Distinct Caspase Cascades Are Initiated in Receptor-mediated and Chemical-induced Apoptosis
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.5053
ISSN1083-351X
AutoresXiaoming Sun, Marion MacFarlane, Jianguo Zhuang, Beni B. Wolf, Douglas R. Green, Gerald M. Cohen,
Tópico(s)Cell death mechanisms and regulation
ResumoRelease of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochromec is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis. Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochromec is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis. Apoptosis is a major form of cell death characterized by a series of stereotypic morphological features. It occurs in two phases, an initial commitment phase followed by an execution phase involving the condensation and fragmentation of nuclear chromatin, dilation of the endoplasmic reticulum, and alterations to the cell membrane resulting in recognition and subsequent phagocytosis of the cell (1Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1394) Google Scholar, 2Takahashi A. Earnshaw W.C. Curr. Opin. Gene & Dev. 1996; 6: 50-55Crossref PubMed Scopus (151) Google Scholar). Caspases, a family of cysteine proteases, play a critical role in the execution phase of apoptosis and are responsible for many of the biochemical and morphological changes associated with apoptosis (3Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 4Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). It has been proposed that “initiator” caspases with long prodomains, such as caspase-8 (MACH/FLICE/Mch5), either directly or indirectly activate “effector” caspases, such as caspases-3, -6, and -7 (3Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar, 5Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). These effector caspases then cleave intracellular substrates, such as poly(ADP-ribose) polymerase (PARP) 1The abbreviations PARPpoly(ADP-ribose)polymeraseZ-VAD.FMKbenzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketoneZ-IETD.CHObenzyloxycarbonyl-Ile-Glu-Thr-Asp aldehydeZ-DEVD.AFCbenzyloxycarbonyl-Asp-Glu-Val-Asp aminofluoromethyl coumarinTNFtumor necrosis factorPSphosphatidylserineDiOC6(3)3,3′-dihexyloxacarbocyanine iodideDEDdeath effector domain and lamins, during the execution phase of apoptosis. Caspase-8 is the most apical caspase in CD95 (Fas/APO-1)-induced apoptosis (7Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 8Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar). Triggering of the CD95 receptor with its cognate ligand or agonistic antibody results in receptor trimerization and recruitment of CD95 receptor-associated protein with death domains (FADD/MORT1), which in turn binds to the death effector domains in the N-terminal region of caspase-8, resulting in its activation. As caspase-8 can activate all known caspasesin vitro (6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar), it is a prime candidate for an initiator caspase in many forms of apoptosis in addition to CD95-induced apoptosis. Procaspase-9 has also been proposed as an initiator caspase; in the presence of dATP and cytochrome c, its long N-terminal domain interacts with Apaf-1 resulting in the activation of caspase-9 (9Li 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 (6261) Google Scholar, 10Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar). Active caspase-9 can then activate the effector caspases-3, -6, and -7 (10Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 11Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). Thus there are at least two major mechanisms by which a caspase cascade resulting in the activation of effector caspases may be initiated as follows: one involving caspase-8 and the other involving caspase-9 as the most apical caspase. poly(ADP-ribose)polymerase benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone benzyloxycarbonyl-Ile-Glu-Thr-Asp aldehyde benzyloxycarbonyl-Asp-Glu-Val-Asp aminofluoromethyl coumarin tumor necrosis factor phosphatidylserine 3,3′-dihexyloxacarbocyanine iodide death effector domain Cytochrome c, which is usually present in the mitochondrial intermembrane space, is released into the cytosol following the induction of apoptosis by many different stimuli including CD95, tumor necrosis factor (TNF), and chemotherapeutic and DNA-damaging agents (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 14Reed J.C. Cell. 1997; 91: 559-562Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). Mitochondria have been proposed to act as an amplifier in CD95-induced apoptosis when activation of caspase-8 cleaves a cytosolic substrate leading to release of cytochrome c (15Kuwana T. Smith J.J. Muzio M. Dixit V. Newmeyer