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• W4248537801 abstract "Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved “Chk1-suppressed” pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved “Chk1-suppressed” pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. The stress-inducible p53 protein acts as a central signal transduction node in the apoptotic response to DNA damage, mainly through its ability to transactivate intrinsic (mitochondrial) and extrinsic (death-receptor) pathway genes (Vousden and Lu, 2002Vousden K.H. Lu X. Live or let die: the cell's response to p53.Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2597) Google Scholar). However, ample evidence supports the existence of p53-independent apoptotic responses to DNA damage. In Drosophila and mouse p53 null embryos, for example, several cell types undergo apoptosis in response to irradiation (IR), but with slower kinetics than p53+/+ cells (Frenkel et al., 1999Frenkel J. Sherman D. Fein A. Schwartz D. Almog N. Kapon A. Goldfinger N. Rotter V. Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.Oncogene. 1999; 18: 2901-2907Crossref PubMed Scopus (38) Google Scholar, Wichmann et al., 2006Wichmann A. Jaklevic B. Su T.T. Ionizing radiation induces caspase-dependent but Chk2- and p53-independent cell death in Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 2006; 103: 9952-9957Crossref PubMed Scopus (64) Google Scholar). Candidate p53-independent apoptotic pathways have surfaced from in vitro studies. ATM/ATR-activated ABL, Chk1, and Chk2 can upregulate p73 protein levels in genotoxically challenged p53-deficient cells, restoring transactivation of PUMA and other proapoptotic p53 targets (Gong et al., 1999Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage.Nature. 1999; 399: 806-809Crossref PubMed Scopus (809) Google Scholar, Roos and Kaina, 2006Roos W.P. Kaina B. DNA damage-induced cell death by apoptosis.Trends Mol. Med. 2006; 12: 440-450Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, Urist et al., 2004Urist M. Tanaka T. Poyurovsky M.V. Prives C. p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2.Genes Dev. 2004; 18: 3041-3054Crossref PubMed Scopus (180) Google Scholar, Yuan et al., 1999Yuan Z.M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage.Nature. 1999; 399: 814-817Crossref PubMed Scopus (524) Google Scholar). p53-independent coupling of DNA damage to mitochondria can also occur through translocation of the nuclear orphan protein Nur77 into the cytosol, activation of nuclear and/or cytosolic caspase-2, or de novo ceramide synthesis by mitochondrial ceramide synthase, all converging on caspase-3 activation (Kolesnick and Fuks, 2003Kolesnick R. Fuks Z. Radiation and ceramide-induced apoptosis.Oncogene. 2003; 22: 5897-5906Crossref PubMed Scopus (373) Google Scholar, Li et al., 2000Li H. Kolluri S.K. Gu J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G. Lu J. Lin F. et al.Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.Science. 2000; 289: 1159-1164Crossref PubMed Scopus (562) Google Scholar, Lin et al., 2004Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3.Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, Zhivotovsky and Orrenius, 2005Zhivotovsky B. Orrenius S. Caspase-2 function in response to DNA damage.Biochem. Biophys. Res. Commun. 2005; 331: 859-867Crossref PubMed Scopus (167) Google Scholar). Other p53-independent processes, involving MAPKs (e.g., SAPK/JNKs, p38) and the transcription factors E2F1, NF-κB, and FOXO1 couple DNA damage to caspase-3 activation by upregulating extrinsic pathway genes including CASP8, whose product activates caspase-3 in a mitochondria-dependent (Bcl-2-inhibitable) or -independent manner (Afshar et al., 2006Afshar G. Jelluma N. Yang X. Basila D. Arvold N.D. Karlsson A. Yount G.L. Dansen T.B. Koller E. Haas-Kogan D.A. Radiation-induced caspase-8 mediates p53-independent apoptosis in glioma cells.Cancer Res. 2006; 66: 4223-4232Crossref PubMed Scopus (43) Google Scholar, Huang et al., 2006Huang H. Regan K.M. Lou Z. Chen J. Tindall D.J. CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage.Science. 