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Release of mitochondrial cytochrome c is upstream of caspase activation in chemical-induced apoptosis in human monocytic tumour cells
Toxicology Letters 102]103 Ž1998. 121]129
Release of mitochondrial cytochrome c is upstream of caspase activation in chemical-induced apoptosis in human monocytic tumour cells Jianguo Zhuang, Gerald M. CohenU MRC Toxicology Unit, Hodgkin Building, Uni¨ ersity of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK
Abstract Apoptosis, induced in human monocytic THP.1 cells by etoposide and N-tosyl-L-phenylalanyl chloromethyl ketone, was accompanied by the processingractivation of caspases, externalisation of phosphatidylserine ŽPS. and reduction in mitochondrial membrane potential Ž DCm .. Activation of caspaseŽs. occurred prior to both PS exposure and reduction in DCm. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp ŽOMe. fluoromethyl ketone ŽZ-VAD.fmk. blocked the activation of caspases, PS exposure and the reduction in DCm as well as the morphological changes associated with apoptosis but it did not inhibit the release of mitochondrial cytochrome c. These results suggest that the execution phase of chemical-induced apoptosis in THP.1 cells may be initiated following mitochondrial damage resulting in release of cytochrome c leading to activation of caspase-9 and then activation of effector caspases-3 and -7. This contrasts to receptor-mediated apoptosis, such as Fas, which results in an initial activation of caspase-8. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Caspases; Mitochondrial membrane potential; Cytochrome c
1. Introduction Apoptosis is a fundamental form of cell death which plays a major role in the development and homeostasis of multicellular organisms ŽArends and Wyllie, 1991.. Disturbances in apoptosis are important in cancer, acquired immunodeficiency syndrome and some neurodegenerative disorders ŽThompson, 1995.. Apoptotic cell death comprises an initial commitment phase followed by an
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Corresponding author.
execution phase, which is characterised by a consistent series of distinct morphological changes ŽEarnshaw, 1995., suggesting the existence of a common execution machinery. In the nematode C. elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence homology to the mammalian cysteine protease, interleukin1 b converting enzyme ŽICE., a family of at least 10 related ICE-like proteases, now known as caspases ŽAlnemri et al., 1996., has been identified. Caspases are characterised by an almost absolute specificity for aspartic acid in the P1 position of
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the substrate ŽThornberry and Molineaux, 1995; Kumar and Lavin, 1996; Cohen, 1997.. They exist in cells as inactive proenzymes and, upon activation, are cleaved at specific aspartate residues, generating a large and small subunit, which together form the active enzyme in the form of a heterotetramer containing two large and two small subunits ŽWilson et al., 1994.. Caspases may be divided into ‘initiator’ caspases with long prodomains Že.g. caspase-8, -9 and -10., which activate ‘effector’ caspases with short prodomains Že.g. caspase-3, -6 and -7., which in turn cleave intracellular proteins, such as poly ŽADP-ribose. polymerase ŽPARP. and lamins, so precipitating the dramatic morphological and biochemical changes of apoptosis ŽCohen, 1997.. Apoptosis induced by CD95 ŽFasrAPO-1. results in the recruitment of the adapter molecule FADD ŽFas-associated protein with death domain., which then binds to the N-terminal death effector domains of caspase-8 resulting in its activation thereby providing a direct link between cell death receptors and the caspases ŽBoldin et al., 1996; Muzio et al., 1996.. Recently, it has been shown that in cells triggered to undergo apoptosis by a variety of stimuli, mitochondrial cytochrome c is released and, in the presence of dATP and Apaf-1, results in the activation of caspase-9, which in turn activates other caspases, such as caspase-3 ŽLiu et al., 1996; Li et al., 1997; Zou et al., 1997.. Therefore execution of the death programme appears to occur following activation of a hierarchy of caspases with caspase-8 and caspase-9 being at the apex of this apoptotic cascade originating at the cell membrane or the mitochondria, respectively. In this study, using human monocytic THP.1 cells we demonstrate that caspases are key executioners of apoptosis induced by the anti-cancer drug, etoposide, and the serine protease inhibitor, N-tosyl-L -phenylalanyl chloromethyl ketone ŽTPCK.. The broad spectrum caspase inhibtor, Z-VAD.fmk, inhibits all the biochemical and morphological features of apoptosis, including activation of caspases, and reduction in mitochondrial membrane potential Ž DCm .. However, ZVAD.fmk does not block the release of cytochrome c, suggesting that following chemical-in-
duced apoptosis activation of caspases is initiated following perturbations of the mitochondria. 2. Materials and methods 2.1. Materials Chemicals were obtained from Sigma Chemical Company ŽPoole, England. except for N-tosyl-Lphenylalanyl chloromethyl ketone ŽTPCK, Boehringer-Mannheim UK, Lewes, England. and benzyloxycarbonyl-Val-Ala-Asp ŽOMe . fluoromethyl ketone ŽZ-VAD.fmk, Enzyme Systems Inc., Dublin, CA, USA.. 2.2. Cell culture and treatment THP.1 cells were maintained as suspension culture and apoptosis induced by etoposide Ž25 m M. or TPCK Ž75 m M. as previously described ŽZhu et al., 1995, 1997.. 2.3. Flow cytometric analysis of apoptosis Cells with externalized phosphatidylserine ŽPS. residues were detected by a FITC-labelled Annexin V binding assay ŽMartin et al., 1995.. For measuring mitochondrial membrane potential Ž DCm . 0.5= 10 6 cells were incubated for 20 min at 378C with 3,39dihexyloxacarbocyanine iodide wDiOC 6 Ž3., 50 nMx ŽMolecular Probes Inc., Eugene, OR, USA., which is retained in mitochondria with a normal membrane potential ŽPetit et al., 1990.. 2.4. Western blot analysis and preparation of cytosolic extracts for assessment of cytochrome c Samples of 0.2= 10 6 cells were prepared for Western blot analysis as previously described ŽMacFarlane et al., 1997.. Isolation of the cytosolic extracts was essentially as described ŽKluck et al., 1997.. Cytosolic protein Ž30 mg. was separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with a mouse monoclonal antibody recognising human cytochrome c Ž7H8.2C12. ŽPharMingen, San Diego, CA, USA. at a dilution of 1:1000. Parallel samples, contain-
