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• W2051258629 abstract "A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochromec, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochromec plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL. A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochromec, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochromec plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL. dihexyloxacarbocyanine iodide carbamoyl cyaniden-chlorophenylhydrazone 4′6-diamino-2-phenylindole dihydrochloride carbonyl cyanidep-(trifluoromethoxy)phenylhydrazone benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone phosphate-buffered saline 1,4-piperazinediethanesulfonic acid. The stereotypical death throes of a cell undergoing apoptosis include DNA fragmentation, nuclear condensation, cell shrinkage, blebbing, and phosphatidylserine externalization (1Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12874) Google Scholar, 2Wyllie A.H. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6725) Google Scholar, 3Martin S.J. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Scie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar), all features that promote the physiologically silent removal of the cell by its phagocytic neighbors. A large body of evidence supports the idea that these events are mediated by the activation of several cytosolic proteases, the caspases, which then orchestrate apoptosis via the cleavage of key substrates (reviewed in Refs. 4Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 5Henkart P.A. Immunity. 1996; 4: 195-201Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 6Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 7Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (290) Google Scholar). For example, specific cleavage of two such substrates, PAK2 and DNA fragmentation factor, activate these proteins, mediating membrane blebbing and DNA fragmentation, respectively, without further requirements for the proteases (for these events) (8Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (604) Google Scholar, 9Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). But how are the caspases activated during apoptosis? Recent studies have delineated one key mechanism responsible for initiating the executioner phase of apoptosis. Early in the process, mitochondria release cytochrome c (10Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar), which upon entry into the cytosol forms a complex with another molecule, Apaf-1 (11Vaux D.L. Cell. 1997; 90: 389-390Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 12Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar), and the unprocessed (and inactive) proform of a caspase, caspase-9 (13Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar). In the presence of dATP or ATP, this complex processes and activates the caspase, which in turn can now trigger a cascade by processing and activating other caspases (in particular, caspases-3, -6, and -7) (13Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar,14Pan G. Humke E.W. Dixit V.M. FEBS Lett. 1998; 426: 151-154Crossref PubMed Scopus (110) Google Scholar). These then cleave key substrates and coordinate the process of apoptotic cell death. Bax is a pro-apoptotic Bcl-2-family protein (15Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5865) Google Scholar, 16Yin X.M. Oltvai Z.N. Veis-Novack D.J. Linette G.P. Korsmeyer S.J. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 387-393Crossref PubMed Scopus (73) Google Scholar) that resides in the cytosol and translocates to mitochondria upon induction of apoptosis (17Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1031) Google Scholar, 18Wolter 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 (1577) Google Scholar). Recently, Bax has been shown to induce cytochromec release and caspase activation in vivo (19Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (797) Google Scholar) andin vitro (20Jürgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar). This release was reportedly dependent upon induction of the mitochondrial permeability transition, an event that is associated with disruption of the mitochondrial inner transmembrane potential (ΔΨm) (21Pastorino 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 (534) Google Scholar) and has been implicated in a variety of apoptotic phenomena (22Zamzami N. Marchetti P. Castedo M. Zamin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1093) Google Scholar, 23Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1431) Google Scholar, 24Zarotti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar, 25Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsch T. Macho A. Haeffner A. Hirsch F. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (784) Google Scholar). Bcl-2 was found to be capable of inhibiting Bax-induced apoptosis but not Bax-induced cytochromec release in cells (19Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (797) Google Scholar). In contrast, Bcl-2 has been shown to be capable of blocking spontaneous cytochrome c release in cell-free extracts and in cells treated with apoptosis-inducing agents (26Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar, 27Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar). In the former, cytochrome c was able to completely bypass the anti-apoptotic effects of Bcl-2 (27Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar). Furthermore, in both cell-free systems and in cells undergoing apoptosis, the release of cytochromec can occur independently of changes in ΔΨm. We therefore examined the ability of Bax to induce the release of cytochrome c and apoptosis and evaluated the relationships between caspase activation, ΔΨm, and the effects of anti-apoptotic Bcl-2-family proteins. Human embryonic kidney cells (293T cells) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 2 mml-glutamine under standard conditions. CEM cells were grown in RPMI medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. Plasmid constructs pcDNA3, pcDNA3.Bax, and pcDNA3-myc-XIAP were generously provided by Dr. John Reed. Green fluorescent protein was purchased fromCLONTECH (Palo Alto, CA). cDNA encoding Bcl-xL was generously provided by Dr. Craig Thompson and cloned into theEcoRI site of pEF.neo. 3,3′-Dihexyloxacarbocyanine iodide (DiOC61(3Martin S.J. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Scie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar)), carbamoyl cyanide n-chlorophenylhydrazone (mCICCP), MitoTracker Orange, and rhodamine green (Rh123) were obtained from Molecular Probes, Inc. (Eugene, OR); DAPI and carbonyl cyanidep-(trifluoromethoxy)phenylhydrazone (FCCP) were from Sigma, and Phiphilux-G6D2 was from OncoImmunin, Inc. (Kensington, MD). Anti-Bax antibody was generously provided by Dr. John Reed (Burnham Institute, La Jolla, CA). Anti-poly(ADP-ribose) polymerase monoclonal antibody and anti-cytochrome c were purchased from Pharmingen (San Diego, CA). Anti-fodrin (nonerythroid spectrin) and anti-β-actin monoclonal antibody were purchased from Chemicon (Temecula, CA) and ICN (Irvine, CA), respectively. Anti-Bcl-xL was purchased from Santa Cruz Inc. (CA). A mixture of protease inhibitors (Complete™) was obtained from Boehringer Mannheim (Indianapolis, IN), and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) was obtained from Kamiya Biomedical Co. (Seattle, WA). Thrombin protease and glutathione-Sepharose-4B columns were purchased from Amersham Pharmacia Biotech. All other chemicals were obtained from Sigma. 293T cells were plated at 1 × 106/100-mm dish on day 0. On day 2, medium was changed, and cells were incubated for an additional 3 h. Cells were then transfected for 6 h using calcium phosphate, after which cells were washed with PBS, and fresh medium was added. Cells were harvested at various time points post-transfection. The amount of DNA in all transfection experiments was made equal by including respective amounts of vector. 293T cells were collected by centrifugation at 200 × g for 10 min at 4 °C. The cells were washed twice with ice-cold PBS, pH 7.2, followed by centrifugation at 200 × g for 5 min. The cell pellet was then resuspended in 500 μl of extraction buffer containing 220 mm mannitol, 68 mm sucrose, 50 mm Pipes-KOH, pH 7.4, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, and protease inhibitors. After a 30-min incubation on ice, cells were homogenized using a glass dounce and a B pestle (80 strokes). Cell homogenates were spun at 14 000 × g for 15 min, and supernatants were removed and stored at −80 °C until analysis by SDS-polyacrylamide gel electrophoresis. Between 15–35 μg of cytosolic protein extract was boiled for 5 min and loaded. Samples were resolved under reducing conditions for 2 h at 80 V on SDS-polyacrylamide gels as described previously. Separated proteins were then blotted onto polyvinylidene difluoride and nitrocellulose membranes at 120 mA overnight. The membranes were blocked for 2 h in PBST (10 mmTris-HCl, pH 7.4, 150 mm NaCl, 0.05% Tween) containing 5% nonfat dried milk and