A Bax/Bak-independent Mechanism of Cytochrome c Release

Abstract

Bax and Bak are multidomain pro-apoptotic members of the Bcl-2 family of proteins that regulate mitochondria-mediated apoptosis by direct modulation of mitochondrial membrane permeability. Since double-knock-out mouse embryonic fibroblasts with deficiency of Bax and Bak are resistant to multiple apoptotic stimuli, Bax and Bak are considered to be an essential gateway for various apoptotic signals. Here we showed that the combination of calcium ionophore A23187 and arachidonic acid induced cytochrome c release and caspase-dependent death of double-knock-out mouse embryonic fibroblasts, indicating that other mechanisms of cytochrome c release exist. Furthermore, A23187/arachidonic acid (ArA)-induced caspase-dependent death was significantly suppressed by the treatment of several serine protease inhibitors including 4-(2-aminoethyl)benzenesulfonylfluoride and l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone but not the overexpression of anti-apoptotic Bcl-2 family of proteins or the inhibition of mitochondrial membrane permeability transition. These results indicate that there are at least two mechanisms of cytochrome c release leading to caspase activation, a Bax/Bak-dependent mechanism and a Bax/Bak-independent, but serine protease(s)-dependent, mechanism. Bax and Bak are multidomain pro-apoptotic members of the Bcl-2 family of proteins that regulate mitochondria-mediated apoptosis by direct modulation of mitochondrial membrane permeability. Since double-knock-out mouse embryonic fibroblasts with deficiency of Bax and Bak are resistant to multiple apoptotic stimuli, Bax and Bak are considered to be an essential gateway for various apoptotic signals. Here we showed that the combination of calcium ionophore A23187 and arachidonic acid induced cytochrome c release and caspase-dependent death of double-knock-out mouse embryonic fibroblasts, indicating that other mechanisms of cytochrome c release exist. Furthermore, A23187/arachidonic acid (ArA)-induced caspase-dependent death was significantly suppressed by the treatment of several serine protease inhibitors including 4-(2-aminoethyl)benzenesulfonylfluoride and l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone but not the overexpression of anti-apoptotic Bcl-2 family of proteins or the inhibition of mitochondrial membrane permeability transition. These results indicate that there are at least two mechanisms of cytochrome c release leading to caspase activation, a Bax/Bak-dependent mechanism and a Bax/Bak-independent, but serine protease(s)-dependent, mechanism. Apoptosis plays a critical role in the regulation of development processes, tissue homeostasis, and elimination of damaged cells. It has been shown that the mitochondria play a crucial role in apoptosis by releasing several apoptogenic molecules, such as cytochrome c, Smac/Diablo, and HtrA2/Omi (1.Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (92) Google Scholar, 2.Wolf B.B. Green D.R. Curr. Biol. 2002; 12: R177-R179Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). After release into the cytosol, cytochrome c binds to Apaf-1 to cause recruitment of caspase-9, which leads to the initiation of a caspase cascade that includes caspase-3 and results in the occurrence of apoptotic cell death. Smac/Diablo and HtrA2/Omi bind directly with members of the inhibitor of apoptosis protein family, which are endogenous caspase inhibitory proteins and thus contribute to caspase activation (1.Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (92) Google Scholar, 2.Wolf B.B. Green D.R. Curr. Biol. 2002; 12: R177-R179Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The best characterized regulators of apoptosis are the Bcl-2 family of proteins, which directly modulate outer mitochondrial membrane permeability during apoptosis. This family of proteins can be categorized into anti-apoptotic members (such as Bcl-2, Bcl-xL, and Mcl-1) and pro-apoptotic members, which consist of multidomain proteins (such as Bax and Bak) and BH3-only proteins (including Bid, Bim, Bik, Bad, Noxa, and Puma) (3.Tsujimoto Y. J. Cell. Physiol. 