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p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release

p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 10, Issue of March 10, pp. 7337–7342, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release* (Received for publication, July 19, 1999, and in revised form, December 1, 1999) Martin Schuler‡, Ella Bossy-Wetzel§, Joshua C. Goldstein, Patrick Fitzgerald, and Douglas R. Green¶ From the Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121 The p53 tumor suppressor gene is critically involved CIP1 (2–5) or GADD45 (6), thus relying on the ability of p53 to act as a sequence-specific transcription factor. In contrast, the in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 tumor-suppressing activity of p53 does not depend on its trans- death signals lead to caspase activation; however, the activational function (7–10). mechanism of caspase activation by p53 still is unclear. It has been shown in several cell types that wild type (wt) Expressing wild type p53 by means of an adenoviral p53 is required for the apoptotic cell death as induced by expression vector, we were able to induce apoptotic cell g-irradiation or a variety of anticancer drugs (11, 12). Yet, the death, as characterized by morphological changes, pathways whereby p53 leads to execution of the apoptosis phosphatidylserine externalization, and internucleoso- program are not well characterized. Possible mechanisms in- null mal DNA fragmentation, in p53 Saos-2 cells. This cell clude transcriptional activation of the proapoptotic Bcl-2 family death was accompanied by caspase activation as well as member Bax (13, 14), the generation of reactive oxygen species by cleavage of caspase substrates and was preceded by (15), and transcriptional up-regulation of death receptors such mitochondrial cytochrome c release. The addition of the as CD95/Fas/APO-1 or DR5/KILLER (16 –19). However, sev- broad-spectrum caspase inhibitor benzyloxycarbonyl- eral lines of evidence imply that the proapoptotic activity of p53 Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly is independent of its function as a transcription factor (20 –22). after transduction almost completely prevented p53-in- The release of cytochrome c from mitochondria is a central duced apoptotic cell death but did not inhibit mitochon- event in the death receptor-independent, “intrinsic,” apoptotic drial cytochrome c release. In contrast, N-acetylcys- pathway (23, 24). Cytochrome c together with ATP and Apaf-1 teine, even at high concentrations, could not prevent facilitates activation by caspase 9 of the effector caspases (25– induction of programmed cell death by p53 expression. 28), which then cleave their substrates, finally leading to the Cytosolic extracts from Saos-2 cells transduced with apoptotic cell death. Furthermore, cytochrome c release can p53, but not from Saos-2 cells transduced with the empty also occur in death receptor-dependent, “extrinsic,” apoptotic adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of pathways by cleavage and activation of the proapoptotic Bcl-2 zVAD-fmk. When Bax was immunodepleted from the cy- family member Bid through caspase 8 (29 –31), possibly serving tosolic extracts of p53-expressing cells before incuba- as an amplification loop. Several studies were undertaken to tion with isolated mitochondria, the in vitro cytochrome establish the involvement of caspase activation in p53-medi- c release was abolished. Thus, we could demonstrate in ated cell death (18, 32–35). Recently, the requirement of Apaf-1 cells and in vitro that p53 activates the apoptotic ma- or caspase 9 for the p53-dependent apoptosis of oncogene-trans- chinery through induction of the release of cytochrome formed murine embryonic fibroblasts has been conclusively c from the mitochondrial intermembrane space. Fur- demonstrated (36). thermore, we provide in vitro evidence for the require- The present study was undertaken to address the hypothesis ment of cytosolic Bax for this cytochrome c-releasing that p53 might induce apoptosis by a death receptor-independ- activity of p53 in Saos-2 cells. ent pathway involving the release of mitochondrial cytochrome c. We show in Saos-2 cells that p53 evokes cytochrome c release prior to caspase activation and prior to the occurrence of apo- The p53 tumor suppressor gene is the central integrator of ptotic cell membrane changes. Furthermore, we biochemically the cellular response to DNA damage, oncogenic transforma- demonstrate the requirement of cytosolic Bax protein for the tion, and growth factor withdrawal (1). The cell cycle regula- cytochrome c-releasing activity of p53. Thereby, in our experi- tory and the DNA repair functions of p53 are largely executed mental system, we provide a link between p53 and the death by transactivation of p53-response genes such as p21/WAF1/ receptor-independent activation of the apoptotic machinery downstream of mitochondria. * This work was supported by National Institutes of Health Grants EXPERIMENTAL PROCEDURES CA69831 and AI40646 (to D. R. G.). This is publication No. 317 of the Cell Lines, Antibodies, and Reagents—The human osteosarcoma cell La Jolla Institute for Allergy and Immunology. The costs of publication line Saos-2 cells was obtained from ATCC (Manassas, VA) and was of this article were defrayed in part by the payment of page charges. maintained in McCoy’s 5A medium containing 15% fetal calf serum, This article must therefore be hereby marked “advertisement”inac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact. penicillin, and streptomycin. ‡ Recipient of a postdoctoral fellowship from the Dr. Mildred Scheel Antibodies against cytochrome c (clone 7H8.2C12), caspase 3 (poly- Stiftung fu ¨ r Krebsforschung, Germany. clonal rabbit antiserum), poly(ADP-ribose) polymerase (clone C2–10), § Supported by Swiss National Science Foundation Fellowship 823A-046638. To whom correspondence should be addressed: La Jolla Institute for The abbreviations used are: wt, wild type; zVAD-fmk, benzyloxy- Allergy and Immunology, 10355 Science Center Dr., San Diego, CA carbonyl-Val-Ala-Asp-fluoromethyl ketone; DEVD-afc, benzyloxycar- 92121. Tel.: 858-558-3500; Fax: 858-558-3526; E-mail: dgreen5240@ bonyl-Asp-Glu-Val-Asp aminofluoromethylcoumarin; PI, propidium aol.com. iodide. This paper is available on line at http://www.jbc.org 7337 This is an Open Access article under the CC BY license. 