D.D. Kornbluth S. J. Biol. Chem. 1998; 273: 16589-16594Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 16Scaffidi 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). Release of mitochondrial cytochrome c and activation of caspase-3 is blocked by anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, and promoted by proapoptotic members, such as Bax and Bak (13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 17Yang 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 (4422) Google Scholar, 18Jurgensmeier 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 (1375) Google Scholar). In chemical- or irradiation-induced apoptosis, cytochrome c release appears to be caspase-independent as it is not inhibited by the broad spectrum cell-permeable caspase inhibitor, Z-VAD.FMK (13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 17Yang 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 (4422) Google Scholar, 19Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar, 20Zhuang J. Dinsdale D. Cohen G.M. Cell Death Differ. 1998; 5: 953-962Crossref PubMed Scopus (88) Google Scholar). Mechanisms for the release of mitochondrial cytochrome cinclude opening of a mitochondrial permeability transition pore, the presence of a specific channel for cytochrome c in the outer membrane, or mitochondrial swelling and rupture of the outer membrane but without loss of mitochondrial membrane potential (14Reed J.C. Cell. 1997; 91: 559-562Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). None of these mechanisms appears generally applicable, as release of cytochromec occurs in cells with normal mitochondrial membrane potential (13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 17Yang 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 (4422) Google Scholar) and by a mechanism independent of rupture of the outer mitochondrial membrane (20Zhuang J. Dinsdale D. Cohen G.M. Cell Death Differ. 1998; 5: 953-962Crossref PubMed Scopus (88) Google Scholar). Two recent studies have highlighted another possible mechanism of mitochondrial cytochrome crelease, involving Bid, a BH3 domain-containing proapoptotic Bcl-2 family member. Cleavage of Bid by caspase-8 results in translocation of the cleaved Bid to the mitochondria where it induces the release of cytochrome c, being 500-fold more potent than Bax (21Li H. Zhu H. Xu C.-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 22Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). The BH3 domain of Bid is essential both for its proapoptotic activity and its ability to induce the release of cytochrome c (22Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar,23Wang K. Yin X.-M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (808) Google Scholar). In this study we address the order in which caspases are activated in receptor-mediated and chemical-induced apoptosis. Our data support the hypothesis that caspase-8 and caspase-9 are the most apical caspases in receptor-mediated and chemical-induced apoptosis, respectively. In chemical-induced apoptosis, cytochrome c release is caspase-independent and is not mediated by cleavage of Bid in contrast to receptor-mediated apoptosis. We propose that caspases act solely as executioners of apoptosis in chemical-induced apoptosis, whereas in receptor-mediated apoptosis they also form an integral part of the cell death-inducing mechanism. Media and serum were purchased from Life Technologies, Inc. (Paisley, UK). Z-VAD.FMK and Z-DEVD.AFC were from Enzyme Systems Inc. (Dublin, CA), and Z-IETD.CHO was kindly provided by Professor L. Rubin (Eisai London Research, London, UK). Anti-CD95 monoclonal antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Annexin V/FITC kit was from Bender Medsystems (Vienna, Austria). DiOC6(3) was purchased from Molecular Probes (Eugene, OR). All other chemicals and human recombinant TNF-α were from Sigma (Poole, UK). Jurkat T cells (clone E6-1) were obtained from ECACC and cultured in RPMI 1640 containing 10% fetal bovine serum and 1% Glutamax. Apoptosis in U937 cells was induced with etoposide (25 μm) or TNF-α (10 ng/ml) and cycloheximide (0.9 μm) (24Zhuang J. Ren Y. Snowden R.T. Zhu H. Gogvadze V. Savill J.S. Cohen G.M. J. Biol. Chem. 1998; 273: 15628-15632Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Some cells were treated for 1 h with Z-VAD.FMK prior to exposure to the apoptotic stimulus. Apoptosis was quantified by PS exposure or by loss of the mitochondrial membrane potential, assessed with DiOC6(3) (20Zhuang J. Dinsdale D. Cohen G.M. Cell Death Differ. 