is that cancer cells deficient in human p53 could have other mutations. Usually they are aneuploid and therefore a mutated human p53 may interact with other mutated genes producing a phenotype on histone modifications. It is relevant to mention that in the Dm p53 null fly used in this study only Dm p53 is affected and therefore the effects that were observed in H3 acetylation are due only to this mutation (Rebollar, 2006).
The fact that a deficiency in Dm p53 produces a phenotype in basal H3 acetylation levels and in the increase of histone acetylation after UV light irradiation, indicates there is an important cross-talk between chromatin modifiers, Dm p53 and the nucleotide excision repair machinery in the fly. A similar network has been suggested to exist in human cells and therefore the fly becomes an interesting model to study the mechanisms that operate between DNA damage, p53 and chromatin dynamics. In contrast, the reduction in the basal levels of K14 acetylation in H3, does not have any effect in viability and fertility of the Dm p53 null flies. However, Dm p53 null organisms are very sensitive to UV light irradiation and a short life span. During development, the organism is exposed to genotoxic stress as consequence of the cell metabolism. Dm p53 may participate in the DNA repair during development and it is possible that the reduction in K14 basal acetylation in the Dmp53 null fly is product of a deficient DNA repair mechanism (Rebollar, 2006).
This work opens several interesting avenues that can be explored exploiting Drosophila genetics. For instance different mutant backgrounds in genes involved in genome stability, including Dm p53 can be used for the analyses of different histone modifications after DNA damage. It can also be interesting to find out if these histone modifications are different depending on the chromatin state. Also, since there are two pathways in nucleotide excision repair, transcription coupled repair and global genome repair it will be interesting to know if the increase in histone acetylation after DNA damage is higher in transcribed regions. However it is difficult to determine differences in histone modifications in specific sequences with this kind of analysis. These questions will be eventually answered by doing chromatin inmunoprecipitations and genetics (Rebollar, 2006).
Ionizing radiation (IR) can induce apoptosis via p53, which is the most commonly mutated gene in human cancers. Loss of p53, however, can render cancer cells refractory to therapeutic effects of IR. Alternate p53-independent pathways exist but are not as well understood as p53-dependent apoptosis. Studies of how IR induces p53-independent cell death could benefit from the existence of a genetically tractable model. In Drosophila, IR induces apoptosis in the imaginal discs of larvae, typically assayed at 4-6 hr after exposure to a LD50 dose. In mutants of Drosophila Chk2 or p53 homologs, apoptosis is severely diminished in these assays, leading to the widely held belief that IR-induced apoptosis depends on these genes. This study shows that IR-induced apoptosis still occurs in the imaginal discs of chk2 and p53 mutant larvae, albeit with a delay. This phenomenon is a true apoptotic response because it requires caspase activity and the chromosomal locus that encodes the pro-apoptotic genes reaper, hid, and grim. Chk2- and p53-independent apoptosis is IR dose-dependent and is therefore probably triggered by a DNA damage signal. It is concluded that Drosophila has Chk2- and p53-independent pathways to activate caspases and induce apoptosis in response to IR. This work establishes Drosophila as a model for p53-independent apoptosis, which is of potential therapeutic importance for inducing cell death in p53-deficient cancer cells (Wichmann, 2006).
The Drosophila homologs of Chk2 and p53 are required, not for induction of apoptosis, but for timely induction of apoptosis in response to irradiation. Radiation-induced cell death still occurs in chk2 and p53 mutants, albeit with a delay. Four lines of evidence support the idea that this delayed cell death is apoptosis rather than necrosis: (1) it is detected by staining with AO, which has been shown to stain apoptotic but not necrotic cells; (2) it accompanies activation of caspases, a hallmark of apoptosis but not necrosis; (3) it requires caspase activity, which is required for apoptosis but not necrosis, and (4) it requires the chromosomal locus encoding the proapoptosis genes rpr, hid, and grim, whose protein products are required to inhibit DIAP1 and activate caspases. These results indicate that there is a Chk2-/p53-independent pathway that commits damaged cells to apoptosis and utilizes many of the same downstream components as the Chk2-/p53-dependent apoptosis pathway (Wichmann, 2006).
