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Caspase-8 Mediates Mitochondrial Release of Pro-apoptotic Proteins in a Manner Independent of Its Proteolytic Activity in Apoptosis Induced by the Protein Synthesis Inhibitor Acetoxycycloheximide in Human Leukemia Jurkat Cells

2008; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês

10.1074/jbc.m808523200

ISSN

1083-351X

Autores

Kimiko Kadohara, Michiko Nagumo, Shun Asami, Yoshinori Tsukumo, Hikaru Sugimoto, Masayuki Igarashi, Kazuo Nagai, Takao Kataoka,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified. Jurkat cells deficient in caspase-8 were more resistant to Ac-CHX than wild-type Jurkat cells and manifested decreased apoptosis induction and caspase activation as well as inefficient release of cytochrome c, Smac/DIABLO, and AIF into the cytosol. In contrast to Fas ligand stimulation, the general caspase inhibitor barely prevented the mitochondrial release of these pro-apoptotic proteins in Ac-CHX-treated cells, suggesting that caspase-8 activity is dispensable for triggering the mitochondrial pathway in Ac-CHX-induced apoptosis. Consistent with this notion, caspase-8-deficient Jurkat cells reconstituted with catalytically inactive caspase-8 became sensitive to Ac-CHX and exhibited apoptosis, caspase activation, the liberation of pro-apoptotic proteins into the cytosol, and Bak conformational change as efficiently as wild-type Jurkat cells. Unlike caspase-3, -6, -7, and -9, a small but significant portion of caspase-8 was found to localize in mitochondria before and after exposure to Ac-CHX. These results clearly demonstrate that caspase-8 is able to mediate the mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in Ac-CHX-induced apoptosis. The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified. Jurkat cells deficient in caspase-8 were more resistant to Ac-CHX than wild-type Jurkat cells and manifested decreased apoptosis induction and caspase activation as well as inefficient release of cytochrome c, Smac/DIABLO, and AIF into the cytosol. In contrast to Fas ligand stimulation, the general caspase inhibitor barely prevented the mitochondrial release of these pro-apoptotic proteins in Ac-CHX-treated cells, suggesting that caspase-8 activity is dispensable for triggering the mitochondrial pathway in Ac-CHX-induced apoptosis. Consistent with this notion, caspase-8-deficient Jurkat cells reconstituted with catalytically inactive caspase-8 became sensitive to Ac-CHX and exhibited apoptosis, caspase activation, the liberation of pro-apoptotic proteins into the cytosol, and Bak conformational change as efficiently as wild-type Jurkat cells. Unlike caspase-3, -6, -7, and -9, a small but significant portion of caspase-8 was found to localize in mitochondria before and after exposure to Ac-CHX. These results clearly demonstrate that caspase-8 is able to mediate the mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in Ac-CHX-induced apoptosis. Caspases are a family of cysteine proteases essential for the regulation of apoptosis (1Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1288) Google Scholar, 2Riedl S.J. Shi Y. Nat. Rev. Mol. Cell Biol. 2004; 5: 897-907Crossref PubMed Scopus (1535) Google Scholar, 3Siegel R.M. Nat. Rev. Immunol. 2006; 6: 308-317Crossref PubMed Scopus (258) Google Scholar). 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Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3417) Google Scholar, 29Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y.J. Sakaki T. Elia A.J. Cheng H.Y.M. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zúñiga-Pflücker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Crossref PubMed Scopus (1140) Google Scholar). The protein synthesis inhibitor cycloheximide (CHX) has been reported to induce apoptosis in a number of cell types (30Martin S.J. Lennon S.V. Bonham A.M. Cotter T.G. J. Immunol. 1990; 145: 1859-1867PubMed Google Scholar, 31Tsuchida H. Takeda Y. Takei H. Shinozawa H. Takahashi T. Sendo F. J. Immunol. 1995; 154: 2403-2412PubMed Google Scholar, 32Tang D. Lahti J.M. Kidd V.J. J. Biol. 