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W2020366336 abstract "Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochromec from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochromec upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells. Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochromec from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochromec upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells. Apoptosis is a process that removes unwanted or damaged cells. The biochemical events that mediate apoptotic cell death are generally initiated in one of two ways. In the first instance, death signals are generated at the cell surface. Activation of such cell surface proteins as the tumor necrosis factor-α or Fas receptors initiate an apoptotic cascade. Alternatively, the deprivation of many trophic growth factors that act through an interaction with a plasma membrane receptor can similarly result in apoptotic cell death. Our current understanding of the events that follow activation of either the tumor necrosis factor-α or Fas receptor envisions an initial premitochondrial phase that involves the Bcl-2 family of pro- and anti-apoptotic proteins and that may or may not require the participation of caspases (1.Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). The mitochondrial phase that follows eventuates in the release of cytochrome c and the consequent activation of caspases, enzymes in which action leads to the variety of phenotypic alterations characteristic of apoptotic cell death. The apoptosis consequent to growth factor deprivation is also held to involve an initial phase mediated by the Bcl-2 family of proteins that again results in cytochrome c release from the mitochondria followed by a caspase-mediated effector phase (2.Deshmukh M. Johnson Jr., E.M. Mol. Pharmacol. 1997; 51: 897-906Crossref PubMed Scopus (192) Google Scholar, 3.Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Signals that result in apoptotic cell death are also generated from within the cell. Staurosporine and taxol are two well known examples of chemicals that induce apoptosis as a result of an interaction with an intracellular target. In most cases, however, the specific target and the earliest events that ensue upon the interaction with the inducing chemical are poorly understood.The best known intracellular target for the induction of apoptosis is, of course, DNA. Physical and chemical agents can damage DNA in a variety of ways and with distinct functional consequences, both immediate and delayed. A number of effects on the integrity of the DNA result in the induction of apoptosis, a response that removes cells that can no longer replicate or that have potentially mutagenic damage. The details as to the mechanism whereby the cell recognizes lesions in the DNA that are not readily repairable and then sets in motion events that result in apoptotic cell death are the subject of considerable research efforts. The most dominant current paradigm places p53 at the center of a process that couples DNA damage to the transcriptional regulation of much of the same pro- and anti-apoptotic machinery that is activated by signals originating from the cell surface.The topoisomerase II inhibitor etoposide is an antineoplastic drug that has been widely used to couple DNA damage to apoptosis (4.Mizumoto K. Rothman R.J. Farber J.L. Mol. Pharmacol. 1994; 46: 890-895PubMed Google Scholar). Topoisomerase II is a nuclear enzyme that functions during both DNA replication and transcription (5.Hande K.R. Eur. J. Cancer. 1998; 34: 1514-1521Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Topoisomerase II prevents “knots” from forming in DNA by allowing the passage of an intact segment of the helical DNA through a transient double strand break (6.Burden A.D. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (491) Google Scholar). Topoisomerase II inhibitors such as etoposide stabilize the complex formed by topoisomerase II and the 5′-cleaved ends of the DNA, thus forming stable (nonrepairable) protein-linked DNA double strand breaks (6.Burden A.D. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (491) Google Scholar). Cells are apparently able to recognize such DNA damage and, in turn, to eliminate the injured cells by apoptosis.A substantial literature details many specific biochemical events that occur upon induction by etoposide in a variety of cell types of the apoptotic cascade. Importantly, many of the same steps that are proposed to be central to the mediation of apoptotic cell death in other models have also been reported to occur upon treatment with etoposide. In most cases, these reports deal with a relatively limited portion of a clearly multistep process. Accordingly, how these individual events are coupled to more proximal and distal ones is not fully understood. In the present study, we document a number of distinct manipulations that prevent the apoptotic death of L929 fibroblasts in response to treatment with etoposide. In turn, we use the mechanism of action of these manipulations to detail a sequence that proceeds from the damage to DNA, through p53 to the release of cytochrome c from the mitochondria, and that eventuates in the apoptotic death of the cell.DISCUSSIONWe have