J. Zhuang, G.M. Cohen r Toxicology Letters 102]103 (1998) 121]129
ing an equal amount of cytosolic protein, were checked by immunoblotting for mitochondrial contamination using the 12C4-F12 mouse monoclonal antibody ŽMolecular Probes. to mitochondrial membrane-bound cytochrome c oxidase Žsubunit II. as described ŽKluck et al., 1997.. 2.5. Measurement of Ac-DEVD.AFC clea¨ age acti¨ ity Cytosolic extracts were prepared as described above and stored at y808C. The proteolytic activity of caspaseŽs. in cytosolic extracts was measured using a continuous fluorimetric assay initially described by Thornberry Ž1994., with minor modifications ŽMacFarlane et al., 1997.. Liberation of fluorogenic 7-amino-4-trifluoromethylcoumarin from Ac-DEVD.AFC ŽEnzyme Systems Inc.. as a measure of caspases-3 and -7 like activity was assayed at excitation and emission wavelengths of 400 and 505 nm, respectively. The protease activity was expressed as pmol AFCrmg proteinrmin. 3. Results 3.1. Etoposide and TPCK-induced apoptosis in THP.1 cells Externalization of PS is considered to be a general feature of apoptosis induced by many different stimuli and can be measured by binding of annexin V, a PS-binding protein ŽKoopman et al., 1994; Martin et al., 1995.. A reduction in DCm has also been reported to be an early event in the induction of apoptosis in many different systems ŽCastedo et al., 1996; Zamzami et al., 1996.. In this study, apoptosis was initially assessed using two flow cytometric methods measuring the externalization of PS and the percentage of cells with decreased DCm . Incubation of THP.1 cells with etoposide, a DNA topoisomerase II inhibitor, or TPCK, a chymotrypsin-like serine protease inhibitor, resulted in a time-dependent induction of apoptosis, as assessed by both PS exposure and decreased DCm ŽFig. 1A.. While a decreased DCm has been proposed to regulate the externalization of PS ŽCastedo et al., 1996., in the present studies
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both these changes demonstrated a similar time dependence, occurring at 2 h after treatment, irrespective of the apoptotic stimulus ŽFig. 1A.. Thus induction of apoptosis in THP.1 cells is accompanied by the externalization of PS and a decreased DCm . It has been shown that during the induction of apoptosis in THP.1 cells at least four caspases are activated, including caspases-2, -3, -6 and -7, and cleave cellular substrates such as PARP and lamins ŽMacFarlane et al., 1997; Zhu et al., 1997.. Therefore the proteolytic activity of caspases was measured by monitoring the Ac-DEVD.AFC cleavage activity of cell lysates following treatment with either etoposide or TPCK. AcDEVD.AFC was chosen as a substrate because the DEVD tetrapeptide sequence mimics the cleavage site in PARP and several other proteins cleaved during apoptosis, i.e. DEVDxG ŽLazebnik et al., 1994; Cohen, 1997.. In addition, caspases-3, -6 and -7 all have Ac-DEVD.AMC cleavage activity ŽFernandes-Alnemri et al., 1995a,b; Nicholson et al., 1995.. The Ac-DEVD.AFC cleaving activity of lysates obtained from control cells was very low even after a 4-h incubation ŽFig. 1B.. In contrast, incubation of THP.1 cells with either etoposide Ž25 m M., or TPCK Ž75 m M., resulted in a time-dependent increase in the Ac-DEVD.AFC cleavage activity ŽFig. 1B.. In lysates prepared from etoposide-treated cells, an increase in Ac-DEVD.AFC cleavage activity was first observed at 2 h after treatment and continued to rise over a 4-h incubation period ŽFig. 1B., paralleling the time course of the induction of apoptosis ŽFig. 1A.. The increase in AcDEVD.AFC cleavage activity in lysates prepared from TPCK-treated cells was similar to that of etoposide treatment in the first 2 h of incubation, but then reached a plateau ŽFig. 1B.. Whether the differences in both the level and pattern of proteolytic activity in lysates obtained from etoposide- and TPCK-treated cells reflected distinct mechanisms employed by the two stimuli to induce apoptosis or were due to some effects of the apoptotic stimulus used on proteolytic activity after the activation of caspases remains to be determined. Next, we examined the temporal sequence of
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Fig. 1. Induction of apoptosis by etoposide and TPCK. THP.1 cells were incubated for up to 4 h with either etoposide Ž25 m M. or TPCK Ž75 m M.. ŽA. Apoptosis was assessed by the number of cells with PS exposure Ž`}`. and decreased DCm Žv}v., respectively. ŽB. Cell lysates were also prepared for measuring Ac-DEVD.AFC cleavage activity as described in Section 2.
activation of caspases-3 and -7 and the proteolysis of the intracellular protein substrate PARP. Untreated control cells showed the presence of the intact ; 32-kDa proform of caspase-3 and ; 35kDa proform of caspase-7 ŽFig. 2A,B, lane 1,
respectively.. Processing of caspase-3 at Asp175 between the large and small subunits yields a p20 fragment, which is further processed at Asp9 and Asp 28 to yield p19 and p17 fragments, respectively ŽFernandes-Alnemri et al., 1996., whereas
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processing of caspase-7 occurs at Asp198 between the large and small subunits, followed by cleavage at Asp 23 to yield the p19 large subunit ŽFernandes-Alnemri et al., 1995b.. Induction of apoptosis with either etoposide or TPCK was accompanied by a time-dependent formation of the catalytically active large subunits of caspases-3 and -7 ŽFig. 2A,B, lanes 2]9, respectively.. Although difficult to precisely quantitate, it was possible that processing of caspase-7 to its catalytically active large subunit was observed prior to that of caspase-3 ŽFig. 2A,B. and also PARP ŽFig. 2C.. Dependent on the apoptotic stimulus, some differences were observed in the formation of the large subunits of caspase-3 ŽFig. 2A., possibly indicating some differences in the mechanisms of activation. Induction of apoptosis by either etoposide or TPCK resulted in a time-dependent loss of intact PARP together with formation of its characteristic signature 85-kDa fragment ŽFig. 2C.. Thus, induction of apoptosis in THP.1 cells was accompanied by PS exposure, reduction in DCm , cleavage of PARP and activation of caspases. The time-course studies suggested that caspase activation occurred upstream of these other events.
