Cell-specific Induction of Apoptosis by Microinjection of Cytochrome c

Abstract

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochromec is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochromec from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochromec release, induction of apoptosis, and Bcl-xLactivity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochromec release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria. Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochromec is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochromec from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochromec release, induction of apoptosis, and Bcl-xLactivity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochromec release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria. Programmed cell death is an important process for the maintenance of homeostasis in multicellular organisms (1Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar, 2White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1325) Google Scholar). Virtually all cells in such organisms have the capacity to undergo this form of death, and thus the onset of the process must be under tight physiological control. Genetic studies in lower organisms have identified several genes that control the onset and execution of programmed cell death (3Hengartner M.E. Ellis R.E. Horvitz H.R. Nature. 1992; 356: 494-499Crossref PubMed Scopus (716) Google Scholar,4White K. Tahaoglu E. Steller H. Science. 1996; 271: 805-807Crossref PubMed Scopus (337) Google Scholar), and additional studies have shown that apoptosis in mammalian cells is controlled by homologous genes that have been conserved throughout evolution (5Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2436) Google Scholar). These studies have demonstrated that apoptosis requires the action of the caspases, a set of death effector cysteine proteases with specificity for Asp-x bonds (6Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2257) Google Scholar, 7Fernandes-Alnemri T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (694) Google Scholar). Another set of proteins, the Bcl-2 family, control the activation of caspases (8Armstrong R. Aja T. Xiang J. Gaur S. Krebs J. Hoang K. Bai X. Korsmeyer S. Karanewsky D. Fritz L. Tomaselli K. J. Biol. Chem. 1996; 271: 16850-16855Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 9Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Helbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, 10Estoppey S. Rodriguez E. Sadoul R. Martinou J.-C. Cell Death Differ. 1997; 4: 34-38Crossref PubMed Scopus (34) Google Scholar). The antiapoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-xL, are localized to the intracellular membranes of the mitochondria, endoplasmic reticulum, and nuclear envelope (11Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar, 12González-Garcia M. Pérez-Ballestero R. Ding L. Duan L. Boise L.H. Thompson C.B. Núñez G. Development. 1994; 120: 3033-3042Crossref PubMed Google Scholar). However, many questions remain concerning the molecular mechanism by which these membrane-bound Bcl-2 family members exert their control over caspase activation. Recent reports have demonstrated that the mitochondrial protein cytochrome c can promote caspase activation in a cell-free apoptosis system (13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Furthermore, it was shown that cytochrome c is released from mitochondria in cells undergoing apoptosis and that this release is prevented by Bcl-2 or Bcl-xL expression (14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 16Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (371) Google Scholar). Bcl-2 has also been shown to regulate cytochrome c release from isolated mitochondria (14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar). These observations led to the hypothesis that cytochromec release is an obligate step in the apoptotic pathway and that antiapoptotic Bcl-2 family members function primarily to regulate cytochrome c release (13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 16Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (371) Google Scholar). However, to date, no data has been presented concerning a causative apoptotic role for cytochromec in intact cells. Here, we utilize cellular microinjection techniques to analyze the effects of cytochrome c on the initiation of apoptosis in living cells and to assess the relationship between cytochrome c release and Bcl-xLfunction. MCF7 cells (17Tewari M. Dixit V.M. J. Biol. Chem. 1995; 270: 3255-3260Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar) stably transfected to express the Fas antigen alone (denoted MCF7F) or both Fas and Bcl-xL (denoted MCF7F/Bcl-xL) (gifts of Dr. V. Dixit, University of Michigan) were grown in RPMI 1640 containing 10% fetal bovine serum, 200 μg/ml G418, and 100 μg/ml hygromycin. Human embryonic kidney 293 and HeLa cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, and 1 × penicillin/streptomycin. Cells were plated on glass cellocate coverslips (Eppendorf) at a density of 40,000/well in 24-well plates for 17–20 h prior to microinjection or cytokine treatment. For experiments utilizing cytokines as death stimuli, cells were treated with either 500 ng/ml anti-Fas (clone CH-11, PanVera Labs, Madison, WI) plus 1 μg/ml cycloheximide, 100 ng/ml TNF 1The abbreviations used are: TNF, tumor necrosis factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; amc, aminomethylcoumarin. (R & D Systems) plus 1 μg/ml cycloheximide, or cycloheximide alone. Following microinjection or cytokine treatment, flat cells (normal) and round/shrunken cells with condensed nuclei (apoptotic) were counted and are presented as percent apoptotic cells. In microinjection experiments, 100–150 cells were counted for each condition; in experiments using cytokines as the death stimulus without microinjection, at