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• W2000601138 abstract "Bcl-2 and Bcl-xL are reported to inhibit CD95-mediated apoptosis in “type II” but not in “type I” cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-xL displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-xL inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-xL proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype. Bcl-2 and Bcl-xL are reported to inhibit CD95-mediated apoptosis in “type II” but not in “type I” cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-xL displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-xL inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-xL proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype. Apoptosis, or programmed cell death, can be induced through two basic and distinct cell death signaling pathways, both of which culminate in the activation of cysteinyl aspartate-specific proteases or caspases. Stimulation of death receptors, such as CD95, TNFR1, and DR5, leads to formation of the death-inducing signaling complex (DISC), 1The abbreviations used are: DISCdeath-inducing signaling complexXIAPX-linked inhibitor-of-apoptosis proteinIAPinhibitor-of-apoptosis proteinBIRbaculoviral repeatCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPARPpoly(ADP-ribose) polymeraseTRAILTNF-related apoptosis-inducing ligand (where TNF is tumor necrosis factor)DEVDaseeffector caspase activity to cleave Z-DEVD.AFC (benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) which minimally contains the receptor, the adapter protein Fas-associated death domain, and caspase-8 (1.Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1043) Google Scholar, 2.Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1131) Google Scholar, 3.Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (586) Google Scholar, 4.Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar, 5.Sprick M.R. Weigand M.A. Rieser E. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). Similarly, various stressors, including toxicants and radiation, can induce mitochondrial release of cytochrome c and formation of the apoptosome, a complex that contains the adapter protein apoptotic protease-activating factor-1 (Apaf-1) and caspase-9 (6.Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7.Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 8.Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 9.Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). All caspases are synthesized as single chain zymogens, possessing a prodomain, a large (∼20 kDa) subunit, and a small (∼10 kDa) subunit. Caspases-8 and -9 are referred to as apical caspases, because they contain long prodomains that allow them to interact with their respective adapter proteins and undergo proximity-induced, autocatalytic activation. In this model, one caspase-8 or caspase-9 molecule within the DISC or apoptosome, respectively, activates another by cleaving between its large and small subunits. The activated apical caspases then propagate the death signal by activating short prodomain effector caspases-3 and/or -7, which proteolytically dismantle the cell (10.Bratton S.B. Cohen G.M. Trends Pharmacol. Sci. 2001; 22: 306-315Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11.Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (773) Google Scholar). death-inducing signaling complex X-linked inhibitor-of-apoptosis protein inhibitor-of-apoptosis protein baculoviral repeat 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid poly(ADP-ribose) polymerase TNF-related apoptosis-inducing ligand (where TNF is tumor necrosis factor) effector caspase activity to cleave Z-DEVD.AFC (benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) Inhibitor-of-apoptosis proteins (IAPs) are a family of proteins that contain baculoviral repeat (BIR) domains and, in some cases, a zinc RING-finger domain (12.Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2285) Google Scholar). X-linked IAP (XIAP), Livin/ML-IAP, cIAP-1, and cIAP-2 are believed to inhibit apoptosis through their direct inhibition of caspases, although some of these proteins are also involved in additional signaling pathways (13.Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1724) Google Scholar, 14.Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1246) Google Scholar, 15.Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar, 16.Kasof G.M. Gomes B.C. J. Biol. Chem. 2001; 276: 3238-3246Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 17.Vucic D. Stennicke H.R. Pisabarro M.T. Salvesen G.S. Dixit V.M. Curr. Biol. 2000; 10: 1359-1366Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 18.Holcik M. Korneluk R.G. Nature Rev. Mol. Cell Biol. 2001; 2: 550-556Crossref PubMed Scopus (234) Google Scholar). 2B. W. M. Richter and C. S. Duckett, www.stke.org/cgi/content/full/OC_sigtrans; 2000/44/pe 1, 1–4. XIAP, the most potent of these caspase inhibitors, selectively inhibits one of the active forms of caspase-9 (p35/p12 heterotetramer) through an interaction involving its BIR3 domain and the small subunit (p12) of caspase-9 (19.Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (682) Google Scholar, 20.Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (863) Google Scholar, 21.Sun C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). In contrast, the BIR2 domain of XIAP, along with a few critical adjacent residues, is required to inhibit active caspases-3 and -7 (22.Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 818-822Crossref PubMed Scopus (298) Google Scholar, 23.Chai J. Shiozaki E. Srinivasula S.M. Wu Q. Datta P. Alnemri E.S. Shi Y. Cell. 2001; 104: 769-780Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 24.Riedl S.J. Renatus M. Schwarzenbacher R. Zhou Q. Sun C. Fesik S.W. Liddington R.C. Salvesen G.S. Cell. 2001; 104: 791-800Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 25.Huang Y. Park Y.C. Rich R.L. Segal D. Myszka D.G. Wu H. Cell. 2001; 104: 781-790Abstract Full Text Full Text PDF PubMed Google Scholar). Consequently, XIAP is thought to inhibit death receptor-induced apoptosis by inhibiting effector caspases and mitochondrial induced apoptosis by inhibiting both apical and effector caspases (26.Bratton S.B. Walker G. Srinivasula S. Sun X.-M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-2001Crossref PubMed Scopus (342) Google Scholar). Recently, a structural homologue of the Drosophila proteins, Reaper, Hid, and Grim, has been identified and termed Smac/DIABLO (27.Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 28.Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar). This protein is normally localized to mitochondria but, like cytochrome c, is released into the cytosol during the early stages of apoptosis, where it appears to promote caspase activity by inhibiting IAPs, particularly XIAP (27.Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 28.Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar, 29.Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (715) Google Scholar, 30.Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (716) Google Scholar, 31.Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (548) Google Scholar, 32.Ekert P.G. Silke J. Hawkins C.J. Verhagen A.M. Vaux D.L. J. Cell Biol. 2001; 152: 483-490Crossref PubMed Scopus (163) Google Scholar). In some cells, stimulation of death receptors can lead to caspase-8-mediated cleavage and activation of the proapoptotic Bcl-2 protein, Bid (33.Gross A. Yin X.M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar, 34.Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 35.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). Truncated Bid stimulates formation of Bax (and/or Bak) pores in the outer mitochondrial membrane, which mediate the release of cytochrome c and, consequently, formation of the Apaf-1/caspase-9 apoptosome (36.Korsmeyer S.J. Wei M.C. Saito M. Weiler S. Oh K.J. Schlesinger P.H. Cell Death Differ. 2000; 7: 1166-1173Crossref PubMed Scopus (851) Google Scholar). In contrast, antiapoptotic Bcl-2 and Bcl-xL proteins, in addition to inhibitors of Bax, such as the viral protein E1B 19K, appear to inhibit cell death by blocking formation of these cytochrome c-releasing pores (36.Korsmeyer S.J. Wei M.C. Saito M. Weiler S. Oh K.J. Schlesinger P.H. Cell Death Differ. 2000; 7: 1166-1173Crossref PubMed Scopus (851) Google Scholar, 37.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Thus, in death receptor-induced apoptosis, protection by Bcl-2 or Bcl-xL is an indicator of the requirement for mitochondrial involvement, and for the sake of convenience, those cells that are protected by Bcl-2 and Bcl-xL have been termed type II cells, whereas those that are not protected are termed type I cells (38.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar, 39.Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). However, the role of mitochondria in death receptor-induced apoptosis has been strongly challenged, in part, because many cells isolated from cytochrome c−/−, apaf-1−/−, and caspase-9−/− mice appear to respond normally to death ligands, such as CD95L (40.Huang D.C. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (278) Google Scholar). In the present study, we provide an explanation for some of the conflicting results previously observed and, in doing so, help to redefine or update the type I/type II cell model. We show that Bcl-2 and Bcl-xL, in addition to inhibiting the release of cytochrome c, also block release of Smac from mitochondria and thus can modulate a second, unique type II signaling pathway. Indeed, XIAP can prevent death receptor-induced apoptosis solely through its inhibition of active caspase-3. Therefore, by inhibiting the release of Smac, Bcl-2 and Bcl-xL can prevent the inactivation of XIAP and inhibit cell death. Jurkat T-cells (J6) and T-lymphoblastic leukemia CEM cells, stably transfected with either empty vector, Bcl-2, or Bcl-xL (41.Belka C. Rudner J. Wesselborg S. Stepczynska A. Marini P. Lepple-Wienhues A. Faltin H. Bamberg M. Budach W. Schulze-Osthoff K. Oncogene. 2000; 19: 1181-1190Crossref PubMed Scopus (119) Google Scholar, 42.Hartmann B.L. Geley S. Loffler M. Hattmannstorfer R. Strasser-Wozak E.M. Auer B. Kofler R. Oncogene. 1999; 18: 713-719Crossref PubMed Scopus (47) Google Scholar), were cultured in RPMI 1640 containing 10% fetal calf serum, 1% Glutamax, and 0.4 mg/ml G418 in 5% CO2 at 37 °C. Cells were treated for 4 h with the agonistic anti-CD95 antibody, CH-11 (10–50 ng/ml) (Upstate Biotechnology, Inc., Lake Placid, NY), or recombinant CD95 ligand (50 ng/ml), cross-linked with M2 anti-FLAG antibody, according to the manufacturer's suggested protocol (Alexis Biochemicals, Nottingham, UK). After treatment, the percentage of annexin V-positive cells (% apoptosis) was determined by fluorescence-activated cell sorter analysis, as described previously (43.Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Annexin V/fluorescein isothiocyanate was purchased from Bender Medsystems (Vienna, Austria). After treatment, cells were pelleted and washed with ice-cold phosphate-buffered saline. Some of the cells were then mixed with Laemmli loading buffer and briefly sonicated. The whole cell protein samples were subsequently resolved by 12–15% SDS-PAGE and immunoblotted for caspase-8, caspase-3, cleaved caspase-6 (recognizes only the processed p18 large subunit; Cell Signaling Technologies, Beverly, MA), and poly(ADP-ribose) polymerase (PARP). The remaining cells were incubated with buffer containing 0.25% digitonin on ice for 20 min. The samples were then centrifuged at 20,000 ×g for 10 min, and the cytosolic fractions (supernatants) were collected. These fractions were separated by SDS-PAGE and immunoblotted for cytochrome c (BD PharMingen) and Smac. The rabbit polyclonal antibodies to caspase-8 (large subunit) (43.Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) and Smacβ (residues 1–186) (44.Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-228Crossref PubMed Scopus (92) Google Scholar) were generated in our laboratory. Lysates prepared from control and CD95-treated Jurkat cells were incubated with protein G-Sepharose beads (Amersham Biosciences), blocked with 3% bovine serum albumin, and precoated with anti-caspase-3 antibodies (BD Transduction Laboratories, Franklin Lakes, NJ). The resulting protein complexes were obtained by centrifugation, washed 4 times in buffer (100 mm HEPES, 0.1% CHAPS, 10 mm dithiothreitol, 10% sucrose, pH 7.0), and immunoblotted for XIAP (Transduction Laboratories, Franklin Lakes, NJ), cIAP-1, or cIAP-2 (R & D Systems, Minneapolis, MN). For the XIAP blots, a secondary antibody specific for mouse IgGκ chains was also utilized, to better distinguish the XIAP band from the heavy chain of the anti-XIAP antibody. Wild-type processed caspase-8 (pET-21b), procaspase-3 (pET-21b), and mature Smac (residues 56–239; pET-15b) were expressed in Escherichia coli BL-21(DE3) and purified on Ni2+-Sepharose beads, as described previously (26.Bratton S.B. Walker G. Srinivasula S. Sun X.-M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-2001Crossref PubMed Scopus (342) Google Scholar). Glutathione S-transferase-tagged XIAP was expressed and purified on GSH-Sepharose beads, as described previously (19.Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (682) Google Scholar). Purified recombinant caspase-8 (p18/p12 form; 5–120 nm), procaspase-3 (200 nm), XIAP (1–40 nm), and Smac (5–100 nm) were incubated in various combinations at 37 °C for 1 h in caspase assay buffer (100 mm HEPES, 0.1% CHAPS, 10 mmdithiothreitol, 10% sucrose, pH 7.0), and caspase-3 was subsequently immunoblotted, as described above. DEVDase activities were determined using the substrate, benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin, as described previously (8.Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). As already noted, during death receptor-induced apoptosis, caspase-8 processes caspase-3 between its large and small subunits. However, recent studies (37.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 45.Rokhlin O.W. Guseva N. Tagiyev A. Knudson C.M. Cohen M.B. Oncogene. 2001; 20: 2836-2843Crossref PubMed Scopus (67) Google Scholar) indicate that the prodomain of caspase-3 is not subsequently removed in cells overexpressing Bcl-2, Bcl-xL, or the viral protein E1B 19K. Because these cells were also resistant to cell death, we found this observation intriguing and thus began to search for a causal relationship. Perez and White (37.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) suggested that maturation of caspase-3 from its p20/p12 to p17/p12 form was required for its catalytic activity and that this second cleavage step was carried out by the Apaf-1/caspase-9 apoptosome. However, this explanation seemed unlikely to us, given that removal of the prodomain from caspase-3 is normally associated with its autocatalytic activity (46.Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (694) Google Scholar, 47.Stennicke H.R. Jurgensmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). Therefore, we initiated studies in which Jurkat T-cells, stably expressing either Bcl-2 or Bcl-xL, were treated with an agonistic CD95 antibody (CH-11). As expected, in vector control-treated cells, CD95 stimulation led to increased apoptosis in a concentration-dependent manner (Fig. 1A). Both procaspase-8 splice forms were processed to their p43 and p41 forms (prodomain + large subunit), respectively (Fig. 1A, lanes 2 and 3), and at the higher concentration of CH-11, some p18 fragment (large subunit only) was also observed (Fig. 1A, lane 3). Caspase-3 was processed primarily to its p19 form or its fully mature p17 form (Fig. 1A, lanes 2 and 3), and the caspase-3 substrates, caspase-6 and poly(ADP-ribose) polymerase (PARP), were significantly processed (Fig. 1A, lanes 2 and 3; Fig. 2A, lane 2). In addition, there was a dramatic increase in caspase-3-like DEVDase activity (data not shown).Figure 2Bcl-2 and Bcl-xL promote XIAP-caspase-3 interactions and inhibit DEVDase activity by preventing release of the IAP antagonist, Smac/DIABLO, from mitochondria. Vector control (VC), Bcl-2-, and Bcl-xL-overexpressing cells were treated with CH-11 antibody (50 ng/ml) for 4 h. Cytosolic lysates from control and treated cells were then prepared and assayed for processing of PARP (A), basal levels of XIAP, cIAP-1, and cIAP-2 (B), and release of cytochrome c (Cyt. c) and Smac/DIABLO from mitochondria (C). D, total caspase-3 (Casp-3) was immunoprecipitated (IP) from each lysate, and the pellets were subsequently Western blotted (WB), for XIAP and caspase-3. For clarity, XIAP blots were also incubated with a mouse anti-IgG k secondary (2°) antibody, because it was difficult to separate the heavy chain (HC) of the mouse polyclonal antibody from XIAP. LC, light chain. E, lysates from untreated vector control (Con) and CD95-stimulated Bcl-2/Bcl-xL-overexpressing cells were also incubated (0–60 min) with exogenous Smac (500 nm) at 37 °C and subsequently assayed for DEVDase activity, as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) In cells overexpressing either Bcl-2 or Bcl-xL, cell death was markedly reduced (Fig. 1A). Caspase-8 was processed normally to its p43 and p41 forms, although to a lesser extent than in control cells, and no p18 fragment was observed (Fig. 1A, lanes 5, 6, 8, and 9). Caspase-8 was active, as it processed caspase-3 between its large and small subunits, but interestingly, almost all of the processed caspase-3 was present in its p20 form (Fig. 1A, lanes 5, 6, 8, and 9). Cleavage of caspase-6 and PARP, as well as DEVDase activity, was significantly reduced in cells overexpressing Bcl-2 or Bcl-xL, indicating that caspase-3 activity was reduced (Fig. 1A, lanes 5, 6, 8, and 9; Fig. 2A, lanes 3 and 4; data not shown). Furthermore, this lack of caspase-3 activity and, consequently, activation of caspase-6 probably explained the observed decrease in caspase-8 processing compared with control cells (Fig. 1A), because caspase-6 is known to process additional procaspase-8 (48.Slee E.A. Harte M.T. Kluck R.M. Wolf B.B. Casiano C.A. Newmeyer D.D. Wang H.G. Reed J.C. Nicholson D.W. Alnemri E.S. Green D.R. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1687) Google Scholar). Thus, similar to previous reports (38.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar, 39.Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), Bcl-2 and Bcl-xL inhibited CD95-mediated apoptosis in Jurkat T-cells, and this appeared to correlate with a block in caspase-3 activity and processing from its p20 to its fully mature p17 form. Although the Jurkat T-cells in the present study clearly underwent death receptor-mediated apoptosis following CD95 ligation with CH-11 antibodies, and did so in a Bcl-2/Bcl-xL-inhibitable manner, there has been considerable debate in the literature as to the existence of type II cells, and particularly the use of anti-CD95 antibodies to induce apoptosis (40.Huang D.C. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (278) Google Scholar, 49.Huang D.C. Tschopp J. Strasser A. Cell Death Differ. 2000; 7: 754-755Crossref PubMed Scopus (31) Google Scholar, 50.Schmitz I. Walczak H. Krammer P.H. Peter M.E. Cell Death Differ. 1999; 6: 821-822Crossref PubMed Scopus (62) Google Scholar). Therefore, to verify that Bcl-2 could influence CD95-mediated processing of caspase-3 and cell death in another cell line, we treated T-cell acute lymphoblastic leukemia CEM cells with either CH-11 antibodies or cross-linked CD95L (50 ng/ml). Remarkably, both treatments induced almost identical levels of apoptosis, and the processing of caspases-8, -3, and -6, in the CH-11 and CD95L-treated cells was virtually indistinguishable (Fig. 1B, lanes 11 and 12). More importantly, however, in Bcl-2 stably transfected CEM cells, cell death was inhibited by ∼50–60% in both treatments, and processing of caspase-8 was significantly reduced (Fig. 1B, lanes 13 and 14). As before, caspase-3 was processed primarily to its p20 form, and there was no autoprocessing of caspase-3 to its fully mature p17 form (Fig. 1B, lanes 13 and 14). Furthermore, the p20 form of caspase-3 was inhibited, as no processing of caspase-6 could be observed (Fig. 1B, lanes l3 and 14). Thus, the inhibition of caspase-3 processing in Bcl-2-transfected cells was not limited to Jurkat T-cells, and the phenomenon could be observed with either anti-CD95 antibody or CD95L. The inability of p20/p12 caspase-3 to remove its prodomain suggested to us that the enzyme might be inhibited. Therefore, because IAPs, including XIAP, cIAP-1, and cIAP-2, are known to inhibit caspase-3 activity with inhibitory constants (Ki) of ∼0.7, ∼108, and ∼35 nm, respectively (13.Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1724) Google Scholar, 15.Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar), we initially investigated whether Bcl-2 or Bcl-xL might influence their expression. The levels of XIAP, cIAP-1, and cIAP-2, however, were not elevated in Bcl-2 or Bcl-xL-transfected cells compared with vector control cells (Fig. 2B, lanes 1–3). Therefore, we next examined if these IAPs might be directly associated with caspase-3 in cells. No interaction between caspase-3 and cIAP-1 or cIAP-2 could be detected (data not shown). However, immunoprecipitation of total caspase-3 from unstimulated and stimulated cells indicated that XIAP was almost exclusively associated with processed caspase-3 (Fig. 2D, compare lane 2 with lanes 3–5). Strikingly, far more XIAP was associated with the partially processed p20 subunit of caspase-3, in cells overexpressing Bcl-2 or Bcl-xL, than with the p17 subunit in vector control cells, even though significantly more of the p17 subunit was immunoprecipitated (Fig. 2D, compare lane 3 with lanes 4 and 5). Because XIAP can bind and inhibit both p17/p12 caspase-3 and p20/p12 caspase-3 in vitro (22.Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 818-822Crossref PubMed Scopus (298) Google Scholar), these data suggested that Bcl-2 and Bcl-xL specifically inhibited the release of some factor from mitochondria that could bind to XIAP and prevent its interaction with processed caspase-3. Smac/DIABLO contains an N-terminal “AVPI” motif, which allows it to bind the BIR3 domain in XIAP and displace caspase-9, as well as a second region that is sufficient to displace processed caspases-3 and -7 from the BIR2 domain in XIAP (29.Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (715) Google Scholar, 30.Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (716) Google Scholar, 31.Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (548) Google Scholar, 51.Srinivasula S.M. Datta P. Fan X.J. Fer" @default.
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