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3.2. Z-VAD.fmk inhibits acti¨ ation of caspases, PS exposure, and reduction in DCm Z-VAD.fmk, a cell membrane permeable inhibitor of caspases, inhibited apoptosis induced by both etoposide and TPCK, as assessed by PS exposure, reduction in DCm and Ac-DEVD.AFC cleavage activity ŽTable 1., providing further evidence of involvement of caspases in the execution phase of apoptosis. Z-VAD.fmk blocked TPCKand etoposide-induced PARP cleavage ŽFig. 3A.. Z-VAD.fmk also clearly blocked the processing of caspases-3 and -7 in cells treated with either TPCK or etoposide ŽFig. 3B,C.. However, a small amount of a slightly larger immunoreactive fragment Ž; p20. of caspase-3 was observed in etoposide co-treated cells ŽFig. 3B, lane 5.. The lack of PARP cleavage ŽFig. 3A, lane 5. indicated that this fragment was catalytically inactive. Thus ZVAD.fmk inhibited apoptosis by inhibiting either the processing of the effector caspases, -3 and -7 or a stageŽs. upstream of this processing, supporting a role for caspases in the execution phase of apoptosis in THP.1 cells in agreement with our previous observations ŽZhu et al., 1995, 1997; Kluck et al., 1997..
Fig. 2. Apoptosis was accompanied by the activation of caspases-3 and -7 and cleavage of PARP. THP.1 cells were incubated for 4 h alone Žlane 1., or up to 4 h with either etoposide Ž25 m M. Žlanes 2]5. or TPCK Ž75 m M. Žlanes 6]9.. At the indicated time points, cells were analysed by Western blot analysis for the cleavage of ŽA. 32 kDa pro-caspase-3, and ŽB. 35 kDa procaspase-7 to their respective catalytically active large subunits, as described in Section 2. Cells were also analysed using a mouse monoclonal antibody ŽC2]10. to detect both intact PARP and its cleaved 85-kDa fragment ŽC..
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Table 1 Z-VAD.fmk inhibits PS exposure, reduction in DCm and Ac-DEVD.AFC cleavage activity
Control Etoposide Etoposideq Z-VAD.fmk TPCK TPCKq Z-VAD.fmk
% PS exposure
% DCm Lo w
pmol AFCrmgrmin
2.8" 0.4 36.6" 2.0 1.9" 0.3 54.0" 3.3 2.3" 0.3
6.0" 2.0 35.5" 6.0 3.7" 1.1 37.6 " 2.6 4.2" 1.5
89 " 63 1584 " 386 65U 464U 81U
THP.1 cells were incubated for 4 h with either etoposide Ž25 m M. or TPCK Ž75 m M. in the presence or absence of Z-VAD.fmk Ž50 m M.. The percentage of apoptotic cells was then determined by the number of cells with PS externalization and decreased DCm , respectively. Cell lysates were also prepared for measuring Ac-DEVD.AFC cleavage activity as described in Section 2. The data represent the mean Ž"S.E.M.. of at least three experiments except for ŽU . where means of two experiments are given.
3.3. Release of cytochrome c occurs prior to the acti¨ ation of caspases Recent studies have implied a key role for the release of mitochondrial cytochrome c in activation of caspases in apoptosis. We therefore investigated the importance of this release in the induction of apoptosis in THP.1 cells and its susceptibility to Z-VAD.fmk. Incubation with either etoposide or TPCK resulted in a time-dependent cytosolic accumulation of cytochrome c ŽFig. 4,
lanes 2]6 and 8]12, respectively, upper blot .. The absence of cytochrome oxidase Žsubunit II. in the cytosolic extracts confirmed that the samples were free from mitochondrial contamination ŽFig. 4, bottom blot .. Following treatment with etoposide, an increase in cytochrome c release was clearly observed at 2 h ŽFig. 4, lane 4, upper blot ., whereas with TPCK this was already evident at 1 h ŽFig. 4, lane 9, upper blot .. However, these data did not distinguish whether cytochrome c release preceded processingractivation of caspases-3 and -7.
Fig. 3. Z-VAD.fmk inhibits PARP cleavage and activation of caspases-3 and -7. THP.1 cells were incubated for 4 h either alone Žlane 1. or with TPCK Ž75 m M. Žlanes 2]3. or etoposide Ž25 m M. Žlanes 4]5., in the presence of Z-VAD.fmk Ž50 m M. Žlanes 3 and 5, respectively.. Cells were analysed by Western blot analysis for ŽA. intact PARP and its cleaved 85-kDa fragment using a rabbit polyclonal antibody Ž318. and for the cleavage of the ŽB. 32-kDa pro-caspase-3 and ŽC. 35-kDa procaspase-7 to their respective catalytically active large subunits, as described in the legend to Fig. 2.
J. Zhuang, G.M. Cohen r Toxicology Letters 102]103 (1998) 121]129
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Fig. 4. Release of cytochrome c is an early event during apoptosis and is not blocked by Z-VAD.fmk. THP.1 cells were incubated for 4 h alone Žlane 1., or up to 4 h with either etoposide Ž25 m M. Žlanes 2]7. or TPCK Ž75 m M. Žlanes 8]13., in the presence of Z-VAD.fmk Ž50 m M. Žlanes 7 and 13, respectively.. At the indicated time points, cytosolic extracts were prepared and analysed by Western blot analysis for cytochrome c Žcyt. c . and also for cytochrome oxidase subunit II Žcyt. oxid.., which serves as a marker of mitochondrial contamination of the cytosolic extracts.