identified five distinct chemicals and one genetic manipulation that significantly reduce cell killing by etoposide. In addition, each of these manipulations prevented a characteristic phenomenon in the evolution of the apoptotic phenotype, namely the release of cytochrome c from the mitochondria. Five distinct biochemical events were identified as the likely respective targets of the action of each of these manipulations. In turn, these events constitute a sequence that proceeds from DNA damage through p53 phosphorylation to Bax up-regulation, its subsequent translocation to the mitochondria with the resultant release of cytochrome c into the cytosol, and ultimately, cell death.The topoisomerase II inhibitor etoposide causes an accumulation of double strand breaks within the nuclei of cells. These breaks are recognized by the multiprotein complex DNA-dependent protein kinase, or more specifically, the heterodimer of Ku subunits that bind to the double-stranded DNA ends (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar, 27.Anderson C.W. Trends Biochem. Sci. 1993; 18: 433-437Abstract Full Text PDF PubMed Scopus (234) Google Scholar). By binding to DNA, Ku recruits and activates the catalytic subunit (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar). The catalytic subunit of DNA-PK is a member of the PI3-kinase family (9.McConnell K. Dynan W.S. Curr. Opin. Cell Biol. 1996; 8: 325-330Crossref PubMed Scopus (27) Google Scholar,28.Jackson S.P. Int. J. Biochem. Cell Biol. 1997; 29: 935-938Crossref PubMed Scopus (91) Google Scholar). The PI3-kinase inhibitor wortmannin significantly reduced the extent of cell killing (Table I). By preventing the DNA-PK catalytic subunit from recognizing the double strand breaks induced by ETO, all downstream manifestations of apoptotic cell death including phosphorylation of p53 are inhibited, thus maintaining the viability of the cells. In the apoptotic cascade, the activation of DNA-PK is pivotal because it provides the necessary link between recognition of DNA damage and the subsequent downstream signaling events.The tumor suppressor protein p53 is a regulator of cell cycle progression and mediator of apoptosis in many cell lines. Activation of p53 occurs through phosphorylation (29.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar). In particular, the Ser-15 in p53 of humans (which corresponds to Ser-18 of mouse) is a substrate for phosphorylation by DNA-PK (30.Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (463) Google Scholar, 31.Wang Y. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4231-4235Crossref PubMed Scopus (73) Google Scholar, 32.Chao C. Saito S. Anderson C.W. Appella E. Xu Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11936-11941Crossref PubMed Scopus (145) Google Scholar). Phosphorylation of p53 occurred after treatment of L929 fibroblasts with 10 μm ETO (Fig. 2 A). By inhibiting DNA-PK (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar), wortmannin prevented the phosphorylation of p53, an effect that, in turn, inhibited the subsequent downstream events that culminate in cell death.It is conceivable that the protection afforded by wortmannin reflects an inhibition of a member of the PI3-kinase family other than DNA-PK. In this regard, it is noteworthy, at least, that the best known member of this family, the PI3-kinase that phosphorylates Akt in the cytosol, is anti-apoptotic (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). That is, this PI3-kinase functions in a pathway that prevents apoptosis. Accordingly, inhibition of this kinase by wortmannin potentiates apoptotic cell death (33.Pastorino J.G. Tafani M. Farber J.L. J. Biol. Chem. 1999; 274: 19411-19416Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the protective effect of wortmannin against cell killing by etoposide in the present study must reflect inhibition of a pro-apoptotic PI3-kinase rather than inhibition of an anti-apoptotic PI3-kinase. In turn, the demonstration here that wortmannin prevents the phosphorylation of p53 is clearly consistent with inhibition of a pro-apoptotic event, namely inhibition of DNA-PK.Phosphorylation of p53 results in the up-regulation of proteins implicated in cell cycle control and apoptosis (34.Burns T.F. El-Deiry W.S. J. Cell. Physiol. 1999; 181: 231-239Crossref PubMed Scopus (200) Google Scholar). In particular, Bax is a pro-apoptotic protein that is transcriptionally regulated by p53 (11.Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 35.Thornborrow E.C. Manfredi J.J. J. Biol. Chem. 2001; 276: 15598-15608Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Treatment of L929 fibroblasts with etoposide increased the content of Bax (Fig. 3 A). By inhibiting protein synthesis and thus the increase in the content of Bax, cycloheximide protected against cell killing by ETO (Table I).The pro-apoptotic action of Bax is believed to be mediated by its interaction with the mitochondria, in particular, its insertion into the outer mitochondrial membrane (36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). Whereas overexpression of Bax leads to mitochondrial permeabilization and cell death (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), evidence suggests that mechanisms in addition to an increase in the content of the protein are necessary for Bax to translocate from the cytosol to the mitochondria (13.Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1565) Google Scholar). It is suspected that a conformational change in Bax results in the exposure of its N-terminal domain, an event that may free the hydrophobic C-terminal membrane-anchoring domain (14.Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (218) Google Scholar, 15.Pawlowski J. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 529-531Crossref PubMed Scopus (180) Google Scholar, 16.Belaud-Rotureau M.A. Leducq N. Macouillard Poulletier de Gannes F. Diolez P. Lacoste L. Lacombe F. Bernard P. Belloc F. Apoptosis. 2000; 5: 551-560Crossref PubMed Scopus (50) Google Scholar). Several mechanisms have been proposed to account for such a Bax conformational change, including an alteration in intracellular pH (an alkalinization of the cytosol) and/or an interaction with the pro-apoptotic protein Bid (38.Eskes R. Desagher S. Antonsson B. Martinou J.C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1011) Google Scholar). In this regard, the data presented above suggest that the increase in content of Bax produced by ETO is not sufficient to induce cell killing.Pretreatment of L929 fibroblasts with the chloride channel inhibitor furosemide reduced the translocation of Bax to the mitochondria (Fig. 4 A). Consequently, furosemide prevented the release of cytochrome c from the mitochondria (Fig. 1) and reduced the extent of cell killing (Table I). Importantly, furosemide did not prevent the increase in the content of Bax. We would argue that, by inhibiting a plasma membrane chloride channel, furosemide alters the ionic strength within the cytosol, an effect that prevents a conformational change in Bax that would otherwise render it susceptible to mitochondrial translocation.The translocation of Bax to the mitochondria in response to treatment with ETO was also prevented by overexpression of Akt (Fig. 6 B), a result that was again reflected in the absence of cytochrome c release (Fig. 6 A) and resistance to cell killing (Table II). Importantly, the cellular content of Bax was not affected by the overexpression of Akt (data not shown). Akt is a serine-threonine kinase that phosphorylates the pro-apoptotic protein Bad (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). Upon its phosphorylation, Bad no longer binds to the anti-apoptotic protein Bcl-X, thereby freeing the latter to bind to Bax and to prevent Bax translocation to the mitochondria.Upon its translocation to the mitochondrion, Bax can cause the release of cytochrome c (12.Narita M. Shimizu S. Ito T. Crittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (867) Google Scholar, 36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). The mechanism by which Bax releases cytochrome c is a matter of some current debate (39.Martinou J.C. Desagher S. Antonsson B. Nat. Cell Biol. 2000; 2: E41-E43Crossref PubMed Scopus (276) Google Scholar, 40.Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. J. Biol. Chem. 2000; 275: 32438-32443Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 41.Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). We have shown that the release of cytochrome c by Bax from both isolated mitochondria in vitro (18.Pastorino J.G. Tafani M. Rothman R.J. Marcineviciute A. Hoek J.B. Farber J.L. J. Biol. Chem. 1999; 274: 31734-31739Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) and from these organelles in the intact cell (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar) is a consequence of the opening of the permeability transition pore. In turn, opening of the permeability transition pore can lead to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of the MPT, prevented the release of cytochrome cin L929 fibroblasts treated with ETO (Fig. 1) and reduced the loss of viability (Table I). However, CyA did not prevent cell killing past 24 h (data not shown), because the protective effects of CyA are transient (20.Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (332) Google Scholar, 21.Broekemeier K.M. Pfeiffer D.R. Biochemistry. 1995; 34: 16440-16449Crossref PubMed Scopus (207) Google Scholar). Whereas CyA is believed to exert its anti-apoptotic effects through binding to cyclophilin D, DUBQ is a potent MPT inhibitor by binding to a ubiquinone binding site that appears to be involved directly in permeability transition pore regulation (19.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Unlike CyA, DUBQ was able to prevent cell killing by ETO over a 3-day time course (Table I).The various manipulations discussed above that modify the response of L929 fibroblasts to etoposide can be summarized by the sequence presented in Fig. 7. ETO induces the accumulation of DNA double strand breaks that are subsequently recognized by DNA-PK. This multiprotein complex then activates p53 through phosphorylation. Upon activation, p53 causes an increase in the transcription of the pro-apoptotic protein Bax. Bax undergoes a conformational change and is able to translocate to the mitochondria. This movement of Bax to the mitochondria induces the MPT, an event that results in the release of cytochrome c and culminates with loss of viability of the cells. Apoptosis is a process