7338 p53-mediated Apoptosis p53 (clones Do-7 and PAb122), p21/WAF1/CIP1 (clone SX118), and Bax (polyclonal rabbit antiserum) were purchased from Pharmingen (San Diego, CA). Polyclonal rabbit antisera against protein kinase C-d and JNK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and an antibody against actin (clone C4) was obtained from ICN Phar- maceuticals (Costa Mesa, CA). A rabbit antiserum against caspase 9 has been described previously (37, 38). The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala- Asp-fluoromethyl ketone (zVAD-fmk) and the fluorochromic caspase substrate benzyloxycarbonyl-Asp-Glu-Val-Asp aminofluoromethylcou- marin (DEVD-afc) were purchased from Enzyme System Products (Livermore, CA). Vectors and Transduction—Replication-deficient adenoviral vectors were generous gifts from Drs. D. C. Maneval and I. Atencio, Canji Inc., San Diego, CA. The vectors either encoded the complete human wt p53 cDNA (Ad.p53) or green fluorescent protein (Ad.GFP) under the control of the cytomegalovirus immediate/early gene promotor (39, 40). The Ad.GFP vector was applied to determine infectibility and maximal tolerated dose of Saos-2 cells, which was found to be 10 particles/ml. For further experiments, the Ad.p53 vector was used for p53 gene transfer, and the empty adenoviral vector (Ad) served as control. Ap- proximately 18 h before transduction, cells were passaged in 10-cm culture dishes or 12-well plates (Fisher) at a density of 200,000/ml or 50,000/ml, respectively. Vectors were diluted in serum-free medium (Optimem I, Life Technologies, Inc.) at the appropriate dose (10 par- ticles/ml). Cells were pulsed with the vectors for 60 min, washed, and supplemented with fresh growth medium containing fetal calf serum. Flow Cytometry—For flow cytometry, cells were harvested by mild trypsinization, followed by washing with growth medium and phos- phate-buffered saline. Cell death was determined by two-color analysis of fluorescein isothiocyanate-labeled annexin V (CLONTECH Labora- tories) binding and propidium iodide (PI) uptake using a Becton Dick- inson FACSCalibur. Cell cycle analysis and quantitation of subdiploid FIG.1. A, Saos-2 cells were transduced in triplicates with the Ad.p53 DNA-containing nuclei was performed by detecting PI staining after vector (triangles) or the empty Ad vector cassette (squares), as described lysing the cells in HFS (0.1% sodium citrate, 0.1% Triton X-100, 50 in the text. Cells were harvested at the indicated time points (hours mg/ml PI) on ice for at least 30 min. from transduction) and were analyzed for annexin V binding and PI Preparation of Mitochondria-free Cytosolic Extracts and of Whole Cell uptake using dual-color flow cytometry. Mean percentages 6S.D. of Extracts—Cytosolic extracts were prepared as described previously (41, viable (annexin V/PI double-negative) cells are given. B, Saos-2 cells 42). In brief, cells were harvested by gently scraping and were incu- were treated with Ad.p53 (triangles) or empty Ad vector (squares) and bated in a buffer containing 220 mM mannitol and 60 mM sucrose on ice were permeabilized and stained with PI on ice for at least 30 min. The for 30 min. Then cells were broken in a Dounce homogenizer by 70 DNA content of 10,000 nuclei was assessed by flow cytometry, and mean values 6S.D. of percentages of nuclei with DNA content below 2N gentle strokes of a type B pestle. The homogenates were centrifuged at (sub-G fraction) of three experiments are given. 16,000 3 g for 15 min, and the mitochondria-free supernatants were 1 frozen at 270 °C until further analysis. Extracts of the pellets as well as whole cell extracts were obtained by dissolving in lysis buffer, followed RESULTS by repetitive vortexing and freeze-thawing. After centrifugation at null Expression of wt p53 Induces Apoptosis in p53 Saos-2 16,000 3 g, the supernatants were stored at 270 °C. Cells—First we assessed whether adenovirus-mediated p53 ex- Isolation of Mitochondria and in Vitro Cytochrome c Release Assay— pression resulted in apoptotic cell death and caspase activation Mitochondria were freshly isolated from the liver of 2-month-old mice in Saos-2 cells. Transduction with Ad.p53 induced phosphati- as described previously (41, 42). Aliquots of the mitochondrial prepara- tion were incubated with cytosolic extracts at 37 °C for various time dylserine externalization as detected by annexin V binding at periods. After centrifugation at 20,000 3 g for 15 min, the supernatants 24 h followed by membrane disruption, leading to PI uptake were analyzed for the presence of cytochrome c by Western blotting. starting at 36 h. No such effects were observed upon transduc- Mitochondrial pellets were resuspended in lysis buffer and were also tion with the same particle number of the empty Ad vector analyzed by Western blotting. cassette (Fig. 1A). Similarly, an increase in subdiploid DNA Western Blotting—The protein content of cytosolic or whole cell ex- content, indicating internucleosomal DNA fragmentation, was tracts was determined by the Bradford assay (Bio-Rad). For each time only observed in Saos-2 cells transduced with Ad.p53 but not in point, 25 mg of total protein were boiled in Laemmli buffer for 5 min Saos-2 cells transduced with Ad (Fig. 1B). followed by centrifugation. The supernatants were subjected to electro- phoresis in 8% or 15% SDS-polyacrylamide gels using a Bio-Rad minigel Caspase activity leading to cleavage of DEVD-afc was de- apparatus, followed by transfer on nitrocellulose membranes (Hybond tected in extracts from Saos-2 cells obtained at 24, 36, and 48 h ECL, Amersham Pharmacia Biotech). After blocking with phosphate- after transduction with Ad.p53 (Fig. 2A). Immunoblotting dem- buffered saline containing 5% nonfat dry milk and 0.1% Tween 20, the onstrated loss of procaspase 9, cleavage of procaspase 3, and membranes were exposed to the primary antibodies overnight at 4 °C cleavage of the caspase substrates poly(ADP-ribose) polymer- on a shaker. Before and after incubation with the horseradish peroxi- ase and protein kinase C-d. Expression of p53 protein as well as dase-conjugated secondary antibodies (Amersham Pharmacia Biotech), induction of the p53 target gene p21/WAF1/CIP1 could be de- the membranes were washed extensively using phosphate-buffered sa- line/Tween. Antibody binding was detected by enhanced chemolumines- tected as early as 12 h after transduction (Fig. 2B). cence (Super Signal; Pierce). p53-mediated Apoptosis in Saos-2 Cells Is Prevented by