Interestingly Z-VAD.fmk did not block the cytosolic accumulation of cytochrome c following treatment with either etoposide ŽFig. 4, lane 7, upper blot . or TPCK ŽFig. 4, lane 13, upper blot ., although it effectively blocked the processingr activation of caspases-3 and -7 as well as PARP cleavage ŽFig. 3.. Thus Z-VAD.fmk prevented all the biochemical changes of apoptosis including the activation of caspases, cleavage of PARP, PS exposure and the fall in DCm except for the release of mitochondrial cytochrome c. This observation strongly suggested that release of cytochrome c occurred upstream of the activation of caspases. 4. Discussion We have studied the induction of apoptosis in THP.1 cells in order to identify the key eventŽs. occurring during the execution phase of apoptotic cell death. We now demonstrate that processingractivation of caspases-3 and -7 accompanies the generation of the apoptotic phenotype induced by either etoposide or TPCK. Several recent studies have highlighted a role for a decrease in DCm as a key committed step in the induction of apoptosis ŽZamzami et al., 1995; Petit et al., 1996.. Our studies showed that the activation of caspase-7 occurred prior to both PS exposure and reduction in DCm ŽFig. 1A,B.. This was further confirmed by the observation that Z-VAD.fmk, the caspase inhibitor, blocked the activation of caspases, PS exposure and reduction
in DCm . Thus, in this model of apoptosis the activation of caspases occurs upstream of both PS exposure and a decrease in DCm . We also demonstrate that Z-VAD.fmk inhibited apoptosis by either blocking the processing of both caspases-3 and -7 or inhibiting a stage upstream of this processing. These results are consistent with recent studies showing that ZVAD.fmk inhibits both the processing of caspases and their proteolytic activities ŽMacFarlane et al., 1997; Zhu et al., 1997.. Z-VAD.fmk completely inhibited the extensive processing of caspase-3 induced by TPCK but was less effective at inhibiting the more modest processing induced by etoposide ŽFig. 3B, lanes 3 and 5., suggesting that the target of Z-VAD.fmk may be different in etoposide- and TPCK-induced apoptosis. Release of mitochondrial cytochrome c appears to be an early event in our study. Pre-treatment with Z-VAD.fmk abolished the activation of caspases, PS exposure, and decrease in DCm , but did not affect the release of cytochrome c ŽFigs. 3 and 4, and Table 1.. These results demonstrate that the release of cytochrome c occurs upstream of the activation of caspases, PS exposure and decrease in DCm or is an independent event, a result consistent with other studies ŽKluck et al., 1997; Yang et al., 1997; Bossy-Wetzel et al., 1998.. Further support for this observation was provided by the finding that TPCK-induced cytochrome c release was clearly observed at 1 h ŽFig. 4., prior to the manifestation of other apoptotic features These data support the hypothesis that in etopo-
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side- and TPCK-induced apoptosis, the activation of caspases occurs after the release of mitochondrial cytochrome c, which most probably acts in concert with Apaf-1 and dATP, resulting in the processing of caspase-9 and the subsequent activation of effector caspases, such as caspase-3 ŽLiu et al., 1996; Li et al., 1997; Zou et al., 1997.. This caspase activation may further damage mitochondria resulting in a dramatic loss of DCm and an amplification of the apoptotic process. In contrast to chemical-mediated apoptosis, Z-VAD.fmk blocks all the features of Fas-induced apoptosis including the release of cytochrome c consistent with an initial activation of caspase-8 at the cell membrane ŽVander Heiden et al., 1997.. In summary, caspases play a key role as executioners of apoptosis and the initial activation of caspase-9 by the complex consisting of cytochrome c, dATP and Apaf-1 may thus represent the point of ‘no return’ in many models of chemical-induced apoptosis. References Alnemri, E.S., Livingston, D.J., Nicholson, D.W., et al., 1996. Human ICErCED-3 protease nomenclature. Cell 87, 171. Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223]254. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V., Wallach, D., 1996. Involvement of MACH, a novel MORT1rFADDinteracting protease, in FasrAPO-1- and TNF receptorinduced cell death. Cell 85, 803]815. Bossy-Wetzel, E., Newmeyer, D.D., Green, D.R., 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37]49. Castedo, M., Hirsch, T., Susin, S.A., et al., 1996. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J. Immunol. 157, 512]521. Cohen, G.M., 1997. Caspases: the executioners of apoptosis. Biochem. J. 326, 1]16. Earnshaw, W.C., 1995. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337]343. Fernandes-Alnemri, T., Litwack, G., Alnemri, E.S., 1995a. Mch2, a new member of the apoptotic Ced-3r Ice cysteine protease gene family. Cancer Res. 55, 2737]2742. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., et al., 1995b. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 55, 6045]6052. Fernandes-Alnemri, T., Armstrong, R.C., Krebs, J., et al.,
1996. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc. Natl. Acad. Sci. 93, 7464]7469. Kluck, R.M., Bossy-Wetzel, E., Green, D.R., Newmeyer, D.D., 1997. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132]1136. Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Keehnen, R.M.J., Pals, S.T., van Oers, M.H.J., 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415]1420. Kumar, S., Lavin, M.F., 1996. The ICE family of cysteine proteases as effectors of cell death. Cell Death Differ. 3, 255]267. Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., Earnshaw, W.C., 1994. Cleavage of polyŽADP-ribose. polymerase by a proteinase with properties like ICE. Nature 371, 346]347. Li, P., Nijhawan, D., Budihardjo, I., et al., 1997. Cytochrome c and dATP-dependent formation of Apaf-1rcaspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479]489. Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: Requirements for dATP and cytochrome c. Cell 86, 147]157. MacFarlane, M., Cain, K., Sun, X.-M., Alnemri, E.S., Cohen, G.M., 1997. Processingractivation of at least four interleukin-1 b converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. J. Cell Biol. 137, 469]479. Martin, S.J., Reutelingsperger, C.P.M., McGahon, A.J., et al., 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545]1556. Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., et al., 1996. FLICE, a novel FADD-homologous ICErCED-3-like protease, is recruited to the CD95 ŽFasrAPO-1. death-inducing signaling complex. Cell 85, 817]827. Nicholson, D.W., Ali, A., Thornberry, N.A., et al., 1995. Identification and inhibition of the ICErCED-3 protease necessary for mammalian apoptosis. Nature 376, 37]43. Petit, P.X., O’Connor, J.E., Grunwald, D., Brown, S.C., 1990. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem. 194, 389]397. Petit, P.X., Susin, S.-A., Zamzami, N., Mignotte, B., Kroemer, G., 1996. Mitochondria and programmed cell death: back to the future. FEBS Lett. 396, 7]13. Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456]1462. Thornberry, N.A., 1994. Interleukin-1 b converting enzyme. Methods Enzymol. 244, 615]631. Thornberry, N.A., Molineaux, S.M., 1995. Interleukin-1 b converting enzyme: A novel cysteine protease required for
J. Zhuang, G.M. Cohen r Toxicology Letters 102]103 (1998) 121]129 IL-1 b production and implicated in programmed cell death. Protein Sci. 4, 3]12. Vander Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T., Thompson, C.B., 1997. Bcl-x L regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627]637. Wilson, K.P., Black, J.-A.F., Thomson, J.A., et al., 1994. Structure and mechanism of interleukin-1 b converting enzyme. Nature 370, 270]274. Yang, J., Liu, X., Bhalla, K., et al., 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129]1132. Zamzami, N., Marchetti, P., Castedo, M., et al., 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661]1672.