1998; 5: 953-962Crossref PubMed Scopus (88) Google Scholar, 25Vanags D.M. Pörn-Ares M.I. Coppola S. Burgess D.H. Orrenius S. J. Biol. Chem. 1996; 271: 31075-31085Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Cell samples were prepared as described (26MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar). Proteins were resolved on 12–15% SDS-polyacrylamide gels and blotted onto nitrocellulose (Hybond-C extra, Amersham, Bucks, UK). Caspases-3 and -7 were detected as described previously (26MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar). A rabbit polyclonal antibody to caspase-8 was raised against the large subunit of caspase-8 (amino acids 210–374). The antibody obtained was characterized by enzyme-linked immunosorbent assay and Western blot analysis, which verified that the antibody recognized intact procaspase-8 and the p43 and p18 subunits. An antibody to caspase-9 was also raised, which recognized both the inactive proform and the activated ∼37- and 35-kDa processed forms. Cytochrome cantibody was purchased from PharMingen (San Diego, CA). The Bid antibody was kindly provided by Dr. X. Wang (22Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). The PARP antibody was obtained from Dr. G. Poirier (Laval University, Quebec, Canada). Cells were collected at the indicated times and washed once in ice-cold phosphate-buffered saline. Cell pellets were resuspended in cytosol extraction buffer, and cytosolic extracts were prepared by the method described previously (19Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar). Jurkat cell lysates were prepared as described previously (26MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar) and activated by addition of dATP (2 mm), cytochrome c (0.25 mg/ml), and MgCl2 (2 mm) (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). The proteolytic activity (cleavage of Z-DEVD.AFC) of the lysate was measured as described previously (26MacFarlane M. Cain K. Sun X.-M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar). Immunodepletion of capsase-8 or -9 from Jurkat cell lysate was performed as described previously (9Li 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 (6261) Google Scholar). Both CD95 antibody and etoposide caused a time-dependent induction of apoptosis in Jurkat T cells, as assessed both by an increase in externalization of phosphatidylserine (PS) or by a decrease in mitochondrial membrane potential (ΔΨm) (Figs.1 and 2). This was accompanied by a similar time-dependent processing of caspase-3, -7, -8, and -9 (Figs. 1 and 2). In Jurkat T cells, caspase-3 was present primarily as its intact 32-kDa proform (Fig.1 A, lane 1). Induction of both chemical- and receptor-mediated apoptosis resulted in loss of the proform of caspase-3 and appearance of three immunoreactive fragments of ∼20 kDa (p20), ∼19 kDa (p19), and ∼17 kDa (p17), following initial cleavage at Asp-175 and then at Asp-9 and Asp-28 (27Fernandes-Alnemri T. Armstrong R. 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). Processing was first detected after 2–3 h treatment with either stimulus (Figs.1 A and 2 A, lanes 2–7).Figure 2Etoposide-induced time-dependent processing of caspases in Jurkat T cells. Jurkat T cells were incubated for the indicated times with etoposide (50 μm) either alone or in the presence of the indicated concentrations of Z-VAD.FMK. Cells were then analyzed by Western blot analysis for the processing of caspase-3 (A), caspase-7 (B), caspase-8 (C), and caspase-9 (D), as described under “Experimental Procedures” and the legend to Fig. 1.View Large Image Figure ViewerDownload (PPT) Caspase-7 was present in control Jurkat T cells primarily as a ∼35-kDa protein (Fig. 1 B, lane 1). Treatment with both anti-CD95 antibody and etoposide resulted in a time-dependent processing of caspase-7 accompanied by the formation of two major products. These were a ∼32-kDa fragment, which probably represents the loss of the prodomain at DSVD23↓A, and a ∼19-kDa (p19) fragment, which corresponds to the catalytically active large subunit (Figs.1 B and 2 B, lanes 2–7) formed following cleavage at IQAD198↓S (27Fernandes-Alnemri T. Armstrong R. 