2006; 314: 294-297Crossref PubMed Scopus (246) Google Scholar, Kasibhatla et al., 1998Kasibhatla S. Brunner T. Genestier L. Echeverri F. Mahboubi A. Green D.R. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1.Mol. Cell. 1998; 1: 543-551Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, Yount et al., 2001Yount G.L. Afshar G. Ries S. Korn M. Shalev N. Basila D. McCormick F. Haas-Kogan D.A. Transcriptional activation of TRADD mediates p53-independent radiation-induced apoptosis of glioma cells.Oncogene. 2001; 20: 2826-2835Crossref PubMed Scopus (26) Google Scholar). Whether the p53-independent pathways identified in vitro operate in vivo remains an active field of investigation. Radio/chemoresistant p53 mutant human cancer cell lines can be induced to die after genotoxic stress by pharmacologic or RNAi targeting of DNA damage-response (DDR) kinases involved in intra-S and/or G2/M checkpoint control, including ATM, ATR, Chk1, Chk2, Polo-like kinases (Plks) (reviewed in Castedo et al., 2004aCastedo M. Perfettini J.L. Roumier T. Andreau K. Medema R. Kroemer G. Cell death by mitotic catastrophe: a molecular definition.Oncogene. 2004; 23: 2825-2837Crossref PubMed Scopus (936) Google Scholar), and most recently, the p38/MAPK-activated kinase MAPKAPK2 (MK-2) (Reinhardt et al., 2007Reinhardt H.C. Aslanian A.S. Lees J.A. Yaffe M.B. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage.Cancer Cell. 2007; 11: 175-189Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Such treatments might spare cells endowed with wild-type p53, presumably because their intact G1 checkpoint enables them to repair and thus survive DNA damage (Zhou and Bartek, 2004Zhou B.B. Bartek J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection.Nat. Rev. Cancer. 2004; 4: 216-225Crossref PubMed Scopus (342) Google Scholar). Although the sensitization of—and selectivity for—p53 mutant cells is at the root of anticancer strategies that target DDR kinases, none of these concepts have been rigorously tested in an animal model, and the underlying cell death mechanism is unclear. To accelerate the discovery of physiologic p53-independent DDRs, we generated p53 mutant zebrafish lines for use in whole organism-based modifier genetic screens (Berghmans et al., 2005Berghmans S. Murphey R.D. Wienholds E. Neuberg D. Kutok J.L. Fletcher C.D. Morris J.P. Liu T.X. Schulte-Merker S. Kanki J.P. et al.tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.Proc. Natl. Acad. Sci. USA. 2005; 102: 407-412Crossref PubMed Scopus (431) Google Scholar). Zebrafish faithfully recapitulate mammalian intrinsic and extrinsic apoptotic signaling (reviewed in Pyati et al., 2007Pyati U.J. Look A.T. Hammerschmidt M. Zebrafish as a powerful vertebrate model system for in vivo studies of cell death.Semin. Cancer Biol. 2007; 17: 154-165Crossref PubMed Scopus (83) Google Scholar). The zebrafish p53M214K allele (or p53e7, for mutation in exon 7) affects a conserved amino acid residue within a region of the DNA-binding domain corresponding to a mutational hotspot in human cancer, producing a transactivation-dead p53 variant. Homozygosity for p53e7 recapitulates key traits associated with p53 deficiency in mammalian systems, including a strong tumor-prone phenotype, lack of G1-checkpoint function, and widespread cellular radioresistance (Berghmans et al., 2005Berghmans S. Murphey R.D. Wienholds E. Neuberg D. Kutok J.L. Fletcher C.D. Morris J.P. Liu