Detection of Caspase Activity—Activity of effector caspases was de- zVAD-fmk but Not by N-Acetylcysteine—Next, testing was con- termined in whole cell extracts using the fluorochromic caspase sub- ducted to determine if the apoptotic cell death observed after strate DEVD-afc by means of a SPECTRAFluor (Tecan, Research Tri- p53 expression in Saos-2 cells was dependent on caspase activ- angle Park, NC), as described previously (37). In brief, cleavage of ity. The addition of the broad spectrum caspase inhibitor DEVD-afc was assessed kinetically at 37 °C every 60 s for 30 min. The zVAD-fmk at a concentration of 100 mM almost completely enzymatic activity was expressed as the maximal velocity of the result- ing fluorescence-time curve. abrogated cell death (Fig. 3A) and DNA fragmentation (Fig. p53-mediated Apoptosis 7339 FIG.2. Whole cells extracts of Saos-2 cells transduced with Ad.p53 were obtained at the indicated time points (hours from transduction). A, the activity of DEVD-cleaving caspases (triangles)in the respective extracts was determined as described in the method section. RFU/s denotes relative fluorescence activity/s (right; y axis). The percentage of annexin V-positive cells was measured for identical time points using fluorescence-activated cell sorter analysis; mean val- ues 1 S.D. are given (gray bars; left, y axis). B, Western blotting of whole cell extracts using the indicated antibodies. 25 mg of total protein were loaded on each lane in 15% (caspase 3, procaspase 9, p21) or 8% (poly(ADP-ribose) polymerase (PARP), protein kinase C-d (PKC-d), p53) SDS-polyacrylamide gels. P21/WAF1/CIP1 is shown as a classical p53- responsive gene. Note the loss of procaspase 9, the occurrence of cleaved caspase 3, as well as cleavage of the caspase substrates poly(ADP- ribose) polymerase and protein kinase C-d at 24 and 36 h. FIG.3. Saos-2 cells were transduced with Ad.p53 or the empty Ad vector as described. Directly after transduction, cells were washed, and either fresh growth medium alone or medium supple- 3B), as induced by expression of wt p53 in Saos-2 cells. Several mented with zVAD-fmk (100 mM)or N-acetylcysteine (NAC,10mM) was mitochondrial genes potentially generating reactive oxygen added. Cells were harvested at the indicated time points and were species were reported to be induced by p53 overexpression (15), analyzed for annexin V binding, PI uptake, and cell cycle distribution. implicating a role of reactive oxygen species as downstream A, the bars indicate the mean percentage 1S.D. of annexin V-positive effectors of p53-mediated apoptosis. However, in Saos-2 cells, cells of three experiments; the black part of the bars indicate the fraction of annexin V-positive/PI-negative cells. B, the mean percentage the addition of N-acetylcysteine up to concentrations of 10 mM 6S.D. of nuclei with sub-G DNA content are given for each time point had no protective effect against p53-induced cell death (Fig. (hours from transduction). Closed triangles denote Saos-2 cells trans- 3A). Moreover, N-acetylcysteine at concentrations of 20 mM or duced with Ad.p53, and open triangles denote Saos-2 cells transduced above induced necrotic cell death in Saos-2 cells (data not with Ad.p53 in the presence of 100 mM zVAD-fmk. shown). null Cytochrome c Is Released from Mitochondria upon p53 Ex- the p53 human lung cancer cell line Calu-6 (data not pression—To assess whether p53 activates caspases by induc- shown). The proapoptotic Bcl-2 family member Bax, which ing the release of cytochrome c from the mitochondrial inter- induces mitochondrial cytochrome c release (44, 48), is reported membrane space into the cytosol (26, 43), subcellular to be transcriptionally activated by p53 in some human cell fractionation of Saos-2 cells was performed at various time lines (13). In concordance, an increase of Bax protein expres- points following transduction with Ad.p53 or with Ad. Cyto- sion was found in Saos-2 cells following p53 overexpression, but chrome c release into the cytosolic fraction of Saos-2 cells could significantly elevated Bax levels were detected only after the be detected as early as 12 h after transduction with Ad.p53; onset of cytochrome c release (Fig. 4A). this corresponded with a depletion of the mitochondria-contain- Ligation of death receptors such as CD95/Fas/APO-1 or tu- ing pellet fraction from cytochrome c (Fig. 4A). No such effect mor necrosis factor receptor 1 also can result in cytochrome c could be observed after transduction of Saos-2 cells with vector release via activation of caspase 8, followed by cleavage and alone (Fig. 4B). Similar kinetics of mitochondrial cytochrome c activation of Bid. Cleaved Bid can then target mitochondria release were observed after adenoviral expression of wt p53 in and induce the release of cytochrome c (29 –31). However, Bid 7340 p53-mediated Apoptosis FIG.4. Saos-2 cells growing in 10-cm dishes were transduced with Ad5.p53 (A)orAd(B, lanes 2 to 4) and cultured either in medium alone (A) or in medium supplemented with 100 mM zVAD-fmk (B, lanes 5 to 7). Cytosolic extracts were obtained at the indicated time points as described under “Experimental Procedures.” Western blotting using the respective antibodies was performed after SDS-polyacrylamide gel electrophoresis in 15% gels. cleavage in death receptor-independent apoptosis as induced by etoposide has been shown to occur downstream of mitochon- drial cytochrome c release and was prevented by the caspase FIG.5. A, freshly isolated murine liver mitochondria were incubated inhibitor zVAD-fmk (46). To address whether the p53-mediated at 37 °C with cytosolic extracts from Saos-2 cells transduced with Ad cytochrome c release observed in our system was dependent on (upper panel) or with cytosolic extracts from non-apoptotic Saos-2 cells the caspase 8 cleavage of Bid, we again applied the broad transduced with Ad.p53 (lower panel). After the indicated periods, spectrum caspase inhibitor zVAD-fmk, which at a concentra- supernatants were analyzed for the release of cytochrome c by Western tion of 1 mM is equally potent against effector caspases such as blotting. For lane Z, the incubation was performed for 90 min in the presence of 10 mM zVAD-fmk. In lane P, the mitochondrial pellet after caspases 3 and 7 and against activator caspases such as the 0-min incubation is given as the internal control. B, aliquots of a caspases 8, 9, and 10 in vitro (47); the addition of 100 mM cytosolic extract from non-apoptotic