that removes unwanted or damaged cells. The biochemical events that mediate apoptotic cell death are generally initiated in one of two ways. In the first instance, death signals are generated at the cell surface. Activation of such cell surface proteins as the tumor necrosis factor-α or Fas receptors initiate an apoptotic cascade. Alternatively, the deprivation of many trophic growth factors that act through an interaction with a plasma membrane receptor can similarly result in apoptotic cell death. Our current understanding of the events that follow activation of either the tumor necrosis factor-α or Fas receptor envisions an initial premitochondrial phase that involves the Bcl-2 family of pro- and anti-apoptotic proteins and that may or may not require the participation of caspases (1.Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). The mitochondrial phase that follows eventuates in the release of cytochrome c and the consequent activation of caspases, enzymes in which action leads to the variety of phenotypic alterations characteristic of apoptotic cell death. The apoptosis consequent to growth factor deprivation is also held to involve an initial phase mediated by the Bcl-2 family of proteins that again results in cytochrome c release from the mitochondria followed by a caspase-mediated effector phase (2.Deshmukh M. Johnson Jr., E.M. Mol. Pharmacol. 1997; 51: 897-906Crossref PubMed Scopus (192) Google Scholar, 3.Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Signals that result in apoptotic cell death are also generated from within the cell. Staurosporine and taxol are two well known examples of chemicals that induce apoptosis as a result of an interaction with an intracellular target. In most cases, however, the specific target and the earliest events that ensue upon the interaction with the inducing chemical are poorly understood. The best known intracellular target for the induction of apoptosis is, of course, DNA. Physical and chemical agents can damage DNA in a variety of ways and with distinct functional consequences, both immediate and delayed. A number of effects on the integrity of the DNA result in the induction of apoptosis, a response that removes cells that can no longer replicate or that have potentially mutagenic damage. The details as to the mechanism whereby the cell recognizes lesions in the DNA that are not readily repairable and then sets in motion events that result in apoptotic cell death are the subject of considerable research efforts. The most dominant current paradigm places p53 at the center of a process that couples DNA damage to the transcriptional regulation of much of the same pro- and anti-apoptotic machinery that is activated by signals originating from the cell surface. The topoisomerase II inhibitor etoposide is an antineoplastic drug that has been widely used to couple DNA damage to apoptosis (4.Mizumoto K. Rothman R.J. Farber J.L. Mol. Pharmacol. 1994; 46: 890-895PubMed Google Scholar). Topoisomerase II is a nuclear enzyme that functions during both DNA replication and transcription (5.Hande K.R. Eur. J. Cancer. 1998; 34: 1514-1521Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Topoisomerase II prevents “knots” from forming in DNA by allowing the passage of an intact segment of the helical DNA through a transient double strand break (6.Burden A.D. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (491) Google Scholar). Topoisomerase II inhibitors such as etoposide stabilize the complex formed by topoisomerase II and the 5′-cleaved ends of the DNA, thus forming stable (nonrepairable) protein-linked DNA double strand breaks (6.Burden A.D. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (491) Google Scholar). Cells are apparently able to recognize such DNA damage and, in turn, to eliminate the injured cells by apoptosis. A substantial literature details many specific biochemical events that occur upon induction by etoposide in a variety of cell types of the apoptotic cascade. Importantly, many of the same steps that are proposed to be central to the mediation of apoptotic cell death in other models have also been reported to occur upon treatment with etoposide. In most cases, these reports deal with a relatively limited portion of a clearly multistep process. Accordingly, how these individual events are coupled to more proximal and distal ones is not fully understood. In the present study, we document a number of distinct manipulations that prevent the apoptotic death of L929 fibroblasts in response to treatment with etoposide. In turn, we use the mechanism of action of these manipulations to detail a sequence that proceeds from the damage to DNA, through p53 to the release of cytochrome c from the mitochondria, and that eventuates in the apoptotic death of the cell. DISCUSSIONWe have identified five distinct chemicals and one genetic manipulation that significantly reduce cell killing by etoposide. In addition, each of these manipulations prevented a characteristic phenomenon in the evolution of the apoptotic phenotype, namely the release of cytochrome c from the mitochondria. Five distinct biochemical events were identified as the likely respective targets of the action of each of these manipulations. In turn, these events constitute a sequence that