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). Processing of caspase-7 was first observed 2 h after treatment with either stimulus. In untreated Jurkat T cells, caspase-8 was present primarily as two isoforms of ∼55 kDa (Fig. 1 C, lane 1), possibly corresponding to caspase-8a and -8b (7Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 28Scaffidi C. Medema J.P. Krammer P.H. Peter M.E. J. Biol. Chem. 1997; 272: 26953-26958Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Exposure to both anti-CD95 antibody and etoposide resulted in a time-dependent processing of caspase-8 initially to two fragments of ∼43 and 41 kDa (p43 and p41, respectively), corresponding to cleavage of both caspase-8a and -8b between the large and small subunits. This was followed by the appearance of a p18 subunit resulting from removal of the death effector domains from the 43- and 41-kDa fragments (Figs.1 C and 2C, lanes 2–7) (6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar, 28Scaffidi C. Medema J.P. Krammer P.H. Peter M.E. J. Biol. Chem. 1997; 272: 26953-26958Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). An increase in the processing of caspase-8 was first observed 2 and 2.5 h after CD95 and etoposide treatment, respectively. Untreated Jurkat cells contained the 46-kDa proform of caspase-9 (Fig.1 D, lane 1), which on induction of apoptosis was processed in a time-dependent manner to yield fragments of ∼37 and 35 kDa (p37 and p35) (Figs. 1 D and 2D, lanes 2–7), resulting from cleavage at both Asp-315 and Asp-330 (6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (483) Google Scholar). The first detectable processing of caspase-9 was evident at 2 h (Figs. 1 D and 2 D). Thus, induction of apoptosis was accompanied by the activation of both the activator caspases -8 and -9 and the effector caspases-3 and-7, which all appeared to occur simultaneously making it extremely difficult to distinguish the order in which they were activated. In order to further address this problem, we used the broad-spectrum caspase inhibitor Z-VAD.FMK, which inhibits apoptosis in many but not all systems (3Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar). Slight inhibition of CD95-induced caspase processing was observed at Z-VAD.FMK (0.1 μm), with marked and almost total inhibition at 1.0 and 10 μm,respectively (Fig. 1, A–D, lanes 8–10). Higher concentrations of Z-VAD.FMK were required to inhibit etoposide-induced processing of these caspases (Fig. 2, A—D, lanes 8–10). Z-VAD.FMK (10 and 25 μm) largely but not completely inhibited the processing of caspases-3, -7, and -9 (Fig. 2, A—D, lanes 8 and 9). As caspase-9 processes caspases-3 and -7 (10Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 29Srinivasan A. Li F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C. Wu J.C. Tomaselli K.J. J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), these data support the suggestion that one of the important targets of Z-VAD.FMK in etoposide-induced apoptosis may be either the processing or the activity of caspase-9. However Z-VAD.FMK (10 μm) completely inhibited the processing of caspase-8, which suggested that caspase-8 was activated downstream of caspases-3 and -7. Most importantly these data strongly suggested that the order of caspase activation and the Z-VAD.FMK target(s) were different in etoposide- and CD95-induced apoptosis in Jurkat cells. The different cellular effects of Z-VAD.FMK on etoposide- and CD95-induced apoptosis in Jurkat cells provided further support for this hypothesis. Both CD95 and etoposide induced apoptosis, as assessed either by an increase in externalization of PS (Fig.3, B and D) or by a decrease in mitochondrial membrane potential (Fig. 3, G andI). Z-VAD.FMK (10 μm) completely inhibited CD95-induced apoptosis assessed by both these criteria (Fig. 3,C and H). However, Z-VAD.FMK (25 μm) did not inhibit etoposide-induced loss of mitochondrial membrane potential (Fig. 3 J) but did inhibit PS exposure (Fig. 3 E). Z-VAD.FMK (25 μm) also did not inhibit etoposide-induced decrease in cell size as measured by forward light scatter (Fig. 3, E and J). Taken together, these data suggest that in CD95-induced apoptosis in Jurkat cells the intracellular target of Z-VAD.FMK is most likely the activator caspase-8 acting upstream of mitochondria, whereas in etoposide-induced apoptosis the target(s) is the activation/processing of caspase-9, which is activated downstream of perturbation of mitochondria. In order to substantiate this hypothesis, we examined the effects of Z-VAD.FMK on the release of mitochondrial cytochromec. In agreement with other studies, CD95 induced a time-dependent increase of cytochromec in the cytosol, most probably due to an increased release of mitochondrial cytochrome c (Fig.4 A). Z-VAD.FMK (10 μm) inhibited this increase (Fig. 4 A), further demonstrating that it inhibited a caspase upstream of the mitochondrial changes. Etoposide also induced a time-dependent increase in cytosolic cytochrome c; however, Z-VAD.FMK (25 μm) did not inhibit this increase (Fig. 4 B) further supporting the hypothesis that the target of Z-VAD.FMK in etoposide-induced apoptosis is downstream of mitochondria. In order to determine if such differences between receptor-mediated and chemically induced apoptosis also occurred in other cells, we examined a human lymphoid tumor cell