T.X. Schulte-Merker S. Kanki J.P. et al.tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.Proc. Natl. Acad. Sci. USA. 2005; 102: 407-412Crossref PubMed Scopus (431) Google Scholar). Here we identify chk1 as a gene whose loss restores IR-induced apoptosis in live p53 mutant zebrafish embryos, and then use in vivo epistasis analyses to dissect the underlying mechanism. Unlike previously identified p53-independent apoptotic pathways, which restore caspase-3 activation downstream of defective p53, Chk1 depletion activates an ATM/ATR-caspase-2 axis that bypasses the mitochondrial and death-receptor pathways. We show that this “Chk1-suppressed” pathway can be triggered in p53-deficient or BCL2-overexpressing human tumor cells, providing a mechanistic rationale for the use of Chk1 inhibitors in cancer therapy. p53e7/e7 mutant zebrafish embryos are refractory to DNA damage-induced cell death, as demonstrated by a nearly complete lack of acridine orange (AO) labeling in the brain and spinal chord of live embryos examined 7.5 hr after whole-body IR delivered at 18 hr postfertilization (hpf) (Figures 1A and 2A). We used morpholino antisense oligonucleotides (MOs) to knock down eight zebrafish S- and G2-checkpoint kinases and two nonkinase checkpoint regulators (p21waf1/cip1 and smc1) in p53e7/e7 mutant embryos. We assessed the ability of each knockdown to restore cell death (AO reactivity) at 7.5 hr post-IR (hpIR). Single knockdowns of all genes tested, excluding plk2, plk3, and aurkb, radiosensitized p53 mutants with variable efficiency (Figures 1B and 1C). Whereas atm, atr, smg-1/atx, and chk2 deficiencies restored only minor AO reactivity averaging 1%–5% of the p53+/+ response, chk1 knockdown resulted in a staining pattern that closely resembled wild-type (87.7% of the p53+/+ response, p < 0.0001; see also Figures 2A and 2B). Enhanced IR-induced cytotoxicity resulted specifically from chk1 knockdown because (1) injections of a chk1 mismatch MO failed to radiosensitize p53 mutants (Figure 2A, bottom panels); (2) the chk1 MO resulted in a robust reduction of the endogenous Chk1 protein pool, correlating with impaired Chk1 activity (Figure 2C); and (3) a specific inhibitor of human Chk1, but not inhibitors of ATM or Chk2, phenocopied the effects of chk1 MO (see Figure 7). As would be expected from Chk1 loss, p53e7/e7;chk1MO embryos lacked the IR-induced G2/M checkpoint (Figures S1A–S1D). chk1 MO also fully radiosensitized p53e6 (p53N168K) homozygotes (Berghmans et al., 2005Berghmans S. Murphey R.D. Wienholds E. Neuberg D. Kutok J.L. Fletcher C.D. Morris J.P. Liu T.X. Schulte-Merker S. Kanki J.P. et al.tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.Proc. Natl. Acad. Sci. USA. 2005; 102: 407-412Crossref PubMed Scopus (431) Google Scholar) and p53 morphants (Langheinrich et al., 2002Langheinrich U. Hennen E. Stott G. Vacun G. Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling.Curr. Biol. 2002; 12: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) lacking p53 protein (Figure 2E), including in mesodermal derivatives (Figures 2F–2H). Together, these results provide in vivo evidence that Chk1 depletion is sufficient to restore IR sensitivity to p53 mutant cells. Chk1 is essential for fly and mouse development, with homozygous null mutants succumbing to major cell cycle defects (Fogarty et al., 1997Fogarty P. Campbell S.D. Abu-Shumays R. Phalle B.S. Yu K.R. Uy G.L. Goldberg M.L. Sullivan W. The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.Curr. Biol. 1997; 7: 418-426Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, Liu et al., 2000Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. et al.Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (185) Google Scholar). We therefore tested whether the cytotoxicity of chk1 knockdown in zebrafish p53 mutants was strictly IR dependent. Indeed, chk1 depletion had no apparent effect on normal zebrafish development and viability, in either the p53+/+ or p53e7/e7 background (Figures 2A and 2D; compare bars 1 and 2 in Figure 2B). Western blots performed with an antizebrafish Chk1 antibody revealed a substantial knockdown of the protein (Figure 2C). Yet chk1 morphants harbored residual levels of Chk1 activity, as shown by weak but persistent levels of phosphorylated Cdc2 (Figure 2C). These results demonstrate that transient depletion, as opposed to persistent total loss (Liu et al., 2000Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. et al.Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (185) Google Scholar), of Chk1 function, is tolerable by vertebrate cells in vivo and compatible with long-term organismal viability. Crucially, however (as already shown above), such transient downregulation is sufficient to restore the IR-induced cell-death response in p53 mutants (Figures 1B, 1C, 2A, and 2B). Chk1 knockdown might restore a wild-type response to IR (that is, classical intrinsic apoptosis) (Kratz et al., 2006Kratz E. Eimon P.M. Mukhyala K. Stern H. Zha J. Strasser A. Hart R. Ashkenazi A. Functional characterization of the Bcl-2 gene family in the zebrafish.Cell Death Differ. 2006; 13: 1631-1640Crossref PubMed Scopus (96) Google Scholar) or trigger a different cell-death program in p53 mutants. To distinguish between these possibilities, we first analyzed two hallmarks of apoptosis: TUNEL-positive DNA fragmentation and cleaved caspase-3 (as well as electron micrographs) in embryos fixed at 7.5 hpIR. AO labeling of irradiated p53e7/e7;chk1MO embryos (Figures 1C and 2A) correlated with high levels of TUNEL labeling throughout the CNS, similar to findings in irradiated p53+/+ embryos (Figure 3A). Multiple cells in the irradiated CNS of p53+/+ and Chk1-depleted p53e7/e7 embryos also showed similar ultrastructural manifestations of apoptosis (e.g., chromatin compaction/segregation and cytoplasmic condensation; Figures 3C and S2). Surprisingly, however, while irradiated p53+/+ embryos exhibited strong immunostaining for active caspase-3, irradiated p53e7/e7;chk1MO embryos did not and showed no increase in active caspase-3 levels compared to p53 single mutants, which were devoid of both TUNEL and active caspase-3 (Figures 3A and 3B). Thus, the p53-independent cell death-inducing DDR triggered by Chk1 depletion is a caspase-3-independent apoptotic pathway (see also Figure S2F). To determine the cell autonomy of the Chk1-antagonized pathway, we generated genetic chimeras (Figures 3D and S3). While p53e7/e7;chk1MO cells grafted into p53e7/e7 hosts often stained TUNEL-positive after IR (39%, n = 102), neighboring host cells did not (Figure 3E, upper panels). In the reciprocal experiment, p53e7/e7 cells transplanted into p53e7/e7;chk1MO hosts remained TUNEL negative within an otherwise TUNEL-positive environment (Figure 3E, lower panels). Therefore, IR-induced TUNEL reactivity of transplanted cells strictly depends on Chk1 dosage, occurs irrespective of the cellular environment, and has very little (if any) influence on neighboring cells. The Chk1-suppressed apoptotic DDR pathway thus functions in a cell-autonomous manner. To molecularly characterize the newly identified apoptotic pathway, we capitalized on the unique advantages of zebrafish embryos for in vivo epistasis analyses. Specifically, we knocked down or forced the expression of candidate pathway contributors in p53e7/e7;chk1MO embryos and assessed the effects on IR-induced cell death using the AO assay. atm and atr single knockdowns severely impaired chk1 knockdown-mediated radiosensitization of zebrafish p53 mutants, indicating that