proceeds from DNA damage through p53 phosphorylation to Bax up-regulation, its subsequent translocation to the mitochondria with the resultant release of cytochrome c into the cytosol, and ultimately, cell death.The topoisomerase II inhibitor etoposide causes an accumulation of double strand breaks within the nuclei of cells. These breaks are recognized by the multiprotein complex DNA-dependent protein kinase, or more specifically, the heterodimer of Ku subunits that bind to the double-stranded DNA ends (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar, 27.Anderson C.W. Trends Biochem. Sci. 1993; 18: 433-437Abstract Full Text PDF PubMed Scopus (234) Google Scholar). By binding to DNA, Ku recruits and activates the catalytic subunit (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar). The catalytic subunit of DNA-PK is a member of the PI3-kinase family (9.McConnell K. Dynan W.S. Curr. Opin. Cell Biol. 1996; 8: 325-330Crossref PubMed Scopus (27) Google Scholar,28.Jackson S.P. Int. J. Biochem. Cell Biol. 1997; 29: 935-938Crossref PubMed Scopus (91) Google Scholar). The PI3-kinase inhibitor wortmannin significantly reduced the extent of cell killing (Table I). By preventing the DNA-PK catalytic subunit from recognizing the double strand breaks induced by ETO, all downstream manifestations of apoptotic cell death including phosphorylation of p53 are inhibited, thus maintaining the viability of the cells. In the apoptotic cascade, the activation of DNA-PK is pivotal because it provides the necessary link between recognition of DNA damage and the subsequent downstream signaling events.The tumor suppressor protein p53 is a regulator of cell cycle progression and mediator of apoptosis in many cell lines. Activation of p53 occurs through phosphorylation (29.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar). In particular, the Ser-15 in p53 of humans (which corresponds to Ser-18 of mouse) is a substrate for phosphorylation by DNA-PK (30.Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (463) Google Scholar, 31.Wang Y. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4231-4235Crossref PubMed Scopus (73) Google Scholar, 32.Chao C. Saito S. Anderson C.W. Appella E. Xu Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11936-11941Crossref PubMed Scopus (145) Google Scholar). Phosphorylation of p53 occurred after treatment of L929 fibroblasts with 10 μm ETO (Fig. 2 A). By inhibiting DNA-PK (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar), wortmannin prevented the phosphorylation of p53, an effect that, in turn, inhibited the subsequent downstream events that culminate in cell death.It is conceivable that the protection afforded by wortmannin reflects an inhibition of a member of the PI3-kinase family other than DNA-PK. In this regard, it is noteworthy, at least, that the best known member of this family, the PI3-kinase that phosphorylates Akt in the cytosol, is anti-apoptotic (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). That is, this PI3-kinase functions in a pathway that prevents apoptosis. Accordingly, inhibition of this kinase by wortmannin potentiates apoptotic cell death (33.Pastorino J.G. Tafani M. Farber J.L. J. Biol. Chem. 1999; 274: 19411-19416Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the protective effect of wortmannin against cell killing by etoposide in the present study must reflect inhibition of a pro-apoptotic PI3-kinase rather than inhibition of an anti-apoptotic PI3-kinase. In turn, the demonstration here that wortmannin prevents the phosphorylation of p53 is clearly consistent with inhibition of a pro-apoptotic event, namely inhibition of DNA-PK.Phosphorylation of p53 results in the up-regulation of proteins implicated in cell cycle control and apoptosis (34.Burns T.F. El-Deiry W.S. J. Cell. Physiol. 1999; 181: 231-239Crossref PubMed Scopus (200) Google Scholar). In particular, Bax is a pro-apoptotic protein that is transcriptionally regulated by p53 (11.Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 35.Thornborrow E.C. Manfredi J.J. J. Biol. Chem. 2001; 276: 15598-15608Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Treatment of L929 fibroblasts with etoposide increased the content of Bax (Fig. 3 A). By inhibiting protein synthesis and thus the increase in the content of Bax, cycloheximide protected against cell killing by ETO (Table I).The pro-apoptotic action of Bax is believed to be mediated by its interaction with the mitochondria, in particular, its insertion into the outer mitochondrial membrane (36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). Whereas overexpression of Bax leads to mitochondrial permeabilization and cell death (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), evidence suggests that mechanisms in addition to an increase in the content of the protein are necessary for Bax to translocate from the cytosol to the mitochondria (13.Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1565) Google Scholar). It is suspected that a conformational change in Bax results in the exposure of its N-terminal domain, an event that may free the hydrophobic C-terminal membrane-anchoring domain (14.Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (218) Google Scholar, 15.Pawlowski J. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 529-531Crossref PubMed Scopus (180) Google Scholar, 16.Belaud-Rotureau M.A. Leducq N. Macouillard Poulletier de Gannes F. Diolez P. Lacoste L. Lacombe F. Bernard P. Belloc F. Apoptosis. 2000; 5: 551-560Crossref PubMed Scopus (50) Google Scholar). Several mechanisms have been proposed to account for such a Bax conformational change, including an alteration in intracellular pH (an alkalinization of the cytosol) and/or an interaction with the pro-apoptotic protein Bid (38.Eskes R. Desagher S. Antonsson B. Martinou J.C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1011) Google Scholar). In this regard, the data presented above suggest that the increase in content of Bax produced by ETO is not sufficient to induce cell killing.Pretreatment of L929 fibroblasts with the chloride channel inhibitor furosemide reduced the translocation of Bax to the mitochondria (Fig. 4 A). Consequently, furosemide prevented the release of cytochrome c from the mitochondria (Fig. 1) and reduced the extent of cell killing (Table I). Importantly, furosemide did not prevent the increase in the content of Bax. We would argue that, by inhibiting a plasma membrane chloride channel, furosemide alters the ionic strength within the cytosol, an effect that prevents a conformational change in Bax that would otherwise render it susceptible to mitochondrial translocation.The translocation of Bax to the mitochondria in response to treatment with ETO was also prevented by overexpression of Akt (Fig. 6 B), a result that was again reflected in the absence of cytochrome c release (Fig. 6 A) and resistance to cell killing (Table II). Importantly, the cellular content of Bax was not affected by the overexpression of Akt (data not shown). Akt is a serine-threonine kinase that phosphorylates the pro-apoptotic protein Bad (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). Upon its phosphorylation, Bad no longer binds to the anti-apoptotic protein Bcl-X, thereby freeing the latter to bind to Bax and to prevent Bax translocation to the mitochondria.Upon its translocation to the mitochondrion, Bax can cause the release of cytochrome c (12.Narita M. Shimizu S. Ito T. Crittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (867) Google Scholar, 36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). The mechanism by which Bax releases cytochrome c is a matter of some current debate (39.Martinou J.C. Desagher S. Antonsson B. Nat. Cell Biol. 2000; 2: E41-E43Crossref PubMed Scopus (276) Google Scholar, 40.Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. J. Biol. Chem. 2000; 275: 32438-32443Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 41.Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). We have shown that the release of cytochrome c by Bax from both isolated mitochondria in vitro (18.Pastorino J.G. Tafani M. Rothman R.J. Marcineviciute A. Hoek J.B. Farber J.L. J. Biol. Chem. 1999; 274: 31734-31739Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) and from these organelles in the intact cell (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar) is a consequence of the opening of the permeability transition pore. In turn, opening of the permeability transition pore can lead to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of the MPT, prevented the release of cytochrome cin L929 fibroblasts treated with ETO (Fig. 1) and reduced the loss of viability (Table I). However, CyA did not prevent cell killing past 24 h (data not shown), because the protective effects of CyA are transient (20.Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (332) Google Scholar, 21.Broekemeier K.M. Pfeiffer D.R. Biochemistry. 1995; 34: 16440-16449Crossref PubMed Scopus (207) Google Scholar). Whereas CyA is believed to exert its anti-apoptotic effects through binding to cyclophilin D, DUBQ is a potent MPT inhibitor by binding to a ubiquinone binding site that appears to be involved directly in permeability transition pore regulation (19.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Unlike CyA, DUBQ was able to prevent cell killing by ETO over a 3-day time course (Table I).The various manipulations discussed above that modify the response of L929 fibroblasts to etoposide can be summarized by the sequence presented in Fig. 7. ETO induces the accumulation of DNA double strand breaks that are subsequently recognized by DNA-PK. This multiprotein complex then activates p53 through phosphorylation. Upon activation, p53 causes an increase in the transcription of the pro-apoptotic protein Bax. Bax undergoes a conformational change and is able to translocate to the mitochondria. This movement of Bax to the mitochondria induces the MPT, an event that results in the release of cytochrome c and culminates with loss of viability of the cells. We have identified five distinct chemicals and one genetic manipulation that significantly reduce cell killing by etoposide. In addition, each of these manipulations prevented a characteristic phenomenon in the evolution of the apoptotic phenotype, namely the release of cytochrome c from the mitochondria. Five distinct biochemical events were identified as the likely respective targets of the action of each of these manipulations. In turn, these events constitute a sequence that proceeds from