line, U937, which is sensitive to tumor necrosis factor (TNF-α). Both TNF/cycloheximide and etoposide caused an induction of apoptosis in U937 cells, assessed by PS externalization, which was accompanied by an increase in cytosolic cytochrome c, processing of caspase-3, and cleavage of PARP, a commonly used measure of caspase-3-like enzymic activity (Fig.5, A–C). Z-VAD.FMK completely inhibited TNF/cycloheximide-induced apoptosis assessed by all these criteria. In contrast, in etoposide-induced apoptosis Z-VAD.FMK completely inhibited PS externalization and the cleavage of PARP but only partially inhibited processing of caspase-3 and did not inhibit the increase in cytosolic cytochrome c. Thus Z-VAD.FMK was more effective at blocking the activity rather than the processing of caspase-3. Taken together, the data from both Jurkat and U937 cells support the hypothesis that Z-VAD.FMK inhibits a target upstream of mitochondria in receptor (CD95 or TNF)-mediated apoptosis, whereas in etoposide-induced apoptosis the Z-VAD.FMK target is downstream of mitochondria. In order to gain further insight into the order of caspase activation in chemical-mediated apoptosis, the processing of various caspases was studied in Jurkat cell lysates, a well established model for understanding postmitochondrial caspase cascades (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Activation of lysates, which resulted in an increased caspase-3-like DEVDase activity, was accompanied by a time-dependent processing of caspases (Fig. 6). Processing of the effector caspases-3 and -7 was first observed after 5 min, and these caspases were almost completely processed after 60 min (Fig. 6, lanes 1–7). Caspase-9 was very rapidly and extensively processed to both its p37 and p35 fragments, initial processing being observed by 1 min and virtually complete processing noted at 30 min (Fig. 6, lanes 1–7). In marked contrast, processing of caspase-8 was first observed at 30 min, when a small amount was processed only to its ∼41- and 43-kDa fragments (Fig. 6,lanes 1–7). Co-incubation of lysates with two different caspase inhibitors, Z-VAD.FMK or benzyloxycarbonyl-Ile-Glu-Thr-Asp-aldehyde (IETD.CHO), resulted in a marked inhibition of the processing of all the caspases with caspase-9 being somewhat less sensitive to inhibition than the other caspases (Fig. 6, lanes 8 and 9). Thus caspase inhibitors may also block the processing of caspases activated by a caspase cascade downstream of mitochondria. To elucidate further the role of caspases-8 and -9 in the postmitochondrial caspase cascade, they were immunodepleted from lysates, and the subsequent ability of the lysate to be activated was assessed. In the presence of dATP/cytochrome c, lysates from control Jurkat cells exhibited a marked DEVDase activity (TableI). Immunodepletion of caspase-8 caused only a very slight decrease in DEVDase activity demonstrating that caspase-8 contributed little to this activity (Table I). Western blot analysis demonstrated that immunodepletion of caspase-8 resulted only in loss of this caspase but not of other caspases (data not shown). In contrast, immunodepletion of caspase-9 resulted in complete inhibition of DEVDase activity (Table I) without loss of caspases-3, -7, and -8 (data not shown) demonstrating the key role of caspase-9 in the postmitochondrial processing of caspases. Taken together these results lend strong support to the hypothesis that caspase-9 is the first caspase activated in a caspase cascade following perturbation of mitochondria and release of cytochrome c.Table IImmunodepletion of caspase-9 but not caspase-8 inhibits cytochrome c/dATP-dependent activationLysate treatmentDEVDasenmol/mg/minControl14.2Preimmune rabbit serum12.2Caspase-8 antibody10.1Caspase-9 antibody0.07Jurkat lysate was prepared and immunodepleted with either preimmune serum, caspase-8, or caspase-9 antibody, and the proteolytic activity (DEVDase) of the control lysate and the immunodepleted lysates was then measured as described under “Experimental Procedures.” Open table in a new tab Jurkat lysate was prepared and immunodepleted with either preimmune serum, caspase-8, or caspase-9 antibody, and the proteolytic activity (DEVDase) of the control lysate and the immunodepleted lysates was then measured as described under “Experimental Procedures.” Cleavage of Bid is important for the release of mitochondrial cytochrome c in CD95-induced apoptosis (21Li H. Zhu H. Xu C.-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 22Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). We wished to investigate whether this mechanism of cytochrome c release is
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