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Moreover, Ac-CHX has been shown to induce strong activation of the c-Jun N-terminal kinase (JNK) pathway and to trigger the release of cytochrome c into the cytosol (42Kadohara K. Tsukumo Y. Sugimoto H. Igarashi M. Nagai K. Kataoka T. Biochem. Pharmacol. 2005; 69: 551-560Crossref PubMed Scopus (10) Google Scholar). In this study, we further investigated the molecular mechanism by which Ac-CHX induces rapid apoptosis. Our results surprisingly provide evidence for the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins. Cells—Human leukemia Jurkat cells, caspase-8-deficient Jurkat cell clone (I9.2) (43Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), FADD-deficient Jurkat cell clone (I2.1) (44Juo P. Woo M.S.A. Kuo C.J. Signorelli P. Biemann H.P. Hannun Y.A. Blenis J. Cell Growth & Differ. 1999; 10: 797-804PubMed Google Scholar), c-FLIPL-transfected Jurkat cell clone (JFL2) (45Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schröter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2205) Google Scholar), and Bcl-xL-transfected Jurkat cell clone (Jurkat-Bcl-xL#1) (42Kadohara K. Tsukumo Y. Sugimoto H. Igarashi M. Nagai K. Kataoka T. Biochem. Pharmacol. 2005; 69: 551-560Crossref PubMed Scopus (10) Google Scholar) were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal calf serum (JRH Bioscience, Lenexa, KS), 100 units/ml penicillin G (Sigma), and 100 μg/ml streptomycin (Sigma). Reagents—Ac-CHX was isolated from the culture broth of an unidentified actinomycete strain designated ML44-113F2, as previously described (41Sugimoto H. Kataoka T. Igarashi M. Hamada M. Takeuchi T. Nagai K. Biochem. Biophys. Res. Commun. 2000; 277: 330-333Crossref PubMed Scopus (26) Google Scholar). CHX and curcumin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) and SP600125 were purchased from the Peptide Institute (Osaka, Japan) and Biomol Research Laboratories (Plymouth Meeting, PA), respectively. Antibodies—Antibodies to AIF (E-1, Santa Cruz Biotechnology, Santa Cruz, CA), Bak (Ab-1, Calbiochem), Bak (NT, Millipore, Lake Placid, NY), β-actin (AC-15, Sigma), Bcl-2 (clone 7, BD Bioscience, Franklin Lakes, NJ), Bid (550365, BD Biosciences), caspase-3 (H-277, Santa Cruz Biotechnology), caspase-6 (3E8, Medical & Biological Laboratories (MBL), Nagoya, Japan), caspase-7 (11E4, Sigma), caspase-8 (5D3, MBL), caspase-8 (5F7, MBL), caspase-9 (5B4, MBL), cytochrome c oxidase (CoxIV) (20E8, Santa Cruz Biotechnology), cytochrome c (7H8.2C12, BD Biosciences), FADD (clone 1, BD Biosciences), Fas (B-10, Santa Cruz Biotechnology), FLAG (M2, Sigma), c-FLIP (Dave-II, Alexis, Lausen, Switzerland), JNK (#9252, Cell Signaling Technology, Inc., Beverly, MA), phospho-JNK (#9251, Cell Signaling), and Smac/DIABLO (clone 7, BD Biosciences) were commercially obtained. Rat anti-mouse caspase-8 (clone 3B10) monoclonal antibody was prepared as described previously (46O'Reilly L.A. Divisekera U. Newton K. Scalzo K. Kataoka T. Puthalakath H. Ito M. Huang D.C. Strasser A. Cell Death Differ. 2004; 11: 724-736Crossref PubMed Scopus (42) Google Scholar). Assay for Apoptosis—Cells were fixed with PBS containing 4% paraformaldehyde at 4 °C overnight, and then stained with 300 μm Hoechst 33342 (Calbiochem). Nuclear morphology was observed under a fluorescence light microscope (Axiovert 200M, Carl Zeiss, Jena, Germany). Apoptotic cells (%) were calculated as (condensed nuclei/total nuclei) × 100. Western Blotting—Cells were washed with PBS and lysed in Triton X-100 lysis buffer consisting of 50 mm Tris-HCl (pH 7.4), 1% Triton X-100, 2 mm dithiothreitol, 2 mm sodium orthovanadate, and the protease inhibitor mixture Complete™ (Roche Diagnostics, Mannheim, Germany). Post-nuclear lysates were collected as supernatants by centrifugation (15,000 × g, 5 min). Protein samples (30 μg/lane) were separated by SDS-PAGE and transferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The transferred membranes were stained with Ponceau S and checked for equal loading of proteins before antibody reaction. The membranes were incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA), followed by analysis using ECL Western blotting detection reagents (GE Healthcare). Subcellular Fractionation—Cells were washed with PBS and lysed with digitonin lysis buffer (10 mm Hepes-KOH (pH 7.2), 100 μm