ATM and ATR are nonredundantly required to activate the pathway after DNA damage (Figures 4A and 4B; 60% and 90% reductions in cell death levels, respectively). In contrast, single or combined knockdowns of p63 and/or p73 led to (at best) a ∼30% reduction in AO staining compared to control p53e7/e7;chk1MO embryos (Figure 4A). This attenuation was reminiscent of the effects of chk2 knockdown (Figure S4A, compare bars 3 and 5) and may reflect a role for p53-independent Chk2-p63/p73 apoptotic pathways (Bernassola et al., 2005Bernassola F. Oberst A. Melino G. Pandolfi P.P. The promyelocytic leukaemia protein tumour suppressor functions as a transcriptional regulator of p63.Oncogene. 2005; 24: 6982-6986Crossref PubMed Scopus (38) Google Scholar, Urist et al., 2004Urist M. Tanaka T. Poyurovsky M.V. Prives C. p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2.Genes Dev. 2004; 18: 3041-3054Crossref PubMed Scopus (180) Google Scholar) in a subset of cell deaths in irradiated p53e7/e7;chk1MO embryos. It is unlikely that these effects result from weaker MO efficiencies, because the chk2, p63, and p73 MOs lead to stronger gene knockdowns than the atm and atr MOs (Figures 2C and S5A–S5C; also see Rentzsch et al., 2003Rentzsch F. Kramer C. Hammerschmidt M. Specific and conserved roles of TAp73 during zebrafish development.Gene. 2003; 323: 19-30Crossref PubMed Scopus (28) Google Scholar). The inability of Chk2, p63, and p73 to account for the majority of cell death events in irradiated p53e7/e7;chk1MO embryos implies that ATM and ATR operate predominantly within a novel apoptotic pathway, which we have designated “Chk1-suppressed pathway” (CS in Figure 7D). To test whether the mitochondrial apoptotic axis contributes to the Chk1-suppressed pathway, we first knocked down the proapoptotic BH3-only family member Puma. puma depletion did not significantly affect AO labeling of irradiated p53e7/e7;chk1MO embryos (Figures 4A and 4B) at a puma MO concentration that is otherwise sufficient to completely block IR-induced apoptosis in p53+/+ zebrafish embryos (Figure S6) (Kratz et al., 2006Kratz E. Eimon P.M. Mukhyala K. Stern H. Zha J. Strasser A. Hart R. Ashkenazi A. Functional characterization of the Bcl-2 gene family in the zebrafish.Cell Death Differ. 2006; 13: 1631-1640Crossref PubMed Scopus (96) Google Scholar). Similarly, a dose of bcl-xl mRNA that completely blocked cell death 7.5 hpIR in wild-type embryos failed to affect the AO reactivity of irradiated p53e7/e7;chk1MO embryos (Figures 4D and 4E; p53+/+ + bcl-xl, 0.035% of the mean p53+/+ response; p53e7/e7;chk1MO + bcl-xl, ∼95% of the mean p53e7/e7;chk1MO response). casp9 knockdown also lacked an effect (Figures 4A, 4B, and S5E). Thus, two major regulators of mitochondrial membrane permeabilization (Puma and Bcl-xL), as well as the main initiator and executioner caspases acting downstream of mitochondria (caspase-9 and caspase-3, see Figure 3), are dispensable for the Chk1-suppressed apoptotic pathway. The death-receptor axis bypasses the requirement for mitochondria and caspase-9, suggesting that it could contribute to the Chk1-suppressed pathway. In addition, a link between Chk1 loss and caspase-8 activation has recently been observed (Xiao et al., 2005Xiao Z. Xue J. Sowin T.J. Rosenberg S.H. Zhang H. A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation.Oncogene. 