Saos-2 cells transduced with zVAD-fmk directly after transduction with Ad.p53 did not pre- Ad.p53 were subjected to three rounds of immunodepletion using a vent mitochondrial cytochrome c release (Fig. 4B). Since normal rabbit serum (NRS), two monoclonal anti-p53 antibodies, a polyclonal anti-Bax antiserum, or a polyclonal anti-JNK1 antiserum caspase activity is required for Bid cleavage and activation via coupled to protein G-Sepharose beads. Upper panel, freshly isolated the death receptor FADD-caspase 8 pathway, this rules out a murine liver mitochondria were incubated at 37 °C for 60 min either role for this pathway in p53-induced cytochrome c release from with buffer, with the complete extract (Untreated), or with the respec- mitochondria in Saos-2 cells. tive immunodepleted extracts (NRS, p53-, bax-, and JNK1-). After centrifugation, supernatants were analyzed for the presence of cyto- p53 Induces Mitochondrial Cytochrome c Release by a Path- chrome c using Western blotting. A mitochondrial pellet is given as way Requiring Cytosolic Bax—To further characterize the positive control for cytochrome c (pellet). Note the reduction of cyto- mechanism of p53-induced cytochrome c release, cytosolic ex- chrome c release into the supernatant following incubation with the tracts from Saos-2 cells either transduced with Ad.p53 or with Bax-immunodepleted extract. Lower panel, untreated (1) and immu- nodepleted (2) extracts were analyzed for expression of p53 (left), Bax the empty Ad vector were prepared and incubated with freshly (middle), or JNK1 (right) using Western blotting. isolated murine liver mitochondria. Only extracts from cells expressing wt p53 contained a cytochrome c-releasing activity DISCUSSION in vitro, which was not inhibited by addition of zVAD-fmk (Fig. 5A), also ruling out the requirement of caspase activity for the From current understanding, there are two pathways trans- in vitro cytochrome c release by p53. When p53 was immunode- ducing a death signal to the apoptotic machinery. The “extrin- pleted from the cytosolic extract, the cytochrome c-releasing sic” pathway involves trimerization of death receptors such as activity was still retained (Fig. 5B). This argued against a CD95/Fas/APO-1 or TNF receptor 1 by binding of their respec- direct effect of p53 on mitochondria in our in vitro system. tive ligands, which leads to recruitment of the activator As p53 has been described to transactivate the proapoptotic caspase 8 via adapter molecules like FADD and TRADD and to Bcl-2 family member Bax in some cell types (13), which was its autoactivation (49). Activated caspase 8 either can directly shown to induce cytochrome c release when added to isolated cleave and activate the effector caspases (50, 51), or it can mitochondria (36, 48), the role of Bax in this in vitro system of cleave Bid to induce the release of mitochondrial cytochrome c, p53-mediated cytochrome c release was further investigated. which also leads to activation of effector caspases via oligomer- Immunodepletion of Bax, but not of p53 or of the control anti- ization with Apaf-1 and caspase 9 in the presence of ATP gen JNK1, abolished the cytochrome c-releasing activity from (29 –31). This caspase-dependent activation of the mitochon- the extract of p53 overexpressing cells. (Fig. 5B). Thus, the drial pathway may be important in cells with low concentra- cytochrome c-releasing activity of p53 was dependent on the tions of death receptors or caspase 8 and might act as an presence of Bax in the cytosol. amplification loop (42). There is evidence for a role of p53 in p53-mediated Apoptosis 7341 regulation of membrane expression of some death receptors in ptotic stimuli, therefore must occur downstream of caspase 3 activation via the cytochrome c-Apaf-1-caspase 9 pathway (46, several cell types (16 –19). In contrast, the death receptor-independent, intrinsic, apo- 62). Whereas no evidence was found for a direct effect of p53 itself on isolated mitochondria in vitro, by immunodepletion we ptotic pathway is directly activated by a death signal, leading could demonstrate the requirement of cytosolic Bax for the to the release of cytochrome c from the mitochondrial inter- cytochrome c-releasing activity of p53. membrane space into the cytosol, which then, in the presence of Based on our results and the results of others (36), we pro- ATP, facilitates oligomerization and activation of Apaf-1 and pose the following model for the induction of apoptosis by p53. caspase 9, leading to activation of caspase 3 and other effector Upon activation, p53 induces the release of mitochondrial cy- caspases (24). The release of cytochrome c is regulated by the tochrome c by a pathway involving cytosolic Bax. Cytosolic various pro- and anti-apoptotic members of the Bcl-2 family cytochrome c facilitates the activation of caspase 3 and other (23, 44, 48, 52–54). effector caspases by caspase 9 oligomerization with the adapter Many inducers of apoptosis, such as g-irradiation, anticancer molecule Apaf-1 in the presence of ATP. Whether p53 induces drugs, staurosporine, and growth factor withdrawal, were cytochrome c release by transcriptionally increasing cytosolic shown to activate apoptotic cell death independent of death Bax levels, which was observed in some human cell lines (13), receptor pathways, thus presumably directly activating this or whether p53 leads to conformational changes and mitochon- intrinsic apoptotic pathway (34, 46, 55–57). Furthermore, mice drial targeting of Bax by a pathway involving other Bcl-2 deficient in Apaf-1 or caspase 9, which in the intrinsic pathway family members (54, 63) or yet unidentified mediators requires act downstream of mitochondrial cytochrome c release, show further analysis. severe developmental defects leading to embryonic lethality, and their cells are resistant to death receptor-independent Acknowledgments—We thank Dr. B. B. Wolf and all other members induction of apoptosis by radiation and a variety of anticancer of the Division of Cellular Immunology for their help and advice. Drs. D. C. Maneval and I. Atencio, Canji Inc., are thanked for their support drugs (58 – 60). Whereas the mitochondrial and the down- and for providing adenoviral vectors. stream events in the intrinsic pathway are well elaborated, the understanding of the events upstream of mitochondria in this REFERENCES pathway is incomplete. A number of studies implies a central 1. Levine, A. J. 