Two lines of evidence support the idea that DNA damage is the signal that induces Chk2-/p53-independent apoptosis after exposure to ionizing radiation. First, the amount of Chk2-/p53-independent apoptosis appears to increase with IR dose. This dose dependence suggests that the amount of DNA damage is what induces Chk2-/p53-independent apoptosis but does not rule out the contribution of other damages that result from IR. Second, higher levels of Chk2-/p53-independent apoptosis are observed when the ability to repair DNA is compromised, as in mei-41, p53 double mutants. Collectively, these data suggest that DNA damage caused by x-rays induces Chk2-/p53-independent apoptosis (Wichmann, 2006).
IR-induced apoptosis in chk2 and p53 mutants shows a temporal delay. IR-induced apoptosis is also delayed in H99 heterozygotes, possibly because H99 heterozygotes contain half the gene dose of the proapoptotic Smac/Diablo orthologs and it may take longer for the proapoptotic gene products to accumulate to the point of an apoptosis-stimulating threshold. IR induced increase in the transcripts of rpr and hid, two of the H99-encoded genes, still occurred in chk2 (rpr and hid) and p53 (hid) mutants, but to lower levels (for rpr) and after a delay. Therefore, apoptosis may be delayed in chk2 and p53 mutants because proapoptotic gene products take longer to accumulate to a threshold in the absence of Chk2 or p53 regulation. The data showing that IR-induced apoptosis is further delayed in a chk2, H99/+ double mutant, compared with a chk2 single mutant, support this claim. Furthermore, the results suggest the existence of at least another signaling pathway that does not operate through Chk2 or p53, but nonetheless links the same signal (DNA damage) to a similar outcome (accumulation of H99-encoded gene products) (Wichmann, 2006).
RT-PCR experiments revealed interesting differences in the identity and onset of induction of proapoptotic genes in chk2 and p53 mutants. rpr and hid are induced at 4 hr after irradiation in chk2 mutants, whereas hid and skl are induced between 12 and 18 hr after irradiation in p53 mutants. The basis for these differences is not understood. More detailed time courses as well as deletion analysis of the H99 locus to determine the contribution of each proapoptotic gene to Chk2-/p53-independent apoptosis needs to be performed to address these issues (Wichmann, 2006).
The data presented in this study establish Drosophila as a model for studying p53-independent apoptosis. p53 is the most commonly mutated gene in human cancers. Loss of p53 poses an immense clinical problem because p53-deficient cancer cells no longer stimulate p53-dependent apoptosis in response to radiation or genotoxic chemotherapy drugs. In this scenario, p53-independent apoptotic pathways become key for inducing cancerous cells to die because they provide potential therapeutic targets. In mammals, a p53-independent apoptosis pathway that is mediated by p73, another member of the p53 family, has been identified. In Drosophila, Dmp53 is the only known p53 family member. Therefore, the p53-independent apoptosis that was identified and characterized in this article is likely to represent a previously unknown process. An important goal in the future will be to dissect the Chk2-/p53-independent pathway that links DNA damage to the proapoptotic genes of the H99 locus (Wichmann, 2006).
Several candidates were tested and eliminated as regulators of Chk2-/p53-independent cell death. Mei-41 (ATR) is not required for Chk2-/p53-independent cell death because mei-41, p53 double mutants actually exhibit more cell death than p53 alone. Recent work showed that ectopic induction of eiger, a TNF ligand homolog, can induce apoptosis in Drosophila. Chk2-/p53-independent cell death still occurs in p53, eiger double mutants, suggesting that the TNF pathway is not involved in the induction of cell death characterized in this study. Work in mammalian cells showed that overexpression of c-Myc can induce p53-independent apoptosis. Chk2-/p53-independent apoptosis still occurs in Dmyc, p53 double mutants, indicating that Dmyc is not required for this response (Wichmann, 2006).
A classical genetic screen may identify components of the Chk2-/p53-independent apoptosis pathway, as well as testing more candidates, such as the transcription factor de2f1, grapes (DmChk1), DmATM, and genes required for autophagy. Autophagic cell death, in which a cell lyses itself, occurs during Drosophila metamorphosis to lyse polyploid tissues such as the salivary glands and the fat body and provide nutrients for diploid cells of the imaginal discs; autophagy has been described in larvae only in the polyploid cells and only in response to starvation. Nonetheless, autophagy shares characteristics with apoptosis, including being detectable by AO staining and being dependent on caspases and the H99 locus, and for this reason remains a formal possibility (Wichmann, 2006).