digitonin, 250 mm sucrose, 1 mm dithiothreitol, 5 mm EGTA, 2 mm MgCl2, 50 mm NaCl, and the protease inhibitor mixture Complete™) on ice for 15 min. After centrifugation (10,000 × g, 5 min), cell lysates containing cytosol were recovered as supernatants. Pellets were washed once with digitonin lysis buffer and treated with Triton X-100 lysis buffer on ice for 15 min. After centrifugation (10,000 × g, 5 min), cell lysates containing mitochondria were collected as supernatants (mitochondrial fraction). Cytosolic and mitochondrial fractions were analyzed by Western blotting. Alternatively, cells were washed with PBS and Dounce-homogenized with sucrose homogenization buffer (10 mm Hepes-NaOH (pH 7.4), 250 mm sucrose, 1 mm EDTA, 1 mm EGTA, Complete™). After repeated centrifugation (800 × g, 5 min) to remove nuclei and unbroken cells, supernatants were further centrifuged (10,000 × g, 10 min). Resultant precipitates were further washed twice with sucrose homogenization buffer to prepare mitochondrial fraction. Cytosolic fractions were recovered as supernatants after centrifugation (100,000 × g, 60 min). Cytosolic and mitochondrial fractions were analyzed by Western blotting. RNAi—Stealth™ RNAi duplexes targeting Bid #1(5′-GGGAAGAAUAGAGGCAGAUUCUGAA-3′) and Bid #2 (5′-AAUAGAGGCAGAUUCUGAAAGUCAA-3′) and negative control duplexes (Low GC duplex #2) were purchased from Invitrogen. Cells were diluted 1 day before transfection, and then suspended with GenePulser electroporation buffer (Bio-Rad). The cells were transfected with 200 nm Stealth™ RNAi duplexes using the GenePulser Xcell electroporation system (Bio-Rad) and incubated for 48 h prior to analysis. Cell Transfection—Cells were diluted 1 day before transfection and washed three times with fetal calf serum-free RPMI 1640 medium. The cells were transfected with pEF pGK puro expression vectors (47Huang D.C.S. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (230) Google Scholar) encoding mouse wild-type caspase-8 or mouse proteolytically inactive caspase-8 mutant (C362G) (48Kataoka T. Tschopp J. Mol. Cell Biol. 2004; 24: 2627-2636Crossref PubMed Scopus (190) Google Scholar) or pEF pGK hygro expression vectors encoding FLAG-tagged human Bcl-2 (47Huang D.C.S. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (230) Google Scholar) using the GenePulser Xcell electroporation system. The cells were cultured for 48 h without selection and then cultured in microtiter plates in the presence of puromycin (5 μg/ml, Sigma) or hygromycin (1 mg/ml, Nacalai Tesque, Inc., Kyoto, Japan). Puromycin- and hygromycin-resistant clones expressing mouse caspase-8 and human Bcl-2 were selected by Western blotting using anti-mouse caspase-8 antibody 3B10 and anti-FLAG antibody M2, respectively. Assay for Mitochondrial Membrane Potential—Cells were incubated with RPMI 1640 medium containing 40 nm 3,3′-dihexyloxacarbocyanine iodide for 15 min. The cells were washed three times with PBS containing 2% fetal calf serum and 0.02% sodium azide, and analyzed with a FACSCalibur™ flow cytometer (BD Biosciences). Detection of Bak Conformational Change—Bak conformational change was detected basically as described previously (49Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (393) Google Scholar, 50Takahashi Y. Karbowski M. Yamaguchi H. Kazi A. Wu J. Sebti S.M. Youle R.J. Wang H.G. Mol. Cell Biol. 2005; 25: 9369-9382Crossref PubMed Scopus (155) Google Scholar, 51Nguyen T.K. Rahmani M. Harada H. Dent P. Grant S. Blood. 2007; 109: 4006-4015Crossref PubMed Scopus (53) Google Scholar). Cells were washed with PBS and lysed with CHAPS lysis buffer (10 mm Hepes-NaOH (pH 7.4), 1% CHAPS, 150 mm NaCl, Complete™) on ice for 15 min. After centrifugation (10,000 × g, 5 min), cell lysates were precleared with isotype-specific control antibody (Invitrogen) and then immunoprecipitated with mouse monoclonal anti-Bak antibody (Ab-1) and protein G Plus-agarose (Calbiochem). The resultant immunoprecipitates containing conformationally changed Bak were separated by SDS-PAGE and analyzed by Western blotting using rabbit polyclonal anti-Bak antibody (NT). Caspase-8 Is Responsible for Apoptosis Induced by Ac-CHX—Ac-CHX induced apoptosis in human leukemia Jurkat cells at concentrations >0.1 μm (Fig. 1A). Upon treatment with Ac-CHX, Jurkat cells started to die within 2 h and steadily underwent apoptosis thereafter (Fig. 1B). The kinetics of Ac-CHX-induced apoptosis is very rapid and seems similar to that of Fas ligand (FasL)-induced apoptosis. Caspase-8 plays an essential role in FasL-induced apoptosis (4Nagata S. Ann. Rev. Genet. 