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p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 10, Issue of March 10, pp. 7337–7342, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release* (Received for publication, July 19, 1999, and in revised form, December 1, 1999) Martin Schuler‡, Ella Bossy-Wetzel§, Joshua C. Goldstein, Patrick Fitzgerald, and Douglas R. Green¶ From the Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121 The p53 tumor suppressor gene is critically involved CIP1 (2–5) or GADD45 (6), thus relying on the ability of p53 to act as a sequence-specific transcription factor. In contrast, the in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 tumor-suppressing activity of p53 does not depend on its trans- death signals lead to caspase activation; however, the activational function (7–10). mechanism of caspase activation by p53 still is unclear. It has been shown in several cell types that wild type (wt) Expressing wild type p53 by means of an adenoviral p53 is required for the apoptotic cell death as induced by expression vector, we were able to induce apoptotic cell g-irradiation or a variety of anticancer drugs (11, 12). Yet, the death, as characterized by morphological changes, pathways whereby p53 leads to execution of the apoptosis phosphatidylserine externalization, and internucleoso- program are not well characterized. Possible mechanisms in- null mal DNA fragmentation, in p53 Saos-2 cells. This cell clude transcriptional activation of the proapoptotic Bcl-2 family death was accompanied by caspase activation as well as member Bax (13, 14), the generation of reactive oxygen species by cleavage of caspase substrates and was preceded by (15), and transcriptional up-regulation of death receptors such mitochondrial cytochrome c release. The addition of the as CD95/Fas/APO-1 or DR5/KILLER (16 –19). However, sev- broad-spectrum caspase inhibitor benzyloxycarbonyl- eral lines of evidence imply that the proapoptotic activity of p53 Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly is independent of its function as a transcription factor (20 –22). after transduction almost completely prevented p53-in- The release of cytochrome c from mitochondria is a central duced apoptotic cell death but did not inhibit mitochon- event in the death receptor-independent, “intrinsic,” apoptotic drial cytochrome c release. In contrast, N-acetylcys- pathway (23, 24). Cytochrome c together with ATP and Apaf-1 teine, even at high concentrations, could not prevent facilitates activation by caspase 9 of the effector caspases (25– induction of programmed cell death by p53 expression. 28), which then cleave their substrates, finally leading to the Cytosolic extracts from Saos-2 cells transduced with apoptotic cell death. Furthermore, cytochrome c release can p53, but not from Saos-2 cells transduced with the empty also occur in death receptor-dependent, “extrinsic,” apoptotic adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of pathways by cleavage and activation of the proapoptotic Bcl-2 zVAD-fmk. When Bax was immunodepleted from the cy- family member Bid through caspase 8 (29 –31), possibly serving tosolic extracts of p53-expressing cells before incuba- as an amplification loop. Several studies were undertaken to tion with isolated mitochondria, the in vitro cytochrome establish the involvement of caspase activation in p53-medi- c release was abolished. Thus, we could demonstrate in ated cell death (18, 32–35). Recently, the requirement of Apaf-1 cells and in vitro that p53 activates the apoptotic ma- or caspase 9 for the p53-dependent apoptosis of oncogene-trans- chinery through induction of the release of cytochrome formed murine embryonic fibroblasts has been conclusively c from the mitochondrial intermembrane space. Fur- demonstrated (36). thermore, we provide in vitro evidence for the require- The present study was undertaken to address the hypothesis ment of cytosolic Bax for this cytochrome c-releasing that p53 might induce apoptosis by a death receptor-independ- activity of p53 in Saos-2 cells. ent pathway involving the release of mitochondrial cytochrome c. We show in Saos-2 cells that p53 evokes cytochrome c release prior to caspase activation and prior to the occurrence of apo- The p53 tumor suppressor gene is the central integrator of ptotic cell membrane changes. Furthermore, we biochemically the cellular response to DNA damage, oncogenic transforma- demonstrate the requirement of cytosolic Bax protein for the tion, and growth factor withdrawal (1). The cell cycle regula- cytochrome c-releasing activity of p53. Thereby, in our experi- tory and the DNA repair functions of p53 are largely executed mental system, we provide a link between p53 and the death by transactivation of p53-response genes such as p21/WAF1/ receptor-independent activation of the apoptotic machinery downstream of mitochondria. * This work was supported by National Institutes of Health Grants EXPERIMENTAL PROCEDURES CA69831 and AI40646 (to D. R. G.). This is publication No. 317 of the Cell Lines, Antibodies, and Reagents—The human osteosarcoma cell La Jolla Institute for Allergy and Immunology. The costs of publication line Saos-2 cells was obtained from ATCC (Manassas, VA) and was of this article were defrayed in part by the payment of page charges. maintained in McCoy’s 5A medium containing 15% fetal calf serum, This article must therefore be hereby marked “advertisement”inac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact. penicillin, and streptomycin. ‡ Recipient of a postdoctoral fellowship from the Dr. Mildred Scheel Antibodies against cytochrome c (clone 7H8.2C12), caspase 3 (poly- Stiftung fu ¨ r Krebsforschung, Germany. clonal rabbit antiserum), poly(ADP-ribose) polymerase (clone C2–10), § Supported by Swiss National Science Foundation Fellowship 823A-046638. To whom correspondence should be addressed: La Jolla Institute for The abbreviations used are: wt, wild type; zVAD-fmk, benzyloxy- Allergy and Immunology, 10355 Science Center Dr., San Diego, CA carbonyl-Val-Ala-Asp-fluoromethyl ketone; DEVD-afc, benzyloxycar- 92121. Tel.: 858-558-3500; Fax: 858-558-3526; E-mail: dgreen5240@ bonyl-Asp-Glu-Val-Asp aminofluoromethylcoumarin; PI, propidium aol.com. iodide. This paper is available on line at http://www.jbc.org 7337 This is an Open Access article under the CC BY license. 