In conclusion, studies have shown that IR-induced apoptosis in two key models for apoptosis, C. elegans and Drosophila, depends on p53. This study has provided evidence that, contrary to the accepted view, Chk2 and p53 are not required for radiation-induced cell death in Drosophila. Furthermore, normal timing of apoptosis that depends on Chk2 and p53 is also not required for ensuring survival after irradiation. Radiation-induced cell death that is independent of Chk2 and p53 depends on radiation dose, has characteristics of apoptosis and is likely to rely on a novel mechanism(s) because no other members of the p53-family are known in Drosophila. This work is the first to establish Drosophila as a model for p53-independent apoptosis. Identification of genes required for Chk2-/p53-independent cell death in Drosophila is of potential therapeutic value because protein products of their human homologs may represent novel targets that can be activated clinically to eliminate p53-deficient cancer cells (Wichmann, 2006).
Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size. Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki). This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although some of the events downstream of the Hpo kinase are understood, its mode of activation remains mysterious. This study shows that Hpo can be activated by Ionizing Radiations (IR) in a p53-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or p53 ectopic expression (Colombani, 2006).
Hpo is the ortholog of the Mammalian Sterile Twenty-like (MST) kinases, which belong to the Ste20 family of kinases. MSTs are highly similar to Hippo (Hpo) in their N-terminal serine/threonine kinase domains as well as in the C-terminal Salvador (Sav) binding region (or SARAH domain). MST1 functions both downstream and upstream of caspases to promote chromatin condensation and nuclear fragmentation, as well as activation of the JNK (Jun N-terminal kinase) and p38 pathways. Like most Ste20 family kinases, MST1/2 auto- or trans-phosphorylates at a number of residues. One of these, T183 in the activation loop, has been shown to be required for full kinase activity and has been used as a useful marker of MST1 activation in cultured cells. In order to study events upstream of Hpo, antibodies that have previously been shown to recognize MST1/2 phosphorylated on T183 were tested for their ability to cross-react with Hpo on the equivalent residue (T195). Interestingly, it was found antibodies that specifically recognized the phosphorylated form of Hpo upon treatment with staurosporine (sts), a known activator of MST1/2. This signal is abolished by RNAi-mediated Hpo depletion and disappears upon phosphatase treatment. Moreover, the antibodies recognize overexpressed tagged Hpo before immunoprecipitation. By contrast, the antibodies did not recognize a nonphosphorylable (T195A) Hpo mutant protein. Myc-tagged wild-type and T195A Hpo were immunoprecipitated and their auto-kinase activity and their activity on an exogenous substrate (Histone H2B, not shown) were measured in both the presence and absence of sts. As has been observed for MST1/2, overexpression of Hpo leads to its activation, presumably via trans-phosphorylation. Sts treatment potently stimulates Hpo kinase activity (5-fold). By contrast, the T195A mutant is severely compromised both in its unstimulated and stimulated activities, suggesting that T195 phosphorylation is crucial to normal Hpo kinase activity. Thus, these phospho-specific antibodies can be used as readouts of Hpo pathway activity (Colombani, 2006).
In the course of testing stimuli that would activate Hpo in tissue culture, it was observed that γ-irradiation potently and rapidly induced Hpo activation. The fly p53 ortholog has been shown to mediate cell death upon ionizing radiation (IR)-induced DNA damage. Although the pro-apoptotic genes reaper (rpr), hid, and sickle are p53 transcriptional targets, removal of these three proteins via chromosomal deficiencies only partially suppresses the cell-death effects of IR in embryos, suggesting that additional death signals act downstream of p53. This prompted an examination of whether the Hpo pathway could function downstream of Drosophila p53 in the response to IR (Colombani, 2006).
Initially, wing imaginal discs (the larval precursors of the adult wing) containing clones of hpo, wts, and sav mutant cells were treated with γ-rays and cell death was examined by staining for activated caspases. Interestingly, although caspase activation was efficiently induced in wild-type tissue or control discs, cell death was severely reduced in hpo, wts, and sav mutant clones and in p53 mutant discs. Quantification of the caspase staining indicated that apoptosis was reduced by 2- to 3-fold in hpo, wts, and sav clones compared to wild-type tissue. This was also true in eye imaginal discs (Colombani, 2006).