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Release of mitochondrial cytochrome c is upstream of caspase activation in chemical-induced apoptosis in human monocytic tumour cells Jianguo Zhuang, Gerald M. CohenU MRC Toxicology Unit, Hodgkin Building, Uni¨ ersity of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK
Abstract Apoptosis, induced in human monocytic THP.1 cells by etoposide and N-tosyl-L-phenylalanyl chloromethyl ketone, was accompanied by the processingractivation of caspases, externalisation of phosphatidylserine ŽPS. and reduction in mitochondrial membrane potential Ž DCm .. Activation of caspaseŽs. occurred prior to both PS exposure and reduction in DCm. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp ŽOMe. fluoromethyl ketone ŽZ-VAD.fmk. blocked the activation of caspases, PS exposure and the reduction in DCm as well as the morphological changes associated with apoptosis but it did not inhibit the release of mitochondrial cytochrome c. These results suggest that the execution phase of chemical-induced apoptosis in THP.1 cells may be initiated following mitochondrial damage resulting in release of cytochrome c leading to activation of caspase-9 and then activation of effector caspases-3 and -7. This contrasts to receptor-mediated apoptosis, such as Fas, which results in an initial activation of caspase-8. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Caspases; Mitochondrial membrane potential; Cytochrome c
1. Introduction Apoptosis is a fundamental form of cell death which plays a major role in the development and homeostasis of multicellular organisms ŽArends and Wyllie, 1991.. Disturbances in apoptosis are important in cancer, acquired immunodeficiency syndrome and some neurodegenerative disorders ŽThompson, 1995.. Apoptotic cell death comprises an initial commitment phase followed by an
U
Corresponding author.
execution phase, which is characterised by a consistent series of distinct morphological changes ŽEarnshaw, 1995., suggesting the existence of a common execution machinery. In the nematode C. elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence homology to the mammalian cysteine protease, interleukin1 b converting enzyme ŽICE., a family of at least 10 related ICE-like proteases, now known as caspases ŽAlnemri et al., 1996., has been identified. Caspases are characterised by an almost absolute specificity for aspartic acid in the P1 position of
0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00296-3
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the substrate ŽThornberry and Molineaux, 1995; Kumar and Lavin, 1996; Cohen, 1997.. They exist in cells as inactive proenzymes and, upon activation, are cleaved at specific aspartate residues, generating a large and small subunit, which together form the active enzyme in the form of a heterotetramer containing two large and two small subunits ŽWilson et al., 1994.. Caspases may be divided into ‘initiator’ caspases with long prodomains Že.g. caspase-8, -9 and -10., which activate ‘effector’ caspases with short prodomains Že.g. caspase-3, -6 and -7., which in turn cleave intracellular proteins, such as poly ŽADP-ribose. polymerase ŽPARP. and lamins, so precipitating the dramatic morphological and biochemical changes of apoptosis ŽCohen, 1997.. Apoptosis induced by CD95 ŽFasrAPO-1. results in the recruitment of the adapter molecule FADD ŽFas-associated protein with death domain., which then binds to the N-terminal death effector domains of caspase-8 resulting in its activation thereby providing a direct link between cell death receptors and the caspases ŽBoldin et al., 1996; Muzio et al., 1996.. Recently, it has been shown that in cells triggered to undergo apoptosis by a variety of stimuli, mitochondrial cytochrome c is released and, in the presence of dATP and Apaf-1, results in the activation of caspase-9, which in turn activates other caspases, such as caspase-3 ŽLiu et al., 1996; Li et al., 1997; Zou et al., 1997.. Therefore execution of the death programme appears to occur following activation of a hierarchy of caspases with caspase-8 and caspase-9 being at the apex of this apoptotic cascade originating at the cell membrane or the mitochondria, respectively. In this study, using human monocytic THP.1 cells we demonstrate that caspases are key executioners of apoptosis induced by the anti-cancer drug, etoposide, and the serine protease inhibitor, N-tosyl-L -phenylalanyl chloromethyl ketone ŽTPCK.. The broad spectrum caspase inhibtor, Z-VAD.fmk, inhibits all the biochemical and morphological features of apoptosis, including activation of caspases, and reduction in mitochondrial membrane potential Ž DCm .. However, ZVAD.fmk does not block the release of cytochrome c, suggesting that following chemical-in-