7338 p53-mediated Apoptosis p53 (clones Do-7 and PAb122), p21/WAF1/CIP1 (clone SX118), and Bax (polyclonal rabbit antiserum) were purchased from Pharmingen (San Diego, CA). Polyclonal rabbit antisera against protein kinase C-d and JNK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and an antibody against actin (clone C4) was obtained from ICN Phar- maceuticals (Costa Mesa, CA). A rabbit antiserum against caspase 9 has been described previously (37, 38). The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala- Asp-fluoromethyl ketone (zVAD-fmk) and the fluorochromic caspase substrate benzyloxycarbonyl-Asp-Glu-Val-Asp aminofluoromethylcou- marin (DEVD-afc) were purchased from Enzyme System Products (Livermore, CA). Vectors and Transduction—Replication-deficient adenoviral vectors were generous gifts from Drs. D. C. Maneval and I. Atencio, Canji Inc., San Diego, CA. The vectors either encoded the complete human wt p53 cDNA (Ad.p53) or green fluorescent protein (Ad.GFP) under the control of the cytomegalovirus immediate/early gene promotor (39, 40). The Ad.GFP vector was applied to determine infectibility and maximal tolerated dose of Saos-2 cells, which was found to be 10 particles/ml. For further experiments, the Ad.p53 vector was used for p53 gene transfer, and the empty adenoviral vector (Ad) served as control. Ap- proximately 18 h before transduction, cells were passaged in 10-cm culture dishes or 12-well plates (Fisher) at a density of 200,000/ml or 50,000/ml, respectively. Vectors were diluted in serum-free medium (Optimem I, Life Technologies, Inc.) at the appropriate dose (10 par- ticles/ml). Cells were pulsed with the vectors for 60 min, washed, and supplemented with fresh growth medium containing fetal calf serum. Flow Cytometry—For flow cytometry, cells were harvested by mild trypsinization, followed by washing with growth medium and phos- phate-buffered saline. Cell death was determined by two-color analysis of fluorescein isothiocyanate-labeled annexin V (CLONTECH Labora- tories) binding and propidium iodide (PI) uptake using a Becton Dick- inson FACSCalibur. Cell cycle analysis and quantitation of subdiploid FIG.1. A, Saos-2 cells were transduced in triplicates with the Ad.p53 DNA-containing nuclei was performed by detecting PI staining after vector (triangles) or the empty Ad vector cassette (squares), as described lysing the cells in HFS (0.1% sodium citrate, 0.1% Triton X-100, 50 in the text. Cells were harvested at the indicated time points (hours mg/ml PI) on ice for at least 30 min. from transduction) and were analyzed for annexin V binding and PI Preparation of Mitochondria-free Cytosolic Extracts and of Whole Cell uptake using dual-color flow cytometry. Mean percentages 6S.D. of Extracts—Cytosolic extracts were prepared as described previously (41, viable (annexin V/PI double-negative) cells are given. B, Saos-2 cells 42). In brief, cells were harvested by gently scraping and were incu- were treated with Ad.p53 (triangles) or empty Ad vector (squares) and bated in a buffer containing 220 mM mannitol and 60 mM sucrose on ice were permeabilized and stained with PI on ice for at least 30 min. The for 30 min. Then cells were broken in a Dounce homogenizer by 70 DNA content of 10,000 nuclei was assessed by flow cytometry, and mean values 6S.D. of percentages of nuclei with DNA content below 2N gentle strokes of a type B pestle. The homogenates were centrifuged at (sub-G fraction) of three experiments are given. 16,000 3 g for 15 min, and the mitochondria-free supernatants were 1 frozen at 270 °C until further analysis. Extracts of the pellets as well as whole cell extracts were obtained by dissolving in lysis buffer, followed RESULTS by repetitive vortexing and freeze-thawing. After centrifugation at null Expression of wt p53 Induces Apoptosis in p53 Saos-2 16,000 3 g, the supernatants were stored at 270 °C. Cells—First we assessed whether adenovirus-mediated p53 ex- Isolation of Mitochondria and in Vitro Cytochrome c Release Assay— pression resulted in apoptotic cell death and caspase activation Mitochondria were freshly isolated from the liver of 2-month-old mice in Saos-2 cells. Transduction with Ad.p53 induced phosphati- as described previously (41, 42). Aliquots of the mitochondrial prepara- tion were incubated with cytosolic extracts at 37 °C for various time dylserine externalization as detected by annexin V binding at periods. After centrifugation at 20,000 3 g for 15 min, the supernatants 24 h followed by membrane disruption, leading to PI uptake were analyzed for the presence of cytochrome c by Western blotting. starting at 36 h. No such effects were observed upon transduc- Mitochondrial pellets were resuspended in lysis buffer and were also tion with the same particle number of the empty Ad vector analyzed by Western blotting. cassette (Fig. 1A). Similarly, an increase in subdiploid DNA Western Blotting—The protein content of cytosolic or whole cell ex- content, indicating internucleosomal DNA fragmentation, was tracts was determined by the Bradford assay (Bio-Rad). For each time only observed in Saos-2 cells transduced with Ad.p53 but not in point, 25 mg of total protein were boiled in Laemmli buffer for 5 min Saos-2 cells transduced with Ad (Fig. 1B). followed by centrifugation. The supernatants were subjected to electro- phoresis in 8% or 15% SDS-polyacrylamide gels using a Bio-Rad minigel Caspase activity leading to cleavage of DEVD-afc was de- apparatus, followed by transfer on nitrocellulose membranes (Hybond tected in extracts from Saos-2 cells obtained at 24, 36, and 48 h ECL, Amersham Pharmacia Biotech). After blocking with phosphate- after transduction with Ad.p53 (Fig. 2A). Immunoblotting dem- buffered saline containing 5% nonfat dry milk and 0.1% Tween 20, the onstrated loss of procaspase 9, cleavage of procaspase 3, and membranes were exposed to the primary antibodies overnight at 4 °C cleavage of the caspase substrates poly(ADP-ribose) polymer- on a shaker. Before and after incubation with the horseradish peroxi- ase and protein kinase C-d. Expression of p53 protein as well as dase-conjugated secondary antibodies (Amersham Pharmacia Biotech), induction of the p53 target gene p21/WAF1/CIP1 could be de- the membranes were washed extensively using phosphate-buffered sa- line/Tween. Antibody binding was detected by enhanced chemolumines- tected as early as 12 h after transduction (Fig. 2B). cence (Super Signal; Pierce). p53-mediated Apoptosis in Saos-2 Cells Is Prevented by Detection of Caspase Activity—Activity of effector caspases was de- zVAD-fmk but Not by