Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

Abstract

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9. The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9. CD95 (APO-1/Fas) is the prototype member of the death receptor family (1Peter M.E. Krammer P.H. Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (894) Google Scholar). Stimulation of CD95 with its cognate ligand or agonistic antibodies results in receptor oligomerization and recruitment of signaling proteins in a death-inducing signaling complex (DISC) 3The abbreviations used are: DISC, death-inducing signaling complex; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; Q-VD-OPh, N-(2-quinolyl)valyl-aspartyl-(2,6-difluoro-phenoxy)-methyl ketone; Bak, Bcl2-antagonist/killer; siRNA, small interfering RNA; CD95L, CD95 ligand; MEF, mouse embryonic fibroblast; 7AAD, 7-aminoactinomycin D; ΔΨM, mitochondrial transmembrane potential. (2Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1786) Google Scholar, 3Schmitz I. Walczak H. Krammer P.H. Peter M.E. Cell Death Differ. 1999; 6: 821-822Crossref PubMed Scopus (62) Google Scholar). Essential for apoptosis signaling through death receptors are the DISC components Fas-associated death domain and caspase-8. Recruitment of caspase-8 into the DISC leads to its dimerization-induced activation and the autoproteolytic release of its active subunits into the cytosol. The active caspase-8 activates downstream caspases, such as caspase-3, -6 and-7, that are responsible for most of the morphological manifestations of cell death (1Peter M.E. Krammer P.H. Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (894) Google Scholar). This death receptor-mediated pathway is also called the extrinsic pathway and is distinguished from the intrinsic mitochondrial death pathway. Apoptosis induction via mitochondria is regulated by the Bcl-2 family and involves early loss of mitochondrial membrane potential (ΔΨM), release of cytochrome c, and other apoptogenic factors (4Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). In the cytosol, cytochrome c binds to Apaf-1 leading to caspase-9 recruitment. At this apoptosome complex, caspase-9 is activated and initiates the caspase cascade and subsequent apoptosis. An involvement of mitochondria has also been demonstrated in CD95 signaling, suggesting a functional role of this organelle in death receptor-mediated apoptosis of certain cell types (5Barnhart B.C. Alappat E.C. Peter M.E. Semin. Immunol. 2003; 15: 185-193Crossref PubMed Scopus (376) Google Scholar, 6Schmitz I. Kirchhoff S. Krammer P.H. Int. J. Biochem. Cell Biol. 2000; 32: 1123-1136Crossref PubMed Scopus (234) Google Scholar). Two types of CD95 signaling pathways have been proposed (7Scaffidi 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 (2631) Google Scholar). CD95 type I cells show efficient DISC formation and strong activation of caspase-8 at the receptor level, which directly triggers the caspase cascade. Type II cells, in contrast, form a weak DISC and produce very little active caspase-8 at the receptor level, which is insufficient to initiate the apoptotic process. The low amounts of active caspase-8 can cleave Bid, a proapoptotic Bcl-2 protein that translocates to mitochondria and induces apoptosome formation (5Barnhart B.C. Alappat E.C. Peter M.E. Semin. Immunol. 2003; 15: 185-193Crossref PubMed Scopus (376) Google Scholar, 6Schmitz I. Kirchhoff S. Krammer P.H. Int. J. Biochem. Cell Biol. 2000; 32: 1123-1136Crossref PubMed Scopus (234) Google Scholar). Thus, type II cells strongly rely on the mitochondrial amplification loop. The two-pathway model is mostly based on the criterion that expression of antiapoptotic Bcl-2 proteins inhibits apoptosis in type II but not type I cells (3Schmitz I. Walczak H. Krammer P.H. Peter M.E. Cell Death Differ. 1999; 6: 821-822Crossref PubMed Scopus (62) Google Scholar, 7Scaffidi 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 (2631) Google Scholar, 8Scaffidi 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 (530) Google Scholar). However, overexpression of Bcl-2 or Bcl-xL only delays or attenuates (but does mostly not abolish) CD95-mediated apoptosis, making an unambiguous classification of CD95 signaling difficult. Moreover, differences in the effects of CD95-stimulating agents, such as agonistic antibodies and recombinant CD95L, have challenged the existence of the two cell types and validity of the type I/II model (9Huang 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 (277) Google Scholar, 10Huang D.C. Tschopp J. Strasser A. Cell Death Differ. 