then probed overnight with an appropriate dilution of the primary antibody. Reactions were detected with suitable secondary antibody conjugated to horseradish peroxidase (The Jackson Laboratory, Bar Harbor, ME and Amersham Pharmacia Biotech) using enhanced chemiluminescence (Pierce). Nuclei from apoptotic cells undergoing DNA fragmentation contain subdiploid amounts of DNA and were therefore quantified by cell cycle analysis as described previously (28Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4426) Google Scholar). Morphological changes such as cell shrinkage, rounding, and membrane blebbing were evaluated by microscopic inspection of cells under phase contrast. Nuclear changes such as chromatin condensation and fragmentation and DEVD-like caspase activity were analyzed by staining with DAPI and Phiphilux-G6D2, respectively. Phiphilux-G6D2 is a fluorogenic substrate that is cleaved in a DEVD-dependent manner to produce rhodamine molecules, which fluoresce red under G2A filter, whereas DAPI stains nuclei (apoptotic or viable) blue under DAPI filter. Briefly, cells were plated on poly(d-lysine)-coated coverslips at 4 × 105/well in a 6-well plate 24 h before transfection. Cells were transfected with 0.5 μg of green fluorescent protein and 2 μg of pcDNA3.Bax or empty vector as described above. Cells were analyzed at 18 h post-transfection. Cells were washed twice in PBS and then incubated in Phiphilux-G6D2 (10 μm) at 37 °C for 1 h in the dark. Cells were then washed twice in PBS and stained for 3 min with DAPI (5 μm) at room temperature in 3.7% paraformaldehyde. Cells were then rinsed twice in PBS, mounted in PBS, and viewied by fluorescence microscopy. Changes in the inner mitochondrial transmembrane potential (ΔΨm) were determined by incubating 1 × 105 cells in 40 nm of DiOC6 (3Martin S.J. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Scie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar) or 150 nm MitoTracker Orange for 20 min at 37 °C. These two fluorochromes incorporate into cells dependent upon their mitochondrial transmembrane potential (29Petit P.X. O'Conner J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Crossref PubMed Scopus (213) Google Scholar). The cells were then scored using FACScan flow cytometery (Becton-Dickinson, Mountain View, CA). Controls were performed in the presence of 50 μm mitochondrial uncoupling agent mCICCP. In all cases, cells were gated to exclude cellular debris associated with necrosis. Assessment of mitochondrial transmembrane potential in isolated mitochondria was carried out by incubating 0.2 μg of mitochondria (prepared as described below) in 80 nm Rh123 and scoring immediately by FACScan flow cytometery. Controls were performed in the presence of FCCP (1 μm). DH-5α bacterial cells containing a pGEX-KG expression vector with the murine Bax protein lacking the C-terminal hydrophobic region (BaxΔC19, amino acids 1–173) were treated with 0.1 mmisopropyl-1-thio-b-d-galactopyranoside for 4–6 h at 30 °C. Bacterial cell pellets were lysed in 0.5 mm EDTA, 1 mm dithiothreitol, 1% Triton X-100, 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin in PBS and sonicated for 4 min on ice (output 6.5; duty 90%). Cell lysates were then centrifuged at 20 000 × g for 20 min at 4 °C. The supernatant was loaded onto a glutathione-Sepharose-4B column and the column washed with PBS. Bound GST-Bax protein was eluted from the column by thrombin protease treatment (10 units/liter). The eluate was incubated with 80 μg/mlN α-p-tosyl-l-lysine chloromethyl ketone protease inhibitor and dialyzed overnight against 20 mm Hepes-KOH pH 7.4, 10 mm KCl, 1.5 MgCl2, 1 mm dithiothreitol, 5 mmEDTA. Mitochondria were isolated from liver tissue of 6-week-old Balb/c mice. Briefly, the livers were taken and homogenized with a Teflon glass potter in Buffer A (0.2 m mannitol, 0.05 m sucrose, 1 mm EDTA, 10 mmKCl, 5 mm succinate, 10 mm Hepes-KOH, pH 7.4, and 0.1% bovine serum albumin). All steps were then carried out at 4 °C. Samples were centrifuged at 1,030 × g for 15 min. The supernatant was transferred to another tube and centrifuged at 3,300 × g for an additional 10 min. Pellets were resuspended in Buffer B (0.3 m mannitol, 5 mmpotassium phosphate, 10 mm Hepes-KOH, pH 7.4, and 0.1% bovine serum albumin) and centrifuged at 1,030 × g for 10 min. The supernatant was collected and centrifuged at 3 300 ×g for 10 min. Finally mitochondrial pellets were resuspended in MSH buffer containing an ATP regenerating system (210 mmmannitol, 70 mm sucrose, 10 mm Hepes-KOH, pH 7.4, 0.2 mm