Overexpression of p53 in the posterior portion of late larval eye imaginal dics was sufficient to induce apoptosis. Loss of function of hpo, wts, and sav decreased cell death in this context, although the effect was less pronounced in sav clones, perhaps as a reflection of the weaker phenotype of the sav mutants. This suggests that the Hpo complex may function as an effector in the p53-mediated response to IR. To test this hypothesis, Hpo activation was measured in cultured cells treated with γ-rays in the presence or absence of dsRNAs directed against p53. Excitingly, depletion of Dmp53 markedly reduced Hpo phosphorylation by IR. The residual level of Hpo activation observed in p53-depleted cells can probably be explained by the fact that the dsRNA-mediated p53 depletion was never complete, as measured by RT-PCR. To check that the increased Hpo phosphorylation observed corresponded to increased activity, IP kinase assays were performed on cells expressing ectopic Hpo. It was observed that IR treatment potently induced Hpo kinase activity. Furthermore, p53 expression alone, in the absence of IR, was sufficient to activate Hpo phosphorylation. Finally, it was determined whether p53-dependent Hpo activation could be observed in vivo by taking advantage of the fact that p53 is not required for viability. Dissected ovaries from p53 mutant and wild-type flies were treated with γ-rays and examin Hpo activity was examined by Western blotting. Interestingly, although γ-rays potently activated Hpo in wild-type flies, this response was abolished in p53 mutant animals. p53 expression in the ovaries was able to induce apoptosis, ovary degeneration, and total loss of fecundity. It is concluded that Hpo is activated as part of a p53-dependent DNA-damage response both in cultured cells and in vivo (Colombani, 2006).
MST1 and 2 are known to be activated by caspase 3 through proteolytic cleavage. Therefore, the possibility exists that the Hpo activation observed is merely a by-product of Rpr-dependent caspase activation. Several lines of evidence suggest that this is not the case. First, reaper overexpression in S2 cells did not increase Hpo activity. Second, depletion of DIAP1 from cultured cells, which potently induces caspase activation, fails to trigger detectable Hpo activation. Third, the phospho-Hpo signal detected corresponds to full-length Hpo rather than a caspase-cleaved fragment. In fact, the caspase cleavage site present in the MSTs is not thought to be conserved in Hpo, and no evidence was seen of Hpo cleavage upon apoptotic stimuli. Fourth, treatment of cultured cells with caspase inhibitors did not affect Hpo activation by IR. Thus, it is unlikely that Hpo is stimulated via p53-dependent caspase activation (Colombani, 2006).
The time course of Hpo activation by IR (2–3 hr for maximal activation) suggests that transcription may be required for this response. Indeed, treatment of cells with IR in the presence of the transcription inhibitor Actinomycin D (ActD) abolishes Hpo activation. Thus, Hpo activation in response to IR requires new gene transcription, which could be mediated, at least in part, by p53. Hpo activity is induced by p53 expression, but Hpo protein itself does not appear to be a target of p53 because Hpo levels are not detectably upregulated when p53 is expressed in the posterior portion of the eye imaginal disc or in Dmp53-expressing clones in the wing disc. Future studies will be aimed at determining the exact mechanism through which Dmp53 promotes Hpo activation (Colombani, 2006).
This study has demonstrate by genetic and biochemical approaches not only that the Hpo pathway is required for the full apoptotic response induced by γ-ray irradiation but also that DNA damage triggers Hpo kinase activity in a p53-dependent manner both in vivo and in vitro. The apoptosis induced by p53 overexpression is strongly affected in hpo, wts, and sav mutant clones and p53 does not modulate Hpo levels. This study constitutes the first description of an upstream activating signal of the Hpo complex in vivo and during organism development (Colombani, 2006).
It is noted that the blockage of p53-induced apoptosis is not complete in hpo clones; this incomplete blockage likely reflects the role of other pro-apoptotic proteins, such as Reaper, Hid, and Sickle, in this process. Thus, it is proposed that, after exposure to ionizing radiations, the ATM, Chk2, p53 signaling pathway is activated and induces apoptosis by targeting expression of pro-apoptotic effectors such as Reaper, as well as by activating the Hpo pathway. This cell-death response to irradiation requires the caspase DRONC and leads to upregulation of JNK activity in a p53-dependent manner. Because Hpo has been shown to induce JNK activation when overexpressed in vivo, it will be interesting to determine whether Hpo is necessary for IR-induced JNK activation (Colombani, 2006).
Several reports have suggested that the mammalian homologs of members of the Hpo pathway might behave as tumor suppressors in humans. In addition, mice lacking the Wts homolog mLats1 are more sensitive to tumor-inducing agents. The current data suggest that one effect of mutations in Hpo-pathway members may be to protect these cells from DNA-damage-induced apoptosis and thus promote tumor progression and the accumulation of additional mutations. Further work on the Hpo pathway should further understanding of the DNA-damage response and its role in the transformation process (Colombani, 2006).
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date revised: 15 July 2008
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