duced apoptosis activation of caspases is initiated following perturbations of the mitochondria. 2. Materials and methods 2.1. Materials Chemicals were obtained from Sigma Chemical Company ŽPoole, England. except for N-tosyl-Lphenylalanyl chloromethyl ketone ŽTPCK, Boehringer-Mannheim UK, Lewes, England. and benzyloxycarbonyl-Val-Ala-Asp ŽOMe . fluoromethyl ketone ŽZ-VAD.fmk, Enzyme Systems Inc., Dublin, CA, USA.. 2.2. Cell culture and treatment THP.1 cells were maintained as suspension culture and apoptosis induced by etoposide Ž25 m M. or TPCK Ž75 m M. as previously described ŽZhu et al., 1995, 1997.. 2.3. Flow cytometric analysis of apoptosis Cells with externalized phosphatidylserine ŽPS. residues were detected by a FITC-labelled Annexin V binding assay ŽMartin et al., 1995.. For measuring mitochondrial membrane potential Ž DCm . 0.5= 10 6 cells were incubated for 20 min at 378C with 3,39dihexyloxacarbocyanine iodide wDiOC 6 Ž3., 50 nMx ŽMolecular Probes Inc., Eugene, OR, USA., which is retained in mitochondria with a normal membrane potential ŽPetit et al., 1990.. 2.4. Western blot analysis and preparation of cytosolic extracts for assessment of cytochrome c Samples of 0.2= 10 6 cells were prepared for Western blot analysis as previously described ŽMacFarlane et al., 1997.. Isolation of the cytosolic extracts was essentially as described ŽKluck et al., 1997.. Cytosolic protein Ž30 mg. was separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with a mouse monoclonal antibody recognising human cytochrome c Ž7H8.2C12. ŽPharMingen, San Diego, CA, USA. at a dilution of 1:1000. Parallel samples, contain-
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ing an equal amount of cytosolic protein, were checked by immunoblotting for mitochondrial contamination using the 12C4-F12 mouse monoclonal antibody ŽMolecular Probes. to mitochondrial membrane-bound cytochrome c oxidase Žsubunit II. as described ŽKluck et al., 1997.. 2.5. Measurement of Ac-DEVD.AFC clea¨ age acti¨ ity Cytosolic extracts were prepared as described above and stored at y808C. The proteolytic activity of caspaseŽs. in cytosolic extracts was measured using a continuous fluorimetric assay initially described by Thornberry Ž1994., with minor modifications ŽMacFarlane et al., 1997.. Liberation of fluorogenic 7-amino-4-trifluoromethylcoumarin from Ac-DEVD.AFC ŽEnzyme Systems Inc.. as a measure of caspases-3 and -7 like activity was assayed at excitation and emission wavelengths of 400 and 505 nm, respectively. The protease activity was expressed as pmol AFCrmg proteinrmin. 3. Results 3.1. Etoposide and TPCK-induced apoptosis in THP.1 cells Externalization of PS is considered to be a general feature of apoptosis induced by many different stimuli and can be measured by binding of annexin V, a PS-binding protein ŽKoopman et al., 1994; Martin et al., 1995.. A reduction in DCm has also been reported to be an early event in the induction of apoptosis in many different systems ŽCastedo et al., 1996; Zamzami et al., 1996.. In this study, apoptosis was initially assessed using two flow cytometric methods measuring the externalization of PS and the percentage of cells with decreased DCm . Incubation of THP.1 cells with etoposide, a DNA topoisomerase II inhibitor, or TPCK, a chymotrypsin-like serine protease inhibitor, resulted in a time-dependent induction of apoptosis, as assessed by both PS exposure and decreased DCm ŽFig. 1A.. While a decreased DCm has been proposed to regulate the externalization of PS ŽCastedo et al., 1996., in the present studies
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both these changes demonstrated a similar time dependence, occurring at 2 h after treatment, irrespective of the apoptotic stimulus ŽFig. 1A.. Thus induction of apoptosis in THP.1 cells is accompanied by the externalization of PS and a decreased DCm . It has been shown that during the induction of apoptosis in THP.1 cells at least four caspases are activated, including caspases-2, -3, -6 and -7, and cleave cellular substrates such as PARP and lamins ŽMacFarlane et al., 1997; Zhu et al., 1997.. Therefore the proteolytic activity of caspases was measured by monitoring the Ac-DEVD.AFC cleavage activity of cell lysates following treatment with either etoposide or TPCK. AcDEVD.AFC was chosen as a substrate because the DEVD tetrapeptide sequence mimics the cleavage site in PARP and several other proteins cleaved during apoptosis, i.e. DEVDxG ŽLazebnik et al., 1994; Cohen, 1997.. In addition, caspases-3, -6 and -7 all have Ac-DEVD.AMC cleavage activity ŽFernandes-Alnemri et al., 1995a,b; Nicholson et al., 1995.. The Ac-DEVD.AFC cleaving activity of lysates obtained from control cells was very low even after a 4-h incubation ŽFig. 1B.. In contrast, incubation of THP.1 cells with either etoposide Ž25 m M., or TPCK Ž75 m M., resulted in a time-dependent increase in the Ac-DEVD.AFC cleavage activity ŽFig. 1B.. In lysates prepared from etoposide-treated cells, an increase in Ac-DEVD.AFC cleavage activity was first observed at 2 h after treatment and continued to rise over a 4-h incubation period ŽFig. 1B., paralleling the time course of the induction of apoptosis ŽFig. 1A.. The increase in AcDEVD.AFC cleavage activity in lysates prepared from TPCK-treated cells was similar to that of etoposide treatment in the first 2 h of incubation, but then reached a plateau ŽFig. 1B.. Whether the differences in both the level and pattern of proteolytic activity in lysates obtained from etoposide- and TPCK-treated cells reflected distinct mechanisms employed by the two stimuli to induce apoptosis or were due to some effects of the apoptotic stimulus used on proteolytic activity after the activation of caspases remains to be determined. Next, we examined the temporal sequence of
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Fig. 1. Induction of apoptosis by etoposide and TPCK. THP.1 cells were incubated for up to 4 h with either etoposide Ž25 m M. or TPCK Ž75 m M.. ŽA. Apoptosis was assessed by the number of cells with PS exposure Ž`}`. and decreased DCm Žv}v., respectively. ŽB. Cell lysates were also prepared for measuring Ac-DEVD.AFC cleavage activity as described in Section 2.