2000; 7: 754-755Crossref PubMed Scopus (31) Google Scholar). We report here a direct genetic proof of the type I/II model using a T cell line with caspase-9 deficiency. Upon CD95 stimulation, caspase-9-deficient cells revealed highly impaired activation of effector caspase-3 and DNA fragmentation, which was restored by reconstitution of caspase-9 expression. Loss of caspase-9 did also not lead to a switch of cell death toward necrosis. Interestingly, caspase-9-deficient cells showed no loss of the mitochondrial transmembrane potential (ΔΨM), although cytochrome c was released. Thus, these findings suggest that both events can be uncoupled and that caspase-9 is required for loss of ΔΨM but not for cytochrome c release. Finally, we demonstrate that different death receptors differentially depend on a caspase-9-triggered mitochondrial amplification loop. Cell Culture and Reagents—All cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum, penicillin, and streptomycin (PAA Laboratories, Linz, Austria). The following antibodies were used: anti-CD95 (2R2) and anti-caspase-8 (12F5; both from BioCheck, Münster, Germany), anti-caspase-3 (catalog number AF-605-NA; R&D Systems, Wiesbaden, Germany), anti-caspase-9 (catalog number 9502; Cell Signaling Technology, Danvers, MA), anti-cytochrome c (7H8.2C12 and 6H2.B4) and anti-Bcl-x (catalog number 610212; BD Biosciences), conformation-specific anti-Bak (Ab-2; Oncogene), anti-Bcl-2 (C2; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-β-actin (AC-74) and anti-tubulin (DM1A; both from Sigma). For detection of murine proteins the following antibodies were used: anti-Bid (catalog number AF860; R&D Systems), anti-caspase-3 (clone 46; BD Biosciences), anti-caspase-8 (1G12; Alexis, Lausen, Switzerland), and anti-caspase-9 (catalog number 9504) and anti-Erk (catalog number 9102; both from Cell Signaling Technology). The horseradish peroxidase-conjugated goat anti-mouse IgG1, IgG2a, and IgG2b were from Southern Biotechnology Associates (Birmingham, AL), the horseradish peroxidase-conjugated goat anti-rabbit IgG was from Santa Cruz Biotechnology. Recombinant human and mouse TRAIL were purchased from R&D Systems (TEC-375) and Alexis (ALX-201-130), respectively. The human cytokines TNF and CD95L were a kind gift from R. U. Jänicke and H. Wajant, respectively. The caspase inhibitor N-(2-quinolyl)valylaspartyl-(2,6-difluoro-phenoxy)methyl ketone (Q-VD-OPh) was purchased from MP Biomedicals (Irvine, CA). Fluorescence-activated Cell Sorter Assays—Surface stainings, DNA fragmentation, and ΔΨM assays were performed as described (11Schmitz I. Weyd H. Krueger A. Baumann S. Fas S.C. Krammer P.H. Kirchhoff S. J. Immunol. 2004; 172: 2194-2200Crossref PubMed Scopus (72) Google Scholar). Flow cytometric analysis of cytochrome c release was carried out as described previously (12Waterhouse N.J. Trapani J.A. Cell Death Differ. 2003; 10: 853-855Crossref PubMed Scopus (141) Google Scholar). Uptake of propidium iodide (2 μg/ml) or 7-aminoactinomycin D (7AAD) (1 μg/ml) into nonfixed cells was evaluated by flow cytometric analyses of the FL2 or FL3 profile, respectively (13Wesselborg S. Engels I.H. Rossmann E. Los M. Schulze-Osthoff K. Blood. 1999; 93: 3053-3063Crossref PubMed Google Scholar). For flow cytometric analysis of Bak conformational change, cells were fixed in phosphate-buffered saline, 0.5% paraformaldehyde on ice for 30 min and subsequently washed three times in phosphate-buffered saline, 1% fetal calf serum. Staining with conformation-specific antibody against Bak and isotype matched control antibody was performed with a 1:50 dilution of the respective antibody in 50 μl of staining buffer (phosphate-buffered saline, 1% fetal calf serum, 50 μg/ml digitonin). Then, cells were washed and resuspended in 50 μl of staining buffer containing 0.1 μg of Alexafluor 488-labeled chicken anti-mouse for 30 min in the dark. Cells were washed again and analyzed on a FACScalibur machine (BD Biosciences). Immunoprecipitation, Western Blot Analysis, and Caspase Activity Assays—Immunoprecipitation of the CD95 DISC and Western blot analyses were performed as described (11Schmitz I. Weyd H. Krueger A. Baumann S. Fas S.C. Krammer P.H. Kirchhoff S. J. Immunol. 