2005; 24: 1403-1411Crossref PubMed Scopus (80) Google Scholar). Even so, the death-receptor pathway converges on caspase-3 activation via caspase-8 (Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (6002) Google Scholar). This caspase-3 recruitment contrasts with the caspase-3 independence of the pathway we identified, which, together with the established cell autonomy of the new pathway (Figure 3E), argues against a role for DNA damage-induced extrinsic signaling downstream of chk1 depletion. Indeed, the AO reactivity of p53e7/e7;chk1MO;casp8MO zebrafish embryos did not differ from that of p53e7/e7;chk1MO specimens (Figures 4A and S5D). Blocking death-receptor signaling with a fadd (Fas Associated protein with Death Domain) MO (Eimon et al., 2006Eimon P.M. Kratz E. Varfolomeev E. Hymowitz S.G. Stern H. Zha J. Ashkenazi A. Delineation of the cell-extrinsic apoptosis pathway in the zebrafish.Cell Death Differ. 2006; 13: 1619-1630Crossref PubMed Scopus (86) Google Scholar) also failed to affect AO staining (Figure 4A). Thus, extrinsic signaling—like mitochondrial signaling—does not appear to play an important role downstream of chk1 loss. The sole caspase whose depletion blocked the Chk1-suppressed pathway was caspase-2, a poorly characterized yet highly conserved caspase with features of both initiator and executioner caspases (Zhivotovsky and Orrenius, 2005Zhivotovsky B. Orrenius S. Caspase-2 function in response to DNA damage.Biochem. Biophys. Res. Commun. 2005; 331: 859-867Crossref PubMed Scopus (167) Google Scholar). In three separate experiments, p53e7/e7;chk1MO;casp2MO1 embryos consistently showed a mean 6-fold decrease in AO labeling compared with p53e7/e7;chk1MO embryos (∼16% of the mean p53e7/e7;chk1MO response, p < 0.0001; Figures 4A and 4B). casp2 MO1, which targets the splice donor site of intron 4, led to marked reductions in casp2 mRNA levels and to aberrant residual transcripts lacking exon 4 (Figure 4C). A second casp2 MO reduced IR-induced death in p53e7/e7;chk1MO embryos (Figure S5F and S5G), and a mismatch version of casp2 MO1 had no effect (data not shown). Altogether, these epistasis analyses identify a novel atm/atr-casp2 apoptotic program as a key mechanism through which Chk1 depletion radiosensitizes p53 mutant zebrafish embryos without recruiting the classical mitochondrial and death-receptor pathways (Figure 7D). We next investigated whether the DNA damage-induced apoptotic pathway suppressed by Chk1 in zebrafish is conserved in human cancer cells defective in p53 signaling. To inhibit Chk1 in these cells, we used the indolocarbazole small molecule Gö6976 (Kohn et al., 2003Kohn E.A. Yoo C.J. Eastman A. The protein kinase C inhibitor Gö6976 is a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints.Cancer Res. 2003; 63: 31-35PubMed Google Scholar), which has greater specificity than the commonly used Chk1 inhibitor UCN-01 (reviewed in Kawabe, 2004Kawabe T. G2 checkpoint abrogators as anticancer drugs.Mol. Cancer Ther. 2004; 3: 513-519PubMed Google Scholar and see below). In HeLa cells (in which the p53 protein pool is depleted by HPV-18 E6), caspase-2 cleavage was readily apparent at 24 hpIR in the presence of Gö6976 (Figure 5A). This effect was synergistic because neither IR nor Gö6976 alone caused substantial increases in cleaved caspase-2 levels compared to basal levels observed in control cells. In addition, caspase-2 cleavage tightly correlated with a strong radiosensitizing effect (∼50% increase in cell death; Figure 5B, compare bars 5 and 6; see also Figures 5J, 5K, and 6C). By contrast, the levels of cleaved caspase-3 in Gö6976-treated cells at 24 hpIR were negligible and did not differ from those observed in irradiated cells not exposed to the inhibitor (Figure 5A). Furthermore, both caspase-2 cleavage and concomitant cellular radiosensitization were insensitive to overexpression of human BCL2, whereas caspase-3 cleavage was completely removed in this context (Figures 5A and 5B, compare bars 7 and 8). Synergistic activation of caspase-2 by Gö6976 and IR did not elicit or involve cytochrome c release from the mitochondria at 24 hpIR (Figure 5C). Together, these findings demonstrate that Chk1 inhibition