2003; 195: 158-167Crossref PubMed Scopus (443) Google Scholar). The multidomain pro-apoptotic proteins Bax/Bak are essential and redundant regulators of a diverse intrinsic mitochondrial cell death pathway; Bax/Bak double-deficient murine embryonic fibroblasts (MEFs) 2The abbreviations used are: MEF, murine embryonic fibroblast; DKO, double-knock-out; TKO, triple-knock-out; ArA, arachidonic acid; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; AEBSA, 4-(2-aminoethyl)benzenesulfonamide; TPCK, l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone; TLCK, l-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone; WT, wild type; mPT, mitochondrial permeability transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DFP, diisopropylfluorophosphate; GFP, green fluorescent protein; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CsA, cyclosporin A; Cyp D, cyclophilin D; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; Ac, acetyl; MCA, methylcoumarylamide.2The abbreviations used are: MEF, murine embryonic fibroblast; DKO, double-knock-out; TKO, triple-knock-out; ArA, arachidonic acid; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; AEBSA, 4-(2-aminoethyl)benzenesulfonamide; TPCK, l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone; TLCK, l-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone; WT, wild type; mPT, mitochondrial permeability transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DFP, diisopropylfluorophosphate; GFP, green fluorescent protein; siRNA, small interfering RNA; PBS, phosphate-buffered saline; CsA, cyclosporin A; Cyp D, cyclophilin D; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; Ac, acetyl; MCA, methylcoumarylamide. are resistant to multiple apoptotic stimuli that increase outer mitochondrial membrane permeability, including staurosporine, ultraviolet radiation, growth factor deprivation, and etoposide (4.Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar). It was recently reported that double-knock-out (DKO) MEFs undergo death in a caspase-dependent manner, although at a lower rate when compared with WT MEFs, after exposure to agents such as arachidonic acid and Ca2+ ionophore (5.Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1213) Google Scholar), raising the possibility that there is a Bax/Bak-independent mechanism that regulates mitochondrial membrane permeability. In this present study, we found that the treatment of the combination of arachidonic acid and A23187 caused cytochrome c release and subsequently caspase activation succeeding the cell death in DKO MEFs. These events were inhibited by the treatment of 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), a serine protease inhibitor, but not the treatment of mitochondrial permeability transition (mPT) inhibitors, deletion of cyclophilin D (mPT component), and overexpression Bcl-2 or Bcl-xL. Antibodies and Chemicals−The following antibodies were used. Numbers in parentheses indicate dilutions used. Anticaspase-3 (1:250) and anti-caspase-9 (1:1000) monoclonal antibodies were purchased from BD Transduction Laboratories and MBL (Nagoya, Japan), respectively. Anti-lamin B1 (1:1000) and anti-GAPDH (1:1000) monoclonal antibodies were obtained from Zymed Laboratories Inc. Inc. (South San Francisco, CA) and BD Biosciences, respectively. Anti-cytochrome c monoclonal antibodies for Western blotting (clone 7H8.2C12, 1:1000) and immunostaining (clone 6H2.B4, 1:500) were purchased from Pharmingen. A23187 and arachidonic acid were obtained from Calbiochem and Sigma, respectively. AEBSF,l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone (TPCK), and l-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone (TLCK) were purchased from Roche Applied Science (Penzberg, Germany). 4-(2-Aminoethyl)benzenesulfonamide (AEBSA) were purchased from Aldrich (Steinheim, Germany). Diisopropylfluorophosphate (DFP) and other chemicals were purchased from Wako Co. (Osaka, Japan). Cell Culture and DNA Transfection−SV40 T antigen-immortalized WT MEFs, Bax/Bak DKO MEFs (kindly provided by Dr. S. J. Korsmeyer), and Bax/Bak/CypD TKO MEFs were grown in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum. Apaf-1-deficient MEFs and control MEFs were kindly provided by Dr. X. Wang and were also grown in the same medium. DNAs encoding human Bax and human Bcl-xL were used in the pUC-CAGGS expression vector. Cells (1 × 106) were transfected with plasmid DNA using the Amaxa electroporation system according to the supplier’s protocol (kit V, program U-20). The transfection efficiency was more than 75% as assessed by co-transfection with DNA expressing green fluorescence protein (GFP). All of the siRNAs were produced by Dharmacon Research. The sequences used were as follows (numbers in parentheses indicate nucleotide positions within the respective open reading frames): mouse cytochrome c siRNA, 5′-GGGAGAAAGGGCAGACCUA-3′ (267-285); mouse HtrA2/Omi siRNA, 5′-GGGGAGUUUGUUGUUGCCA-3′ (760-778); and GFP siRNA, 5′-GGCUACGUCCAGGAGCGCA-3′ (274-292). Mouse caspase-9 siRNA SMARTpool™ was also purchased from