activation of caspases-3 and -7 and the proteolysis of the intracellular protein substrate PARP. Untreated control cells showed the presence of the intact ; 32-kDa proform of caspase-3 and ; 35kDa proform of caspase-7 ŽFig. 2A,B, lane 1,
respectively.. Processing of caspase-3 at Asp175 between the large and small subunits yields a p20 fragment, which is further processed at Asp9 and Asp 28 to yield p19 and p17 fragments, respectively ŽFernandes-Alnemri et al., 1996., whereas
J. Zhuang, G.M. Cohen r Toxicology Letters 102]103 (1998) 121]129
processing of caspase-7 occurs at Asp198 between the large and small subunits, followed by cleavage at Asp 23 to yield the p19 large subunit ŽFernandes-Alnemri et al., 1995b.. Induction of apoptosis with either etoposide or TPCK was accompanied by a time-dependent formation of the catalytically active large subunits of caspases-3 and -7 ŽFig. 2A,B, lanes 2]9, respectively.. Although difficult to precisely quantitate, it was possible that processing of caspase-7 to its catalytically active large subunit was observed prior to that of caspase-3 ŽFig. 2A,B. and also PARP ŽFig. 2C.. Dependent on the apoptotic stimulus, some differences were observed in the formation of the large subunits of caspase-3 ŽFig. 2A., possibly indicating some differences in the mechanisms of activation. Induction of apoptosis by either etoposide or TPCK resulted in a time-dependent loss of intact PARP together with formation of its characteristic signature 85-kDa fragment ŽFig. 2C.. Thus, induction of apoptosis in THP.1 cells was accompanied by PS exposure, reduction in DCm , cleavage of PARP and activation of caspases. The time-course studies suggested that caspase activation occurred upstream of these other events.
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3.2. Z-VAD.fmk inhibits acti¨ ation of caspases, PS exposure, and reduction in DCm Z-VAD.fmk, a cell membrane permeable inhibitor of caspases, inhibited apoptosis induced by both etoposide and TPCK, as assessed by PS exposure, reduction in DCm and Ac-DEVD.AFC cleavage activity ŽTable 1., providing further evidence of involvement of caspases in the execution phase of apoptosis. Z-VAD.fmk blocked TPCKand etoposide-induced PARP cleavage ŽFig. 3A.. Z-VAD.fmk also clearly blocked the processing of caspases-3 and -7 in cells treated with either TPCK or etoposide ŽFig. 3B,C.. However, a small amount of a slightly larger immunoreactive fragment Ž; p20. of caspase-3 was observed in etoposide co-treated cells ŽFig. 3B, lane 5.. The lack of PARP cleavage ŽFig. 3A, lane 5. indicated that this fragment was catalytically inactive. Thus ZVAD.fmk inhibited apoptosis by inhibiting either the processing of the effector caspases, -3 and -7 or a stageŽs. upstream of this processing, supporting a role for caspases in the execution phase of apoptosis in THP.1 cells in agreement with our previous observations ŽZhu et al., 1995, 1997; Kluck et al., 1997..
Fig. 2. Apoptosis was accompanied by the activation of caspases-3 and -7 and cleavage of PARP. THP.1 cells were incubated for 4 h alone Žlane 1., or up to 4 h with either etoposide Ž25 m M. Žlanes 2]5. or TPCK Ž75 m M. Žlanes 6]9.. At the indicated time points, cells were analysed by Western blot analysis for the cleavage of ŽA. 32 kDa pro-caspase-3, and ŽB. 35 kDa procaspase-7 to their respective catalytically active large subunits, as described in Section 2. Cells were also analysed using a mouse monoclonal antibody ŽC2]10. to detect both intact PARP and its cleaved 85-kDa fragment ŽC..
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Table 1 Z-VAD.fmk inhibits PS exposure, reduction in DCm and Ac-DEVD.AFC cleavage activity
Control Etoposide Etoposideq Z-VAD.fmk TPCK TPCKq Z-VAD.fmk
% PS exposure
% DCm Lo w
pmol AFCrmgrmin
2.8" 0.4 36.6" 2.0 1.9" 0.3 54.0" 3.3 2.3" 0.3
6.0" 2.0 35.5" 6.0 3.7" 1.1 37.6 " 2.6 4.2" 1.5
89 " 63 1584 " 386 65U 464U 81U
THP.1 cells were incubated for 4 h with either etoposide Ž25 m M. or TPCK Ž75 m M. in the presence or absence of Z-VAD.fmk Ž50 m M.. The percentage of apoptotic cells was then determined by the number of cells with PS externalization and decreased DCm , respectively. Cell lysates were also prepared for measuring Ac-DEVD.AFC cleavage activity as described in Section 2. The data represent the mean Ž"S.E.M.. of at least three experiments except for ŽU . where means of two experiments are given.
3.3. Release of cytochrome c occurs prior to the acti¨ ation of caspases Recent studies have implied a key role for the release of mitochondrial cytochrome c in activation of caspases in apoptosis. We therefore investigated the importance of this release in the induction of apoptosis in THP.1 cells and its susceptibility to Z-VAD.fmk. Incubation with either etoposide or TPCK resulted in a time-dependent cytosolic accumulation of cytochrome c ŽFig. 4,
lanes 2]6 and 8]12, respectively, upper blot .. The absence of cytochrome oxidase Žsubunit II. in the cytosolic extracts confirmed that the samples were free from mitochondrial contamination ŽFig. 4, bottom blot .. Following treatment with etoposide, an increase in cytochrome c release was clearly observed at 2 h ŽFig. 4, lane 4, upper blot ., whereas with TPCK this was already evident at 1 h ŽFig. 4, lane 9, upper blot .. However, these data did not distinguish whether cytochrome c release preceded processingractivation of caspases-3 and -7.