and IR synergize to activate caspase-2 and trigger BCL2- and mitochondria-independent cell death in p53-defective human cells, consistent with our zebrafish data. Before testing whether caspase-2 is required for cell-death induction, we verified the specificity of Gö6976 as an inhibitor of Chk1. CHK1 siRNA, but not a LACZ control siRNA, induced caspase-2 cleavage in concert with IR at 24 hr posttreatment but did not stimulate caspase-3 processing at this stage, in accord with the effects of Gö6976 (Figure 5D). Furthermore, while Gö6976 inhibited Chk1 in a dose-dependent manner, it did not impair MK-2 activity (Figure 5E), in contrast with UCN-01 (Reinhardt et al., 2007Reinhardt H.C. Aslanian A.S. Lees J.A. Yaffe M.B. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage.Cancer Cell. 2007; 11: 175-189Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). To test whether caspase-2 is required for Gö6976-mediated HeLa cell killing after IR, we used three independent CASP2 shRNAs that produced strong and specific knockdowns (Figure 5G). Each shRNA significantly reduced apoptosis induction at 48 hr after IR + Gö6976 treatment, but not after IR treatment alone (Figure 5H; see also Figures 5J and 5K). In contrast, the reduction in apoptosis observed upon CASP3 knockdown at 48 hr (stage at which caspase-3 is eventually cleaved) was independent of Gö6976, as CASP3 shRNA led to a similar attenuation after IR treatment alone (Figure 5H). The severity of the apoptotic blockades caused by the CASP2 shRNAs (20%–45% reductions, p < 0.01 for each) correlated with their respective knockdown efficiencies (Figures 5G and 5H). Altogether, these results demonstrate that caspase-2—but not caspase-3—is specifically required for the increase in IR-induced apoptosis observed in Chk1-inhibited human cancer cells, similar to its requirement in irradiated p53e7/e7;chk1MO zebrafish embryos. If the ATM/ATR-caspase-2 apoptotic axis in zebrafish is well conserved in human cells, ATM and ATR should be activated after Chk1 inhibition in irradiated HeLa cells, similar to caspase-2. Indeed, IR + Gö6976 treatment led to synergistic increases in phosphorylated Chk2 at Thr68 (readout for ATM activity) and phosphorylated Chk1 at Ser317 (readout for ATR activity) (Figure 5I). Elevated ATM and ATR activities correlated with increased levels of DNA damage in the IR + Gö6976-treated cells, as indicated by an increased abundance of phosphorylated H2A.X (Figure 5I). Even though Chk2 was strongly activated in this context (Figure 5I), a specific CHK2 siRNA failed to block caspase-2 activation (Figures S4B and S4C). This result substantiates our prediction (based on epistasis analyses in zebrafish) that the Chk1-suppressed pathway is Chk2 independent. Taken together, our experiments in HeLa cells show that apoptosis after IR + Gö6976 treatment of human cells involves ATM and ATR activation, is independent of Chk2, Bcl-2, mitochondria, and caspase-3, but requires caspase-2 activation and function (Figure 7D). Thus, the zebrafish Chk1-suppressed pathway is evolutionarily conserved in human cancer cells. MK-2 depleted Tp53−/− MEFs (mouse embryonic fibroblasts) undergo DNA damage-induced apoptosis exclusively during mitosis (Reinhardt et al., 2007Reinhardt H.C. Aslanian A.S. Lees J.A. Yaffe M.B. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage.Cancer Cell. 2007; 11: 175-189Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). In contrast, pH3/TUNEL double-labeling of irradiated p53e7/e7;chk1MO zebrafish embryos indicates that Chk1-suppressed apoptosis operates pre" @default.
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• W4248537801 title "Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3" @default.
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