1999; 33: 29-55Crossref PubMed Scopus (666) Google Scholar, 5Siegel R.M. Chan F.K.M. Chun H.J. Lenardo M.J. Nat. Immunol. 2000; 1: 469-474Crossref PubMed Scopus (348) Google Scholar, 6Peter M.E. Krammer P.H. Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (884) Google Scholar, 7Krammer P.H. Arnold R. Lavrik I.N. Nat. Rev. Immunol. 2007; 7: 532-542Crossref PubMed Scopus (472) Google Scholar). To determine if caspase-8 is responsible for Ac-CHX-induced apoptosis, Jurkat cells deficient in caspase-8 were compared with wild-type cells for the induction of apoptosis following exposure to Ac-CHX. Caspase-8-deficient cells were more resistant to Ac-CHX-induced apoptosis (Fig. 1A) and manifested slower induction of apoptosis upon treatment with Ac-CHX (Fig. 1B), although they were completely resistant to FasL-induced apoptosis (Fig. 1C). Approximately 50% of caspase-8-deficient cells underwent apoptosis when they were treated with Ac-CHX (1 μm) for 5 h. These results indicate that Ac-CHX is able to induce apoptosis in a manner independent of caspase-8. Nevertheless, especially at early time points upon exposure to Ac-CHX, it seems most likely that caspase-8 is required for accelerated induction of apoptosis. Caspase-8 Is Responsible for Caspase Activation Induced by Ac-CHX—In wild-type Jurkat cells, Ac-CHX induced processing of caspase-3 and caspase-9 into their active forms at concentrations >0.1 μm (Fig. 2A) and within 2 h (Fig. 2B). In caspase-8-deficient cells exposed to Ac-CHX, processing of caspase-3 and caspase-9 was greatly decreased (Fig. 2, A and B). Consistent with these results, in caspase-8-deficient cells, Ac-CHX induced little processing of caspase-6 and caspase-7 into their active forms (Fig. 2C). Thus, these results indicate that caspase-8 is responsible for caspase activation induced by Ac-CHX. Caspase-8 Is Responsible for Mitochondrial Release of Pro-apoptotic Proteins in Ac-CHX-induced Apoptosis—Mitochondria play a central role in the induction of apoptosis via the release of pro-apoptotic proteins into the cytosol (16Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (92) Google Scholar, 17Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar, 18Chipuk J.E. Bouchier-Hayes L. Green D.R. Cell Death Differ. 2006; 13: 1396-1402Crossref PubMed Scopus (413) Google Scholar, 19Garrido C. Galluzzi L. Brunet M. Puig P.E. Didelot C. Kroemer G. Cell Death Differ. 2006; 13: 1423-1433Crossref PubMed Scopus (811) Google Scholar). Upon treatment with Ac-CHX, cytochrome c and Smac/DIABLO were released into the cytosol within 2 h, accompanied by their profound reduction in mitochondria (Fig. 3). AIF was also released into the cytosol in Ac-CHX-treated cells, although a large part of AIF was still retained in mitochondria (Fig. 3). In caspase-8-deficient cells, these apoptotic factors were only weakly released into the cytosol upon exposure to Ac-CHX (Fig. 3). These data indicate that caspase-8 is responsible for the mitochondrial release of pro-apoptotic proteins in Ac-CHX-induced apoptosis. However, because the general caspase inhibitor z-VAD-fmk prevented apoptosis induced by Ac-CHX (42Kadohara K. Tsukumo Y. Sugimoto H. Igarashi M. Nagai K. Kataoka T. Biochem. Pharmacol. 2005; 69: 551-560Crossref PubMed Scopus (10) Google Scholar), it seems that the release of Smac/DIABLO and AIF is not essential for induction of apoptosis by Ac-CHX. However, these changes are considered to be useful markers for the mitochondrial activation during Ac-CHX-induced apoptosis. Caspase Activity Is Dispensable for Mitochondrial Release of Pro-apoptotic Proteins in Ac-CHX-induced Apoptosis—Caspase-8 is known to cleave Bid into its truncated form that translocates to mitochondria and induces the release of pro-apoptotic proteins into the cytosol in FasL-induced apoptosis (13Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 14Li H. Zhu H. Xu C. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar). In contrast to FasL stimulation, z-VAD-fmk barely prevented the release of cytochrome c, Smac/DIABLO, and AIF into the cytosol in Ac-CHX-treated cells (Fig. 4A). The possibility that z-VAD-fmk is insufficient to block caspase activity is excluded by the observations that z-VAD-fmk at the same concentration totally prevented the release of cytochrome c, Smac/DIABLO, and AIF into the cytosol in FasL-treated cells (Fig. 4A) and that z-VAD-fmk suppressed caspase activity induced by Ac-CHX to background levels as measured using fluorescent caspase substrates (42Kadohara K. Tsukumo Y. Sugimoto H. Igarashi M. Nagai K. Kataoka T. Biochem. Pharmacol. 2005; 69: 551-560Crossref PubMed Scopus (10) Google Scholar). Full-length Bid (p24) was cleaved into its truncated form (p15) in wild-type Jurkat cells but not caspase-8-deficient cells when treated with FasL (Fig. 4B). However, Bid processing was still observed in caspase-8-deficient cells during Ac-CHX-induced apoptosis (Fig. 4B). Because Ac-CHX-induced Bid processing in caspase-8-deficient cells was prevented by z-VAD-fmk (Fig. 4C), it seems that Bid is cleaved not by caspase-8 but by other caspases in Ac-CHX-treated cells. To exclude the possibility that Bid is required for Ac-CHX-induced mitochondrial release of pro-apoptotic proteins, wild-type Jurkat cells were transiently transfected with the Stealth™ RNAi duplexes targeting Bid (Fig. 4D). Knockdown of Bid failed to affect the release of cytochrome c, Smac/DIABLO, and AIF into the cytosol upon 2-h treatment with Ac-CHX (Fig. 4E). Taken together, these data suggest that caspase activity is dispensable for the mitochondrial release of pro-apoptotic proteins in Ac-CHX-induced apoptosis. Caspase-8 Is Able to Induce Apoptosis Without Its Enzymatic Activity in Ac-CHX-treated Cells—To determine if the enzymatic activity of caspase-8 is necessary for Ac-CHX-induced apoptosis, caspase-8-deficient Jurkat cells were reconstituted with wild-type caspase-8 or catalytically inactive caspase-8 mutant (C362G). Three independent transfectants expressing wild-type caspase-8 (JC8WT1, JC8WT2, and JC8WT3) and inactive caspase-8 (JC8IN1, JC8IN2, and JC8IN3) at endogenous levels comparable to those in B lymphoma A20 cells were established (Fig. 5A). These transfectants expressed FADD and c-FLIPL at similar levels to wild-type and caspase-8-deficient cells, whereas caspase-8-deficient cells and their transfectants expressed slightly decreased levels of Fas compared with wild-type cells (Fig. 5A). Caspase-8 was processed into its active form containing a large subunit (p20) only when wild-type caspase-8 transfectants (JC8WT1, JC8WT2, and JC8WT3), but not inactive caspase-8 transfectants (JC8IN1, JC8IN2, and JC8IN3), were exposed to FasL (Fig. 5B). Consistent with these results, FasL was able to induce apoptosis in wild-type caspase-8 transfectants (JC8WT1, JC8WT2, and JC8WT3) but not inactive caspase-8 transfectants (JC8IN1, JC8IN2, and JC8IN3) (Fig. 5D). Therefore, these data clearly indicate that the enzymatic activity of caspase-8 is essential for FasL-induced apoptosis. By contrast, caspase-8 was processed into the p43 intermediate form but not the p20 large subunit when both wild-type caspase-8 transfectants (JC8WT1, JC8WT2, and JC8WT3) and inactive caspase-8 transfectants (JC8IN1, JC8IN2, and JC8IN3) were treated with Ac-CHX (Fig. 5C). Therefore, it seems that caspase-8 is cleaved not by self-processing but by other downstream caspases in Ac-CHX-treated cells. This notion was further supported by the observation that caspase-8 processing did not occur in Bcl-xL-overexpressing cells during Ac-CHX-induced apoptosis, whereas FasL stimulation induced processing of caspase-8 into the active form containing the large subunit (supplemental Fig. S1 and Ref. 42Kadohara K. Tsukumo Y. Sugimoto H. Igarashi M. Nagai K. Kataoka T. Biochem. Pharmacol. 2005; 69: 551-560Crossref PubMed Scopus (10) Google Scholar). As shown in Figs. 1 and 5E, compared with wild-type cells, Ac-CHX did not efficiently induce apoptosis in caspase-8-deficient cells. In contrast to FasL

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