N-Acetylcysteine—Next, testing was con- termined in whole cell extracts using the fluorochromic caspase sub- ducted to determine if the apoptotic cell death observed after strate DEVD-afc by means of a SPECTRAFluor (Tecan, Research Tri- p53 expression in Saos-2 cells was dependent on caspase activ- angle Park, NC), as described previously (37). In brief, cleavage of ity. The addition of the broad spectrum caspase inhibitor DEVD-afc was assessed kinetically at 37 °C every 60 s for 30 min. The zVAD-fmk at a concentration of 100 mM almost completely enzymatic activity was expressed as the maximal velocity of the result- ing fluorescence-time curve. abrogated cell death (Fig. 3A) and DNA fragmentation (Fig. p53-mediated Apoptosis 7339 FIG.2. Whole cells extracts of Saos-2 cells transduced with Ad.p53 were obtained at the indicated time points (hours from transduction). A, the activity of DEVD-cleaving caspases (triangles)in the respective extracts was determined as described in the method section. RFU/s denotes relative fluorescence activity/s (right; y axis). The percentage of annexin V-positive cells was measured for identical time points using fluorescence-activated cell sorter analysis; mean val- ues 1 S.D. are given (gray bars; left, y axis). B, Western blotting of whole cell extracts using the indicated antibodies. 25 mg of total protein were loaded on each lane in 15% (caspase 3, procaspase 9, p21) or 8% (poly(ADP-ribose) polymerase (PARP), protein kinase C-d (PKC-d), p53) SDS-polyacrylamide gels. P21/WAF1/CIP1 is shown as a classical p53- responsive gene. Note the loss of procaspase 9, the occurrence of cleaved caspase 3, as well as cleavage of the caspase substrates poly(ADP- ribose) polymerase and protein kinase C-d at 24 and 36 h. FIG.3. Saos-2 cells were transduced with Ad.p53 or the empty Ad vector as described. Directly after transduction, cells were washed, and either fresh growth medium alone or medium supple- 3B), as induced by expression of wt p53 in Saos-2 cells. Several mented with zVAD-fmk (100 mM)or N-acetylcysteine (NAC,10mM) was mitochondrial genes potentially generating reactive oxygen added. Cells were harvested at the indicated time points and were species were reported to be induced by p53 overexpression (15), analyzed for annexin V binding, PI uptake, and cell cycle distribution. implicating a role of reactive oxygen species as downstream A, the bars indicate the mean percentage 1S.D. of annexin V-positive effectors of p53-mediated apoptosis. However, in Saos-2 cells, cells of three experiments; the black part of the bars indicate the fraction of annexin V-positive/PI-negative cells. B, the mean percentage the addition of N-acetylcysteine up to concentrations of 10 mM 6S.D. of nuclei with sub-G DNA content are given for each time point had no protective effect against p53-induced cell death (Fig. (hours from transduction). Closed triangles denote Saos-2 cells trans- 3A). Moreover, N-acetylcysteine at concentrations of 20 mM or duced with Ad.p53, and open triangles denote Saos-2 cells transduced above induced necrotic cell death in Saos-2 cells (data not with Ad.p53 in the presence of 100 mM zVAD-fmk. shown). null Cytochrome c Is Released from Mitochondria upon p53 Ex- the p53 human lung cancer cell line Calu-6 (data not pression—To assess whether p53 activates caspases by induc- shown). The proapoptotic Bcl-2 family member Bax, which ing the release of cytochrome c from the mitochondrial inter- induces mitochondrial cytochrome c release (44, 48), is reported membrane space into the cytosol (26, 43), subcellular to be transcriptionally activated by p53 in some human cell fractionation of Saos-2 cells was performed at various time lines (13). In concordance, an increase of Bax protein expres- points following transduction with Ad.p53 or with Ad. Cyto- sion was found in Saos-2 cells following p53 overexpression, but chrome c release into the cytosolic fraction of Saos-2 cells could significantly elevated Bax levels were detected only after the be detected as early as 12 h after transduction with Ad.p53; onset of cytochrome c release (Fig. 4A). this corresponded with a depletion of the mitochondria-contain- Ligation of death receptors such as CD95/Fas/APO-1 or tu- ing pellet fraction from cytochrome c (Fig. 4A). No such effect mor necrosis factor receptor 1 also can result in cytochrome c could be observed after transduction of Saos-2 cells with vector release via activation of caspase 8, followed by cleavage and alone (Fig. 4B). Similar kinetics of mitochondrial cytochrome c activation of Bid. Cleaved Bid can then target mitochondria release were observed after adenoviral expression of wt p53 in and induce the release of cytochrome c (29 –31). However, Bid 7340 p53-mediated Apoptosis FIG.4. Saos-2 cells growing in 10-cm dishes were transduced with Ad5.p53 (A)orAd(B, lanes 2 to 4) and cultured either in medium alone (A) or in medium supplemented with 100 mM zVAD-fmk (B, lanes 5 to 7). Cytosolic extracts were obtained at the indicated time points as described under “Experimental Procedures.” Western blotting using the respective antibodies was performed after SDS-polyacrylamide gel electrophoresis in 15% gels. cleavage in death receptor-independent apoptosis as induced by etoposide has been shown to occur downstream of mitochon- drial cytochrome c release and was prevented by the caspase FIG.5. A, freshly isolated murine liver mitochondria were incubated inhibitor zVAD-fmk (46). To address whether the p53-mediated at 37 °C with cytosolic extracts from Saos-2 cells transduced with Ad cytochrome c release observed in our system was dependent on (upper panel) or with cytosolic extracts from non-apoptotic Saos-2 cells the caspase 8 cleavage of Bid, we again applied the broad transduced with Ad.p53 (lower panel). After the indicated periods, spectrum caspase inhibitor zVAD-fmk, which at a concentra- supernatants were analyzed for the release of cytochrome c by Western tion of 1 mM is equally potent against effector caspases such as blotting. For lane Z, the incubation was performed for 90 min in the presence of 10 mM zVAD-fmk. In lane P, the mitochondrial pellet after caspases 3 and 7 and against activator caspases such as the 0-min incubation is given as the internal control. B, aliquots of a caspases 8, 9, and 10 in vitro (47); the addition of 100 mM cytosolic extract from non-apoptotic Saos-2 cells transduced with zVAD-fmk directly after transduction with Ad.p53 did not pre- Ad.p53 