Fig. 3. Z-VAD.fmk inhibits PARP cleavage and activation of caspases-3 and -7. THP.1 cells were incubated for 4 h either alone Žlane 1. or with TPCK Ž75 m M. Žlanes 2]3. or etoposide Ž25 m M. Žlanes 4]5., in the presence of Z-VAD.fmk Ž50 m M. Žlanes 3 and 5, respectively.. Cells were analysed by Western blot analysis for ŽA. intact PARP and its cleaved 85-kDa fragment using a rabbit polyclonal antibody Ž318. and for the cleavage of the ŽB. 32-kDa pro-caspase-3 and ŽC. 35-kDa procaspase-7 to their respective catalytically active large subunits, as described in the legend to Fig. 2.
J. Zhuang, G.M. Cohen r Toxicology Letters 102]103 (1998) 121]129
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Fig. 4. Release of cytochrome c is an early event during apoptosis and is not blocked by Z-VAD.fmk. THP.1 cells were incubated for 4 h alone Žlane 1., or up to 4 h with either etoposide Ž25 m M. Žlanes 2]7. or TPCK Ž75 m M. Žlanes 8]13., in the presence of Z-VAD.fmk Ž50 m M. Žlanes 7 and 13, respectively.. At the indicated time points, cytosolic extracts were prepared and analysed by Western blot analysis for cytochrome c Žcyt. c . and also for cytochrome oxidase subunit II Žcyt. oxid.., which serves as a marker of mitochondrial contamination of the cytosolic extracts.
Interestingly Z-VAD.fmk did not block the cytosolic accumulation of cytochrome c following treatment with either etoposide ŽFig. 4, lane 7, upper blot . or TPCK ŽFig. 4, lane 13, upper blot ., although it effectively blocked the processingr activation of caspases-3 and -7 as well as PARP cleavage ŽFig. 3.. Thus Z-VAD.fmk prevented all the biochemical changes of apoptosis including the activation of caspases, cleavage of PARP, PS exposure and the fall in DCm except for the release of mitochondrial cytochrome c. This observation strongly suggested that release of cytochrome c occurred upstream of the activation of caspases. 4. Discussion We have studied the induction of apoptosis in THP.1 cells in order to identify the key eventŽs. occurring during the execution phase of apoptotic cell death. We now demonstrate that processingractivation of caspases-3 and -7 accompanies the generation of the apoptotic phenotype induced by either etoposide or TPCK. Several recent studies have highlighted a role for a decrease in DCm as a key committed step in the induction of apoptosis ŽZamzami et al., 1995; Petit et al., 1996.. Our studies showed that the activation of caspase-7 occurred prior to both PS exposure and reduction in DCm ŽFig. 1A,B.. This was further confirmed by the observation that Z-VAD.fmk, the caspase inhibitor, blocked the activation of caspases, PS exposure and reduction
in DCm . Thus, in this model of apoptosis the activation of caspases occurs upstream of both PS exposure and a decrease in DCm . We also demonstrate that Z-VAD.fmk inhibited apoptosis by either blocking the processing of both caspases-3 and -7 or inhibiting a stage upstream of this processing. These results are consistent with recent studies showing that ZVAD.fmk inhibits both the processing of caspases and their proteolytic activities ŽMacFarlane et al., 1997; Zhu et al., 1997.. Z-VAD.fmk completely inhibited the extensive processing of caspase-3 induced by TPCK but was less effective at inhibiting the more modest processing induced by etoposide ŽFig. 3B, lanes 3 and 5., suggesting that the target of Z-VAD.fmk may be different in etoposide- and TPCK-induced apoptosis. Release of mitochondrial cytochrome c appears to be an early event in our study. Pre-treatment with Z-VAD.fmk abolished the activation of caspases, PS exposure, and decrease in DCm , but did not affect the release of cytochrome c ŽFigs. 3 and 4, and Table 1.. These results demonstrate that the release of cytochrome c occurs upstream of the activation of caspases, PS exposure and decrease in DCm or is an independent event, a result consistent with other studies ŽKluck et al., 1997; Yang et al., 1997; Bossy-Wetzel et al., 1998.. Further support for this observation was provided by the finding that TPCK-induced cytochrome c release was clearly observed at 1 h ŽFig. 4., prior to the manifestation of other apoptotic features These data support the hypothesis that in etopo-
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side- and TPCK-induced apoptosis, the activation of caspases occurs after the release of mitochondrial cytochrome c, which most probably acts in concert with Apaf-1 and dATP, resulting in the processing of caspase-9 and the subsequent activation of effector caspases, such as caspase-3 ŽLiu et al., 1996; Li et al., 1997; Zou et al., 1997.. This caspase activation may further damage mitochondria resulting in a dramatic loss of DCm and an amplification of the apoptotic process. In contrast to chemical-mediated apoptosis, Z-VAD.fmk blocks all the features of Fas-induced apoptosis including the release of cytochrome c consistent with an initial activation of caspase-8 at the cell membrane ŽVander Heiden et al., 1997.. In summary, caspases play a key role as executioners of apoptosis and the initial activation of caspase-9 by the complex consisting of cytochrome c, dATP and Apaf-1 may thus represent the point of ‘no return’ in many models of chemical-induced apoptosis. References Alnemri, E.S., Livingston, D.J., Nicholson, D.W., et al., 1996. Human ICErCED-3 protease nomenclature. Cell 87, 171. Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223]254. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V., Wallach, D., 1996. Involvement of MACH, a novel MORT1rFADDinteracting protease, in FasrAPO-1- and TNF receptorinduced cell death. Cell 85, 803]815. Bossy-Wetzel, E., Newmeyer, D.D., Green, D.R., 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37]49. Castedo, M., Hirsch, T., Susin, S.A., et al., 1996. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J. Immunol. 157, 512]521. Cohen, G.M., 1997. Caspases: the executioners of apoptosis. Biochem. J. 326, 1]16. Earnshaw, W.C., 1995. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337]343. Fernandes-Alnemri, T., Litwack, G., Alnemri, E.S., 1995a. Mch2, a new member of the apoptotic Ced-3r Ice cysteine protease gene family. Cancer Res. 55, 2737]2742. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., et al., 1995b. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 55, 6045]6052. Fernandes-Alnemri, T., Armstrong, R.C., Krebs, J., et al.,
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