Dharmacon Research. Cells (1 × 106) were transfected twice on alternate days with 10 μg of siRNA using the Amaxa electroporation system. Twenty-four hours after the second transfection with siRNA, cells were used for experiments. Cell Viability and DEVDase Activity Assay−Cells (2 × 105/well) were seeded into 6-well dishes. After 24 h, the cells were treated with 10 μm A23187/100 μm arachidonic acid or 10 μm etoposide in the presence or absence of 100 μm Z-VAD-fmk or 100 μg/ml AEBSF. Cells were harvested and stained with 1 μm propidium iodide, 1 μm Cy3-conjugated annexin V, or 1 μm Hoechst 33342 for 5 min at room temperature and were analyzed with a flow cytometer (BD Biosciences, FACS Caliber) or under a fluorescence microscope (Olympus, BX50). For DEVDase assay, cells were washed three times with phosphate-buffered saline (PBS) and suspended in 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, and 10 mm EGTA. After the addition of Triton-X to 0.1%, cells were incubated for 30 min at on ice. Lysates were clarified by centrifugation at 8000 rpm for 3 min, and cleared lysates containing 50 μg of protein were incubated with 100 μm enzyme substrate Ac-DEVD-MCA at 37 °C for 1 h. Levels of released 7-amino-4-methlycoumarin were measured using a spectrofluorometer (Hitachi F-3000) with excitation at 380 nm and emission at 460 nm. Preparation of the Cytosolic Fraction and Total Cell Lysate−For the detection of released cytochrome c, the cytosolic fraction was collected from MEFs after incubation with 0.1 mg/ml digitonin for 5 min at 37 °C in isotonic buffer (20 mm potassium-Hepes (pH 7.4), 10 mm KCl, 1.5 mm MgCl2, 250 mm sucrose, and 1 mm Na2+-EDTA). After centrifugation at 8000 rpm for 5 min, aliquots of the supernatant (cytosolic fraction) and the pellet (mitochondrial fraction) were analyzed by Western blotting with an anti-cytochrome c antibody. In some experiments, cells were lysed with radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate (sodium salt), and 150 mm NaCl). Immunofluorescence Staining−Cells were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 15 min at room temperature. After incubation with 2% fetal bovine serum in PBS for 1 h, the cells were incubated with anti-cytochrome c for 1 h. After washing three times with PBS, the cells were incubated with the secondary antibody (Alexa Fluor 488-conjugated anti-mouse IgG, 1:1000) for 1 h. Then fluorescence was detected under a confocal microscope (Zeiss, LSM 510). Microinjection−DKO MEFs (1.5 × 104) were plated on 35-mm dishes 1 day before use. PBS or PBS containing 400 μm AEBSF, 400 μm AEBSA, 100 μm DFP, 100 μm TLCK, or 200 μm TPCK was microinjected through a glass capillary into the cytoplasm with a Narishige micromanipulator. For identification of injected cells, samples were mixed with green fluorescent protein. Cells were then treated with A23187/ArA at 1 h. Cell morphology was examined 20 h after A23187/ArA treatment under a fluorescence microscope. We added various apoptotic reagents to Bax/Bak DKO MEFs, Apaf-1-/- MEFs, and their control MEFs, all of which were immortalized, and then measured DEVDase activity. In both DKO MEFs (Fig. 1A) and Apaf-1-/- MEFs (Fig. 1B), activation of DEVDase was not observed after exposure to etoposide that induced mitochondria-mediated apoptosis, whereas in the control MEFs, activation of DEVDase was readily observed, confirming previous observations that Bax/Bak is essential for such apoptosis (4.Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar, 6.Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar). Time-dependent decrease of DEVDase activities was due to disruption of plasma membrane. Similar results were also obtained when these cells were treated with staurosporine, UV, and X-rays (data not shown). In contrast, when the cells were treated with A23187 plus ArA, activation of DEVDase was observed in DKO MEFs but not in Apaf-1-/- MEFs (Fig. 1, C and D). It has previously been reported that ionomycin (another Ca2+ ionophore) plus ArA could induce DEVDase activation in DKO MEFs (5.Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1213) Google Scholar). Consistent with the elevation of caspase activation, A23187/ArA treatment induced cleavage of caspase-9, caspase-3, and lamin B1 in WT and DKO MEFs but not in Apaf-1-/- MEFs (Fig. 1, E and F). Furthermore, A23187/ArA induced the death of DKO MEFs, as assessed by staining with annexin V (Fig. 1G) and propidium iodide (data not shown), whereas such cell death was not completely inhibited by Z-VAD-fmk, a pan-caspase inhibitor (Fig. 1G and data not shown), indicating that A23187/ArA induced both caspase-dependent and caspase-independent death of DKO MEFs. It is known that not a few reagents induce both apoptosis and necrosis, such as oxidative stress (7.Pompeia C. Lima T. Curi R. Cell Biochem. Funct. 