DNA damage through p53 phosphorylation to Bax up-regulation, its subsequent translocation to the mitochondria with the resultant release of cytochrome c into the cytosol, and ultimately, cell death. The topoisomerase II inhibitor etoposide causes an accumulation of double strand breaks within the nuclei of cells. These breaks are recognized by the multiprotein complex DNA-dependent protein kinase, or more specifically, the heterodimer of Ku subunits that bind to the double-stranded DNA ends (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar, 27.Anderson C.W. Trends Biochem. Sci. 1993; 18: 433-437Abstract Full Text PDF PubMed Scopus (234) Google Scholar). By binding to DNA, Ku recruits and activates the catalytic subunit (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar). The catalytic subunit of DNA-PK is a member of the PI3-kinase family (9.McConnell K. Dynan W.S. Curr. Opin. Cell Biol. 1996; 8: 325-330Crossref PubMed Scopus (27) Google Scholar,28.Jackson S.P. Int. J. Biochem. Cell Biol. 1997; 29: 935-938Crossref PubMed Scopus (91) Google Scholar). The PI3-kinase inhibitor wortmannin significantly reduced the extent of cell killing (Table I). By preventing the DNA-PK catalytic subunit from recognizing the double strand breaks induced by ETO, all downstream manifestations of apoptotic cell death including phosphorylation of p53 are inhibited, thus maintaining the viability of the cells. In the apoptotic cascade, the activation of DNA-PK is pivotal because it provides the necessary link between recognition of DNA damage and the subsequent downstream signaling events. The tumor suppressor protein p53 is a regulator of cell cycle progression and mediator of apoptosis in many cell lines. Activation of p53 occurs through phosphorylation (29.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (709) Google Scholar). In particular, the Ser-15 in p53 of humans (which corresponds to Ser-18 of mouse) is a substrate for phosphorylation by DNA-PK (30.Lees-Miller S.P. Sakaguchi K. Ullrich S.J. Appella E. Anderson C.W. Mol. Cell. Biol. 1992; 12: 5041-5049Crossref PubMed Scopus (463) Google Scholar, 31.Wang Y. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4231-4235Crossref PubMed Scopus (73) Google Scholar, 32.Chao C. Saito S. Anderson C.W. Appella E. Xu Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11936-11941Crossref PubMed Scopus (145) Google Scholar). Phosphorylation of p53 occurred after treatment of L929 fibroblasts with 10 μm ETO (Fig. 2 A). By inhibiting DNA-PK (26.Smith G.C.M. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar), wortmannin prevented the phosphorylation of p53, an effect that, in turn, inhibited the subsequent downstream events that culminate in cell death. It is conceivable that the protection afforded by wortmannin reflects an inhibition of a member of the PI3-kinase family other than DNA-PK. In this regard, it is noteworthy, at least, that the best known member of this family, the PI3-kinase that phosphorylates Akt in the cytosol, is anti-apoptotic (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). That is, this PI3-kinase functions in a pathway that prevents apoptosis. Accordingly, inhibition of this kinase by wortmannin potentiates apoptotic cell death (33.Pastorino J.G. Tafani M. Farber J.L. J. Biol. Chem. 1999; 274: 19411-19416Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the protective effect of wortmannin against cell killing by etoposide in the present study must reflect inhibition of a pro-apoptotic PI3-kinase rather than inhibition of an anti-apoptotic PI3-kinase. In turn, the demonstration here that wortmannin prevents the phosphorylation of p53 is clearly consistent with inhibition of a pro-apoptotic event, namely inhibition of DNA-PK. Phosphorylation of p53 results in the up-regulation of proteins implicated in cell cycle control and apoptosis (34.Burns T.F. El-Deiry W.S. J. Cell. Physiol. 1999; 181: 231-239Crossref PubMed Scopus (200) Google Scholar). In particular, Bax is a pro-apoptotic protein that is transcriptionally regulated by p53 (11.Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 35.Thornborrow E.C. Manfredi J.J. J. Biol. Chem. 2001; 276: 15598-15608Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Treatment of L929 fibroblasts with etoposide increased the content of Bax (Fig. 3 A). By inhibiting protein synthesis and thus the increase in the content of Bax, cycloheximide protected against cell killing by ETO (Table I). The pro-apoptotic action of Bax is believed to be mediated by its interaction with the mitochondria, in particular, its insertion into the outer mitochondrial membrane (36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). Whereas overexpression of Bax leads to mitochondrial permeabilization and cell death (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), evidence suggests that mechanisms in addition to an increase in the content of the protein are necessary for Bax to translocate from the cytosol to the mitochondria (13.Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1565) Google Scholar). It is suspected that a conformational change in Bax results in the exposure of its N-terminal domain, an event that may free the hydrophobic C-terminal membrane-anchoring domain (14.Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (218) Google Scholar, 15.Pawlowski J. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 529-531Crossref PubMed Scopus (180) Google Scholar, 16.Belaud-Rotureau M.A. Leducq N. Macouillard Poulletier de Gannes F. Diolez P. Lacoste L. Lacombe F. Bernard P. Belloc F. Apoptosis. 2000; 5: 551-560Crossref PubMed Scopus (50) Google Scholar). Several mechanisms have been proposed to account for such a Bax conformational change, including an alteration in intracellular pH (an alkalinization of the cytosol) and/or an interaction with the pro-apoptotic protein Bid (38.Eskes R. Desagher S. Antonsson B. Martinou J.C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1011) Google Scholar). In this regard, the data presented above suggest that the increase in content of Bax produced by ETO is not sufficient to induce cell killing. Pretreatment of L929 fibroblasts with the chloride channel inhibitor furosemide reduced the translocation of Bax to the mitochondria (Fig. 4 A). Consequently, furosemide prevented the release of cytochrome c from the mitochondria (Fig. 1) and reduced the extent of cell killing (Table I). Importantly, furosemide did not prevent the increase in the content of Bax. We would argue that, by inhibiting a plasma membrane chloride channel, furosemide alters the ionic strength within the cytosol, an effect that prevents a conformational change in Bax that would otherwise render it susceptible to mitochondrial translocation. The translocation of Bax to the mitochondria in response to treatment with ETO was also prevented by overexpression of Akt (Fig. 6 B), a result that was again reflected in the absence of cytochrome c release (Fig. 6 A) and resistance to cell killing (Table II). Importantly, the cellular content of Bax was not affected by the overexpression of Akt (data not shown). Akt is a serine-threonine kinase that phosphorylates the pro-apoptotic protein Bad (24.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar). Upon its phosphorylation, Bad no longer binds to the anti-apoptotic protein Bcl-X, thereby freeing the latter to bind to Bax and to prevent Bax translocation to the mitochondria. Upon its translocation to the mitochondrion, Bax can cause the release of cytochrome c (12.Narita M. Shimizu S. Ito T. Crittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (867) Google Scholar, 36.Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (546) Google Scholar). The mechanism by which Bax releases cytochrome c is a matter of some current debate (39.Martinou J.C. Desagher S. Antonsson B. Nat. Cell Biol. 2000; 2: E41-E43Crossref PubMed Scopus (276) Google Scholar, 40.Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. J. Biol. Chem. 2000; 275: 32438-32443Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 41.Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). We have shown that the release of cytochrome c by Bax from both isolated mitochondria in vitro (18.Pastorino J.G. Tafani M. Rothman R.J. Marcineviciute A. Hoek J.B. Farber J.L. J. Biol. Chem. 1999; 274: 31734-31739Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) and from these organelles in the intact cell (37.Pastorino J.G. Chen S-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar) is a consequence of the opening of the permeability transition pore. In turn, opening of the permeability transition pore can lead to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of the MPT, prevented the release of cytochrome cin L929 fibroblasts treated with ETO (Fig. 1) and reduced the loss of viability (Table I). However, CyA did not prevent cell killing past 24 h (data not shown), because the protective effects of CyA are transient (20.Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (332) Google Scholar, 21.Broekemeier K.M. Pfeiffer D.R. Biochemistry. 1995; 34: 16440-16449Crossref PubMed Scopus (207) Google Scholar). Whereas CyA is believed to exert its anti-apoptotic effects through binding to cyclophilin D, DUBQ is a potent MPT inhibitor by binding to a ubiquinone binding site that appears to be involved directly in permeability transition pore regulation (19.Fontaine E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 25734-25740Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Unlike CyA, DUBQ was able to prevent cell killing by ETO over a 3-day time course (Table I). The various manipulations discussed above that modify the response of L929 fibroblasts to etoposide can be summarized by the sequence presented in Fig. 7. ETO induces the accumulation of DNA double strand breaks that are subsequently recognized by DNA-PK. This multiprotein complex then activates p53 through phosphorylation. Upon activation, p53 causes an increase in the transcription of the pro-apoptotic protein Bax. Bax undergoes a conformational change and is able to translocate to the mitochondria. This movement of Bax to the mitochondria induces the MPT, an event that results in the release of cytochrome c and culminates with loss of viability of the cells." @default.
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W2020366336 title "The Course of Etoposide-induced Apoptosis from Damage to DNA and p53 Activation to Mitochondrial Release of Cytochromec" @default.
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