EGTA, 5 mm succinate, pH 7.0, 0.15% bovine serum albumin, 2 mm ATP, 1 mmdATP, 10 μm phosphocreatine, 50 μg/ml creatine kinase, 10 μg/ml leupeptin, 10 μg/ml aprotinin). The freshly isolated mitochondria were then incubated with recombinant Bax protein in the presence or absence of S-100 cytosolic extract. After 5 to 60 min at 37 °C, mitochondria were removed by centrifugation at 20 000 ×g, and supernatants were analyzed by immunoblotting as described above. Jurkat cells were grown for 3 days. Cell pellets were then resuspended in 20 mmextraction buffer (Hepes-KOH, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 1 mm sodium-EDTA, 1 mm sodium-EGTA, 1 mm dithiothreitol, 250 mm sucrose, 10 mm succinate, 10 μg/ml leupeptin, 10 μg/ml aprotinin), incubated for 30 min on ice, and lysed by homogenization using a glass dounce (40 strokes/B-pestle). Cell debris were removed by centrifugation at 20,000 ×g in an Eppendorf centrifuge for 15 min at 4 °C. Supernatants were re-centrifuged at 100 000 × g for 1 h in an Ultracentrifuge, and the resulting S-100 extracts were stored at −70 °C. 100 μg of freshly isolated mitochondria protein was incubated with various amounts of Bax protein in 500 μl of MSH buffer containing an ATP regenerating system, andA 520 was measured over time (20Jürgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 30Halestrap A.P. Quinlan P.T. Whipps D.E. Armston A.E. Biochem. J. 1986; 236: 779-789Crossref PubMed Scopus (68) Google Scholar). A decrease in light scattering is consistent with an increase in mitochondrial volume. As controls for mitochondrial swelling atractyloside (5 mm in Me2SO) and CaCl2 (100 μm) were used. Bax promotes apoptosis induced by removal of growth factors and other stimuli (15Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5865) Google Scholar, 18Wolter 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 (1577) Google Scholar, 31Deckwerth T.L. Elliott J.L. Knudson C.M. Johnson E.M. Snider W.D. Korsmeyer S.J. Neuron. 1996; 17: 401-411Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 32Sakakura C. Sweeney E.A. Shirahama T. Igarashi Y. Hakomori S. Tsujimoto H. Imanishi T. Ohyama T. Yamazaki J. Hagiwara A. Yamaguchi T. Sawai K. Takahashi T. Surg. 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Trends Genet. 1995; 11: 326-327Abstract Full Text PDF PubMed Scopus (95) Google Scholar). We monitored caspase activation in individual cells using a cell-permeable, fluorescent substrate (as described under “Materials and Methods”). As shown in Fig. 1 A, Bax-transfected cells exhibited both morphological and biochemical characteristics of apoptosis. Cells induced to die upon Bax expression appeared rounded and blebbed while displaying condensed chromatin, fragmented nuclei, and active DEVD-cleaving caspases. We then examined whether expression of Bax could induce the release of mitochondrial cytochromec. Fig. 1 B shows that as the levels of Bax protein increased, cytochrome c could be detected in the cytosol. In other experiments including earlier time points before 9 h (not shown), negligible Bax expression was observed with neither cytochrome c nor caspase activation detectable. We previously observed that the release of cytochrome cinduced by staurosporine or UVB irradiation occurs before and independently of caspase activation and subsequent apoptosis (38Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1997; 17: 37-49Crossref Scopus (1107) Google Scholar). To examine this with respect to Bax-induced cytochrome crelease, we treated Bax-transfected cells with the pan-caspase inhibitor zVAD-fmk. As described by others (39Loddick S.A. MacKenzie A. Rothwell N.J. Neuroreport. 1996; 7: 1465-1468Crossref PubMed Scopus (193) Google Scholar, 40Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 184: 2067-2072Crossref PubMed Scopus (271) Google Scholar, 41Jacobsen M.D. Weil M. Raff M.C. J. Cell Biol. 1996; 133: 1041-1051Crossref PubMed Scopus (366) Google Scholar, 42Milligan C.E. Prevette D. Yaginuma H. Homma S. Cardwell C. Fritz L.C. Tomaselli K.J. Oppenheim R.W. Schwartz L.M. Neuron. 