2003; 21: 97-104Crossref PubMed Scopus (99) Google Scholar) and Ca2+ overload (8.Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2345) Google Scholar). Staining with Hoechst 33342 revealed that A23187/ArA induced nuclear pyknosis, which was partially inhibited by Z-VAD-fmk (Fig. 1H). A23187/ArA-induced caspase activation and death of DKO MEFs were slightly delayed when compared with these processes in WT MEFs (Fig. 1, C and G). Primary cultures of DKO MEFs tended to mainly undergo caspase-independent death (data not shown). These results indicated that A23187/ArA induced caspase-dependent death of immortalized MEFs in both a Bax/Bak-dependent and a Bax/Bak-independent manner. To examine whether mitochondria were involved in the A23187/ArA-induced caspase-dependent death of DKO MEFs, we used gene silencing with siRNA to down-regulate caspase-9 and cytochrome c. Caspase-9 is activated by forming a complex called the apoptosome with Apaf-1 and cytochrome c in the presence of (d)ATP (9.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). activation of caspase-9 leads to activation of caspase-3 (9.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-9 and cytochrome c were in DKO MEFs by the respective siRNAs (Fig. and of caspase-9 and cytochrome c in DKO MEFs A23187/ArA-induced as assessed by annexin V but not completely (Fig. and D). silencing of caspase-9 partially inhibited the activation of DEVDase (Fig. due to silencing (Fig. silencing of cytochrome c inhibited more the activation of DEVDase (Fig. These results indicated that A23187/ArA-induced caspase activation the presence of c. inhibition of cell death after silencing of cytochrome c was mainly due to the occurrence of caspase-independent death the addition of Z-VAD-fmk not A23187/ArA-induced as shown in the of mitochondrial in the with silencing of cytochrome c, we also the in the presence of inhibitor of although cell results were obtained (Fig. These results indicated that A23187/ArA-induced caspase-dependent death the presence of c but not We examined whether cytochrome c was released from the mitochondria in DKO MEFs. revealed that etoposide induced cytochrome c release in WT MEFs but not DKO MEFs (data not shown), whereas A23187/ArA caused cytochrome c release from both WT and DKO MEFs in the presence of Z-VAD-fmk (Fig. Consistent with this cytochrome c was in DKO MEFs, but not DKO MEFs, as assessed by (Fig. All these indicated that A23187/ArA induced cytochrome c release in a Bax/Bak-independent and subsequently activated to induce cell death. was cytochrome c released from the mitochondria in a Bax/Bak-independent after treatment with we examined whether Bcl-xL could this Bax/Bak-independent of cytochrome c shown in a pan-caspase inhibitor, but not overexpression of Bcl-xL, inhibited A23187/ArA-induced death of DKO MEFs. Consistent with this activation of both and cytochrome c release was not by Bcl-xL (Fig. and the expression of Bcl-xL inhibited apoptosis in DKO MEFs (Fig. ArA and Ca2+ ionophore A23187 are to induce the (8.Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2345) Google Scholar, V. D. L. P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, L. D. V. P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), which is a cyclosporin increase of mitochondrial membrane permeability that various to the S.J. 2002; PubMed Scopus Google Scholar). Since the has been to a role in apoptotic cytochrome c we whether was also involved in A23187/ArA-induced Bax/Bak-independent cytochrome c release and cell death. CsA, and all of which are known to the S.J. 2002; PubMed Scopus Google Scholar, V. P. A. Biochem. PubMed Scopus Google Scholar), were added to cultures of DKO MEFs. shown in of these reagents inhibited A23187/ArA-induced whereas Z-VAD-fmk caused that the was not involved in this of cell death. To this we cyclophilin D in which the is completely T. S. T. T. Y. PubMed Scopus Google Scholar). produced from which immortalized MEFs were shown in A23187/ArA induced the death of TKO and DKO MEFs, with cell death partially inhibited by Consistent with these cytochrome c release was also observed in TKO MEFs (Fig. indicating that the was not involved in Bax/Bak-independent cytochrome c release and cell death induced by To some into the mechanisms of A23187/ArA-induced cytochrome c we the of various such as protease inhibitors and inhibitors, on DKO MEFs and found that