2004; 172: 2194-2200Crossref PubMed Scopus (72) Google Scholar). Caspase-3-like activity was measured in triplicates using Asp-Glu-Val-Asp-aminomethylcoumarin (Biomol, Hamburg, Germany) as a substrate (14Sohn D. Schulze-Osthoff K. Janicke R.U. J. Biol. Chem. 2005; 280: 5267-5273Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Transfection and Reverse Transcriptase-PCR Analysis— Caspase-9-deficient Jurkat cells were electroporated at 250 V and 950 μF with an N-terminally FLAG-tagged procaspase-9 construct and selected for hygromycin resistance. RNA was isolated and PCR amplified for caspase-9 and glyceraldehyde-3-phosphate dehydrogenase using standard protocols. The 3′ and 5′ primers used for amplification were 5′-ATG GAC GAA GCG GAT CGG and 5′-CCC TGG CCT TAT GAT GTT-3′ for caspase-9 and 5′-GTG GAA GGA CTC ATG ACC ACA G-3′ and 5′-CTG GTG CTC AGT GTA GCC CAG-3′ for glyceraldehyde-3-phosphate dehydrogenase. siRNA Transfection—siRNAs targeting CASP9 and control siRNAs directed against luciferase were obtained from Dharmacon RNA Technologies, Inc. (Lafayette, CO). Transfection into HT1080 and SKW6.4 cells was performed with Dharmafect 4 according to manufacturer's procedures (Dharmacon). At 48 cells were with CD95L or for cell death was by staining with and flow cell death was as apoptosis apoptosis We different Jurkat for their toward cell this Jurkat cells were with different of anti-CD95 antibody for and apoptosis was measured by of DNA in a flow efficient of the CD95 death we protein to the We that Jurkat a toward CD95-mediated apoptosis with or other Jurkat At a of of cells showed apoptosis with in very of antibody were μg/ml a certain number of cells showed DNA However, the of apoptosis was in cells that of cells was not by of we analyzed CD95 cell expression by flow in both Jurkat of CD95 on the cell suggesting a in the signaling cascade. and in line with this cells showed no expression of caspase-9 protein or as analyzed by Western and protein expression of Bcl-2 and Bcl-xL was in and cells of other caspases, Bcl-2 family and inhibitor of apoptosis proteins were the two Jurkat not CD95 is to cell death certain D. M. J. 1998; PubMed Scopus Google Scholar, R. M. A. S. Tschopp J. Immunol. 2000; PubMed Scopus Google Scholar). cells switch to necrosis upon CD95 stimulation, we analyzed cell death by the propidium iodide cells were with of the cells propidium which to and 48 of CD95 In contrast, cells showed only a cell death of at the overexpression of Bcl-2 that only a caspase-9 of cells a and against anti-CD95 We findings and cells were for with anti-CD95 and subsequently analyzed by most of the cells showed an apoptotic upon CD95 stimulation, cells Thus, cells show highly impaired apoptosis upon CD95 and not switch to cell caspase-9 activation of other caspases, we and cells with recombinant CD95L for various by Western blot the of caspase-8 was in caspase-9-deficient cells the active of caspase-8 was in cells of CD95 stimulation, in cells only 4 caspase-3 activation was in cells at and that of caspase-9. In contrast, caspase-3 was at the in the caspase-9 Jurkat caspase-8 in the not its of activity (14Sohn D. Schulze-Osthoff K. Janicke R.U. J. Biol. Chem. 2005; 280: 5267-5273Abstract Full Text Full Text PDF PubMed Scopus (89) Google we its recruitment to CD95 by DISC in and cells, Fas-associated death domain and caspase-8 were to the DISC at Thus, DISC formation is not by the loss of caspase-9. However, caspase-9 activity is required for efficient activation of the effector caspase-3 CD95-mediated apoptosis in Jurkat or not caspase-9 the CD95 pathway only in Jurkat cells, we an RNA in type I and type II HT1080 and SKW6.4 cells were with siRNAs or control 48 caspase-9 expression was in both cell lines with siRNAs with control cells HT1080 type II cells were for with CD95L, of caspase-9 resulted in CD95-mediated apoptosis In SKW6.4 type I cells, however, no in apoptosis induction was control and caspase-9 cells the of caspase-9 was in these cells in HT1080 cells Thus, caspase-9 expression is required for efficient induction via CD95 in type II but not in type I that the in cells were by the of we cells with caspase-9. were selected that procaspase-9 at with cells showed toward anti-CD95 and CD95L as cells suggesting that caspase-9 is the only the CD95 pathway in cells Furthermore, as for two CD95 expression was not in the of these was for and cells were with recombinant CD95L for various and caspase was analyzed by Western that the caspase-9 cells caspase-8 and as as caspase-3 of with At this cells revealed activation of caspase-3, in the caspase-9 cells no caspase-3 was the of caspase-9 we and cells with anti-CD95 for and analyzed apoptotic measured by DNA fragmentation, cells showed of apoptosis as cells, the cells were toward anti-CD95 cells also for propidium iodide and showed Asp-Glu-Val-Asp-aminomethylcoumarin as cells, cells in both Thus, reconstitution of caspase-9 expression in cells restored toward CD95-mediated apoptosis. of proapoptotic factors from such as cytochrome c, is to be with loss of ΔΨM H. H. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the these two events is and we to apoptotic events at the mitochondria in we measured the conformational of Bak, a of mitochondrial Korsmeyer S.J. 2001; PubMed Scopus Google using a conformation-specific antibody and flow Interestingly, of CD95 Bak was activated and a conformational in and cells In line, upon CD95 activation, as by Western was in the three cell lines not Flow cytometric of cytochrome c release revealed that of anti-CD95 apoptogenic factors were from the mitochondria of and cells however, loss of ΔΨM was only in and cells but not in caspase-9-deficient cells show that caspase-9 can cytochrome c release and loss of ΔΨM as two events in apoptosis. we only CD95-mediated apoptosis or also and cell death on caspase-9 in Jurkat this and cells were with different of CD95L, or TRAIL for were analyzed for cell death at the of DNA and membrane cells were to CD95L, and cells apoptosis and Thus, the of cells is against agonistic antibodies as as against recombinant cells were against apoptosis and Interestingly, and cells showed different on the both cell lines were toward cell death with cells and In to CD95L and TRAIL, caspase-9-deficient and cells to a was used as a death and Thus, death apoptosis differentially on caspase-9 in a cell of caspase-9 death receptor signaling also in cells, we analyzed and caspase-9-deficient mouse embryonic for their toward CD95-mediated apoptosis. has been not to CD95 in or activated R. A. M. A. D. J. R. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. H. 1998; Full Text Full Text PDF PubMed Scopus Google but no were performed in type II cells such as we and caspase-9-deficient with of CD95L or in a upon of cell death in caspase-9-deficient was In contrast, both cell types upon of Thus, as in the Jurkat model, caspase-9 but not apoptosis. We also the of caspase Upon with CD95L for cell death was only in caspase-9-deficient of cells of the inhibitor cell death to in caspase-9-deficient also strongly was cell death in cells in the of the caspase Finally, we analyzed caspase and in and caspase-9-deficient by Western The caspase-8 was in both cell although was in caspase-9-deficient procaspase-9 was in cells, expression in the was and caspase-3 were in However, caspase-9-deficient showed amounts of the of and caspase-3 also in type II cells the amplification to be required for caspase activation and efficient cell In this we a caspase-9-deficient tumor cell line to the of the mitochondrial pathway in death receptor-mediated apoptosis. By using caspase-9-deficient Jurkat cells and siRNAs we provide direct genetic evidence that type II cells depend on the mitochondrial amplification to CD95-mediated apoptosis. In contrast, caspase-9 is in type I cells Cells from caspase-9-deficient have been to have various R. A. M. A. D. J. R. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. H. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, these analyzed only type I cells, such as and activated but not type II cells from caspase-9 for their CD95 sensitivity. Thus, the two pathway model of apoptosis has been directly in a components of the apoptosome are we show that mouse embryonic from caspase-9-deficient are toward CD95-mediated apoptosis their Furthermore, in the overexpression of Bcl-2 has been to the of however, has This is by the that Bcl-2 only delays or attenuates but CD95-mediated apoptosis. In line, also against apoptosis Bcl-2 only or at low of the death ligand J. K. S. C. 2005; PubMed Scopus Google Scholar). the other cells for or Apaf-1 show highly toward CD95-mediated apoptosis F. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. S. Korsmeyer S.J. 1999; PubMed Scopus Google Scholar). In the we the role of the mitochondrial pathway in a that Jurkat cells depend on caspase-9 for efficient via CD95 genetic evidence for the validity of the two pathway model in CD95-mediated apoptosis. has been that differences type I and type II cells are only using agonistic antibodies but not CD95L (9Huang 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 (277) Google Scholar, 10Huang D.C. Tschopp J. Strasser A. Cell Death Differ. 2000; 7: 754-755Crossref PubMed Scopus (31) Google suggesting that these two cell types not at a In contrast, we report here that CD95-mediated apoptosis in type II cells is on mitochondria also recombinant CD95L is We have previously that type I and type II cells differences also in DISC formation upon with CD95L (3Schmitz I. Walczak H. Krammer P.H. Peter M.E. Cell Death Differ. 1999; 6: 821-822Crossref PubMed Scopus (62) Google Scholar). Moreover, other that this be also in cell For and activated T cells as type I cells, and type II cells (5Barnhart B.C. Alappat E.C. Peter M.E. Semin. Immunol. 2003; 15: 185-193Crossref PubMed Scopus (376) Google Scholar). was also that activated T cells are type II cells, which switch to a type I upon activation I. Krueger A. Baumann S. H. Krammer P.H. Kirchhoff S. J. Immunol. 2003; PubMed Scopus Google Scholar). In DISC analyses we that the recruitment of caspase-8 to CD95 was not by the or of caspase-9. However, the of caspase-8 was analyzed in cell caspase-8 was strongly impaired in caspase-9-deficient This that of caspase-8 does not with the receptor-mediated activation of caspase-8 and its of be that the detection of caspase-8 in cell a substrate in a but caspase-8 mitochondrial a number of apoptogenic factors are from mitochondria cytochrome c, apoptosis-inducing and (4Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). c release leads to formation of the apoptosome and activation of from by inhibitor of apoptosis was that CD95-mediated apoptosis in Jurkat cells be by overexpression of Bcl-2 or apoptogenic factors have to be from mitochondria or pathway is results show that apoptosis are highly on suggesting that at in the cell types analyzed here apoptogenic factors from the mitochondrial other cytochrome c only a role in cell death by these death at very of CD95L apoptosis be in cells suggesting that at of the death ligand amounts of are activated of the mitochondrial amplification loop. Interestingly, caspase-9 did not with apoptosis, suggesting that distinct signaling components be in this pathway. The model for cell death that of and activation of are required for cell death to by a number of Tschopp J. 2004; Full Text Full Text PDF PubMed Scopus Google and by activation E. F. S. R. J. R. 2001; PubMed Scopus Google Scholar, Li M. A. 2001; PubMed Scopus Google Scholar). In the of activation, signaling and leads to of via the in caspase activation at the receptor H. S. K. M. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, cell death by be of This is in line with a report that apoptosis and is not by a Bcl-2 in R. A. Strasser A. S. Cell Death Differ. PubMed Scopus Google Scholar). is also that a cell type be of type I for death receptor type II for Interestingly, a caspase-9 inhibitor apoptosis in cells were not K. 2000; Google Scholar). also that the of caspase-9 is by different receptor or formation of distinct receptor signaling as was proposed for and also for apoptosis E. H. D. D. M. D. A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, Tschopp J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). also show that cytochrome c release and loss of ΔΨM, which are to in can be as two We that loss of ΔΨM only in the of active and not in cells in which caspase activation is highly impaired upon death receptor This caspase requirement for the loss of ΔΨM is with findings that cytochrome c release is of caspase activation and of mitochondrial depolarization E. EMBO J. 1998; 17: PubMed Scopus Google Scholar). Interestingly, cytochrome c is to ΔΨM its release into the N.J. M. J. Cell Biol. 2001; PubMed Scopus Google which cells of the CD95 death Moreover, loss of ΔΨM be by the of proteins complex I of the J. Cell Biol. 2003; PubMed Scopus Google Scholar, C. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of these were in or cells, these findings to cells and for the that loss of ΔΨM can be uncoupled from cytochrome c release death receptor-mediated apoptosis. The caspase-9-deficient Jurkat line a very for analysis of cell death in the of apoptosome We C. and D. for and R. U. A. and H. for