1995; 15: 385-393Abstract Full Text PDF PubMed Scopus (298) Google Scholar, 43Slee E.A. Zhu H. Chow S.C. MacFarlane M. Nicholson D.W. Cohen G.M. Biochem. J. 1996; 315: 21-24Crossref PubMed Scopus (405) Google Scholar), zVAD-fmk significantly inhibited apoptosis during the period studied (24 h) (Fig. 2 A). As shown in Fig.2 B, this was not because of any effect on Bax expression levels. Bax-induced apoptosis corresponded to the activation of caspases and the subsequent cleavage of fodrin and poly(ADP-ribose) polymerase, two caspase substrates previously shown to be cleaved during apoptosis (44Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar), whereas the caspase inhibitor zVAD-fmk efficiently blocked this. Nevertheless, Bax-induced cytochromec release proceeded with the same kinetics with or without caspase inhibition (Fig. 2 B) as measured by cell fractionation and immunoblot analysis. This was confirmed by densitometric analysis of the cytochrome c immunoblots. At all time points, the cytosolic cytochrome c in the presence of Bax plus zVAD-fmk was ≥ that of Bax alone (data not shown).Figure 2Caspase inhibitors block Bax-induced apoptosis but have no effect on the kinetics of cytochrome crelease. A, 293T cells were treated with 100 μm zVAD-fmk and then transfected with 2 μg of Bax. Cells were harvested at various time points, and DNA fragmentation was assessed by cell cycle subdiploid DNA content. ○, Bax; •, Bax + zVAD-fmk. B, Western blot analysis of the time-course release of cytochrome c after Bax expression in the presence or absence of 100 μm zVAD-fmk. Fodrin and poly(ADP-ribose) polymerase (PARP) cleavage, substrates of caspases associated with apoptosis, were also analyzed. Actin was used as loading control. C, 293T cells were co-transfected with 2 μg of Bax and 2 μg XIAP and samples harvested at specific time points post-transfection and analyzed for DNA fragmentation. ○, Bax; •, Bax + XIAP. D, cells were either treated with zVAD-fmk or co-transfected with XIAP and analyzed by immunoblot for cytochromec release and poly(ADP-ribose) polymerase (PARP) cleavage after 18 h of Bax expression. 5 μg of isolated mitochondria fraction (Mito. Fr.) was used as a positive control for cytochrome c staining. Actin was used as loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As another approach to inhibiting caspase activation, we co-transfected Bax and a construct for expression of XIAP. Recent studies have shown that this molecule is a potent inhibitor of caspase function, including caspase activation by cytochrome c (45Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar). As shown in Fig. 2, XIAP coexpression completely blocked apoptotic cell death (Fig.2 C) and caspase substrate cleavage (Fig. 2 D) triggered by Bax. Nevertheless, XIAP expression had no effect on Bax-induced cytochrome c release (Fig. 2 D). Once again zVAD-fmk was demonstrated to be capable of blocking caspase activation but had no effect on cytochrome c release. Cell-free systems have been extremely valuable for the analysis of apoptosis mechanisms, including cytochrome c release from mitochondria (9Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 10Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 13Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar, 26Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar, 27Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar, 46Newmeyer D.D. Farschon D.M. Reed J.C. Cell. 1994; 79: 353-364Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 47Kluck R.M. Martin S.J. Hoffman B.M. Zhou J.S. Green D.R. Newmeyer D.D. EMBO J. 1997; 16: 4639-4649Crossref PubMed Scopus (360) Google Scholar). We therefore asked whether recombinant Bax protein can induce cytochrome c releasein vitro and whether this is a direct or indirect effect of the protein. As shown in Fig. 3, the addition of Bax to cytosolic extracts containing mitochondria induced a rapid release of cytochrome c. Similarly, the addition of Bax to isolated mitochondria rapidly induced cytochrome crelease, which was even more pronounced than that seen in the presence of cytosol. It appears, therefore, that this release is a direct effect of Bax on the mitochondria, as observed by others (19Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (797) Google Scholar, 20Jürgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar). Interestingly, the presence of cytosol appeared to delay the Bax-induced release of cytochrome c from mitochondriain vitro. It is possible that this can be simply explained by levels of inhibitors in the cytosol, such as Bcl-2 or other Bax-binding proteins, which may sequester Bax and thereby interfere with its activity. In many systems, apoptosis is associated with a loss of mitochondrial inner membrane p" @default.
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• W2051258629 title "Bax-induced Caspase Activation and Apoptosis via Cytochromec Release from Mitochondria Is Inhibitable by Bcl-xL" @default.
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