AEBSF, which serine through of the serine J. P. Biochem. PubMed Scopus Google Scholar) and has been to some of cell death S. C. T. A. A. Y. Y. Y. Mol. Cell. Biol. PubMed Scopus Google Scholar, D. de J. I. Cell 2003; PubMed Scopus Google Scholar, L. J. C. M. J. C. Cell 2003; PubMed Scopus Google Scholar, M. R. L. 2001; PubMed Scopus Google Scholar), could A23187/ArA-induced caspase activation and cell death (Fig. inhibited A23187/ArA-induced cell death in a manner, whereas AEBSA, an of AEBSF, not at (Fig. To the of a serine we examined other serine protease inhibitors, DFP, TLCK, and The addition of DFP, TLCK, and TPCK into medium not A23187/ArA-induced death (data not shown), which could be due to their membrane we microinjected into cells. shown in all the serine protease inhibitors inhibited A23187/ArA-induced cell death. Since inhibitors are to × in the cytoplasm Y. S. T. Y. J. Cell PubMed Google Scholar), the of are to nm. not inhibited A23187/ArA-induced cell death but also cytochrome c release (Fig. and G). These results indicated that a serine is involved in the of A23187/ArA-induced cell death of cytochrome c was a more inhibitor of A23187/ArA-induced cell death than Z-VAD-fmk (Fig. that at a in the or that inhibited both caspase-dependent and caspase-independent cell death. We examined the of a serine protease in the mitochondria that is known to be involved in apoptosis Y. Y. R. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Srinivasula S.M. R. R. L. Y. T. Alnemri E.S. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in silencing of HtrA2/Omi on A23187/ArA-induced although significantly apoptosis of DKO MEFs (data not shown). This revealed the following the combination of Ca2+ ionophore A23187 and arachidonic acid caspase-dependent cell death following cytochrome c release as as caspase-independent death in Bax/Bak double-knock-out MEFs. The Bcl-2 family of proteins and the are not involved in this of cell death. this of death is by an serine These the of a mechanism of cytochrome c release that is by serine but not by the Bcl-2 Bax/Bak-dependent and Bax/Bak-independent mechanisms to to After treatment with UV, or staurosporine, cytochrome c release in a Bcl-2 manner, whereas A23187/ArA not induced cytochrome c release in a Bcl-2 but also in a Bcl-2 serine manner. This be to the of of the Since apoptosis induced by BH3-only proteins in a Bax/Bak-dependent E.H. M.C. S. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, W.X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: PubMed Scopus Google Scholar), A23187/ArA apoptotic in addition to BH3-only A23187/ArA with Ca2+ (5.Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1213) Google Scholar), Ca2+ or proteins be involved in the mechanism of A23187/ArA-induced cell death. the Ca2+ was a that cytochrome c release we this possibility by MEFs in and Cyp D (Fig. and G). This was with obtained in Cyp MEFs and T. S. T. T. Y. PubMed Scopus Google Scholar) that cell death the is but not apoptosis, due to the of caused by a than Ca2+ be the as an of Bax/Bak-independent cytochrome c We could not the possibility that A23187/ArA-induced cytochrome c release was by the Since A23187/ArA-induced death of DKO MEFs was inhibited by the treatment of serine protease inhibitors including and TPCK, of a serine to be involved in this of cell death. is to be serine protease inhibitors suppressed A23187/ArA-induced the possibility was that inhibited A23187/ArA-induced death by mitochondrial Ca2+ by using as an of mitochondrial Ca2+ S. and Y. serine or more in the mitochondria that are involved in the regulation of death due to For a mitochondrial protein be by a serine in that cytochrome c but of the mechanism serine but not cytochrome c release and caspase-dependent cell death were observed in immortalized MEFs. other cells also the to undergo these of It has previously been that the of a of the that cells from these are resistant to apoptosis T. Ross A.J. A. Zong W.X. P. Y. M. M.C. A. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Zong W.X. J. Thompson C.B. 2003; PubMed Google Scholar). that present in cell cultures from are as to cell death as wild cells T. Zong W.X. J. Thompson C.B. 2003; PubMed Google Scholar). The cell death has both as as apoptotic M. J.M. Zhivotovsky B. S. S.A. Nicotera P. 15: Full Text PDF PubMed Scopus Google Scholar), and some induce which can cause cell death with apoptotic B. Cell 2003; PubMed Scopus Google Scholar). In death of cells, apoptotic which are Bax/Bak-independent, be In we the of a mechanism of cytochrome c release that a serine but not the or Bcl-2 family