were subjected to three rounds of immunodepletion using a vent mitochondrial cytochrome c release (Fig. 4B). Since normal rabbit serum (NRS), two monoclonal anti-p53 antibodies, a polyclonal anti-Bax antiserum, or a polyclonal anti-JNK1 antiserum caspase activity is required for Bid cleavage and activation via coupled to protein G-Sepharose beads. Upper panel, freshly isolated the death receptor FADD-caspase 8 pathway, this rules out a murine liver mitochondria were incubated at 37 °C for 60 min either role for this pathway in p53-induced cytochrome c release from with buffer, with the complete extract (Untreated), or with the respec- mitochondria in Saos-2 cells. tive immunodepleted extracts (NRS, p53-, bax-, and JNK1-). After centrifugation, supernatants were analyzed for the presence of cyto- p53 Induces Mitochondrial Cytochrome c Release by a Path- chrome c using Western blotting. A mitochondrial pellet is given as way Requiring Cytosolic Bax—To further characterize the positive control for cytochrome c (pellet). Note the reduction of cyto- mechanism of p53-induced cytochrome c release, cytosolic ex- chrome c release into the supernatant following incubation with the tracts from Saos-2 cells either transduced with Ad.p53 or with Bax-immunodepleted extract. Lower panel, untreated (1) and immu- nodepleted (2) extracts were analyzed for expression of p53 (left), Bax the empty Ad vector were prepared and incubated with freshly (middle), or JNK1 (right) using Western blotting. isolated murine liver mitochondria. Only extracts from cells expressing wt p53 contained a cytochrome c-releasing activity DISCUSSION in vitro, which was not inhibited by addition of zVAD-fmk (Fig. 5A), also ruling out the requirement of caspase activity for the From current understanding, there are two pathways trans- in vitro cytochrome c release by p53. When p53 was immunode- ducing a death signal to the apoptotic machinery. The “extrin- pleted from the cytosolic extract, the cytochrome c-releasing sic” pathway involves trimerization of death receptors such as activity was still retained (Fig. 5B). This argued against a CD95/Fas/APO-1 or TNF receptor 1 by binding of their respec- direct effect of p53 on mitochondria in our in vitro system. tive ligands, which leads to recruitment of the activator As p53 has been described to transactivate the proapoptotic caspase 8 via adapter molecules like FADD and TRADD and to Bcl-2 family member Bax in some cell types (13), which was its autoactivation (49). Activated caspase 8 either can directly shown to induce cytochrome c release when added to isolated cleave and activate the effector caspases (50, 51), or it can mitochondria (36, 48), the role of Bax in this in vitro system of cleave Bid to induce the release of mitochondrial cytochrome c, p53-mediated cytochrome c release was further investigated. which also leads to activation of effector caspases via oligomer- Immunodepletion of Bax, but not of p53 or of the control anti- ization with Apaf-1 and caspase 9 in the presence of ATP gen JNK1, abolished the cytochrome c-releasing activity from (29 –31). This caspase-dependent activation of the mitochon- the extract of p53 overexpressing cells. (Fig. 5B). Thus, the drial pathway may be important in cells with low concentra- cytochrome c-releasing activity of p53 was dependent on the tions of death receptors or caspase 8 and might act as an presence of Bax in the cytosol. amplification loop (42). There is evidence for a role of p53 in p53-mediated Apoptosis 7341 regulation of membrane expression of some death receptors in ptotic stimuli, therefore must occur downstream of caspase 3 activation via the cytochrome c-Apaf-1-caspase 9 pathway (46, several cell types (16 –19). In contrast, the death receptor-independent, intrinsic, apo- 62). Whereas no evidence was found for a direct effect of p53 itself on isolated mitochondria in vitro, by immunodepletion we ptotic pathway is directly activated by a death signal, leading could demonstrate the requirement of cytosolic Bax for the to the release of cytochrome c from the mitochondrial inter- cytochrome c-releasing activity of p53. membrane space into the cytosol, which then, in the presence of Based on our results and the results of others (36), we pro- ATP, facilitates oligomerization and activation of Apaf-1 and pose the following model for the induction of apoptosis by p53. caspase 9, leading to activation of caspase 3 and other effector Upon activation, p53 induces the release of mitochondrial cy- caspases (24). The release of cytochrome c is regulated by the tochrome c by a pathway involving cytosolic Bax. Cytosolic various pro- and anti-apoptotic members of the Bcl-2 family cytochrome c facilitates the activation of caspase 3 and other (23, 44, 48, 52–54). effector caspases by caspase 9 oligomerization with the adapter Many inducers of apoptosis, such as g-irradiation, anticancer molecule Apaf-1 in the presence of ATP. Whether p53 induces drugs, staurosporine, and growth factor withdrawal, were cytochrome c release by transcriptionally increasing cytosolic shown to activate apoptotic cell death independent of death Bax levels, which was observed in some human cell lines (13), receptor pathways, thus presumably directly activating this or whether p53 leads to conformational changes and mitochon- intrinsic apoptotic pathway (34, 46, 55–57). Furthermore, mice drial targeting of Bax by a pathway involving other Bcl-2 deficient in Apaf-1 or caspase 9, which in the intrinsic pathway family members (54, 63) or yet unidentified mediators requires act downstream of mitochondrial cytochrome c release, show further analysis. severe developmental defects leading to embryonic lethality, and their cells are resistant to death receptor-independent Acknowledgments—We thank Dr. B. B. Wolf and all other members induction of apoptosis by radiation and a variety of anticancer of the Division of Cellular Immunology for their help and advice. Drs. D. C. Maneval and I. Atencio, Canji Inc., are thanked for their support drugs (58 – 60). Whereas the mitochondrial and the down- and for providing adenoviral vectors. stream events in the intrinsic pathway are well elaborated, the understanding of the events upstream of mitochondria in this REFERENCES pathway is incomplete. A number of studies implies a central 1. Levine, A. J. 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Published: Mar 1, 2000

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