least 300 cells were counted for each condition. Cell microinjection was performed on the stage of a Nikon Diaphot 300 inverted microscope using an Eppendorf pressure injector (model 5246) and (model were from glass using a and using Eppendorf MCF7F or cells were plated on glass cellocate coverslips (Eppendorf) h prior to the in alone or plus cytochromec from in and for each were into the cytoplasm of cells Cells were into medium The intracellular of are to a of the on using microinjection and an cellular of G. J. G. Cell Res. PubMed Scopus Google Scholar). we that the of cytochrome c is to the and that are the is 1 with a of we that cell are cells were for apoptosis as for apoptosis, was to the medium to a of Cells were at for and under In experiments, caspase or (8Armstrong R. Aja T. Xiang J. Gaur S. Krebs J. Hoang K. Bai X. Korsmeyer S. Karanewsky D. Fritz L. Tomaselli K. J. Biol. Chem. 1996; 271: 16850-16855Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) were with to the at a of 1 and with cytochrome c. and and and were from MCF7F cells were plated on glass cellocate coverslips (Eppendorf) at a density of and in of a 24-well cells were with of protein and of either or to express pro-caspase 3, pro-caspase 3 which cysteine was to using the the pro-caspase or pro-caspase using For each pro-caspase expression coding was by utilizing was in to h cells were with alone or plus with or without 1 mm caspase cells were for apoptosis as and MCF7F cells were by at × for at The cell were with and with of mm mm mm 1 mm 1 mm 1 mm and mm containing mm The cells were with of a and the were at × for at The were at × for at and the mitochondrial were The of the × were at × for 1 h at and the were used for caspase the of cytochrome c of cytochrome and cell from or 293 cells were by in and to were with and with that cytochrome c were with to and by with in the was by to of the the ability of cytochrome c to caspases in of from each cell was with cytochrome c and 1 mm at for 1 h in a of of A. the of the the was to a and was to a of in of mm 1 mm CHAPS, 10% 3 was at using a were performed in and the was as in a of cytochromec for cellular microinjection, we the of cytochrome c cell in the 293 and MCF7F cell this the for cytochrome c in an intact a that human and cytochrome were performed on from of and was with of cytochrome c. The results that and cells each of cytochrome c between and 100 not we that of is and thus with a 3 cytochrome a of cytochrome c is This the cytochromec cell for 293 and MCF7F cells. We the effects of cytochrome c into the human embryonic kidney 293 cell Cells were with of of cytochrome c with and for the onset of apoptosis We a dose-dependent induction of cell death with a at The onset of apoptosis was with effects between 2 and 3 h apoptosis was as as 1 h the of 3 cytochrome c not The cytochrome apoptosis of 293 cells was caspase it was inhibited by of the caspase T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (694) Google Scholar) or (8Armstrong R. Aja T. Xiang J. Gaur S. Krebs J. Hoang K. Bai X. Korsmeyer S. Karanewsky D. Fritz L. Tomaselli K. J. Biol. Chem. 1996; 271: 16850-16855Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) and The induction of apoptosis was for cytochrome c microinjection no was of other proteins, or were not was also that the cytochrome c effects were due to However, microinjection of the and not induce apoptosis not that the cytochrome c was demonstrated that cytochrome c microinjection was sufficient to induce caspase-dependent apoptosis in 293 we the effects of cytochrome into other cell the human breast carcinoma cell MCF7 stably transfected to express Fas on and the human carcinoma cell MCF7F cells were to 3 cytochrome 2 HeLa cells a response between that with 293 cells and MCF7F cells. We also the ability of in the of 1 mm (13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar), to in from and HeLa cells 2 The ability of cytochrome c plus to in these with the of the intact cells to cytochrome c. These data suggested that the cytochrome death of intact 293 and HeLa cells was due to activation of caspases that were either or inhibited in MCF7F analyze the for the of MCF7F cells to cytochrome we it into MCF7F cells that been transfected with expression for MCF7F cells express caspases and can undergo caspase-dependent apoptosis (17Tewari M. Dixit V.M. J. Biol. Chem. 1995; 270: 3255-3260Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, H. A.M. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) but caspase 3 C. A. L. C. K. J. we expression of pro-caspase 3 in MCF7F cells to cytochrome c The results that MCF7F cells transfected with a control remain insensitive to cytochrome microinjection of cells transfected with the pro-caspase 3 led rapidly to apoptosis 3, This death was due to activation of the caspase death was inhibited by of Furthermore, expression of a pro-caspase 3, at the not the cells to cytochrome c. with pro-caspase 3 alone was not sufficient to induce apoptosis, and cells with cytochrome c 3 cytochrome c was by expression of pro-caspase 3 but not by expression of either pro-caspase T. Armstrong R.C. Krebs J. Srinivasula S.M. Wang L. Bullrich F. Fritz L.C. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7464-7469Crossref PubMed Scopus (694) Google Scholar, E.S. G. Yuan J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) or pro-caspase E.S. G. Yuan J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. T. J. Armstrong R.C. Wang L. Trapani J.A. Tomaselli K.J. Litwack G. Alnemri E.S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) expression of and not apoptosis in these cells Duan H. K. A.M. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The that MCF7F cells were not by cytochrome but are of undergoing apoptosis in response to other (17Tewari M. Dixit V.M. J. Biol. Chem. 1995; 270: 3255-3260Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, M.F. V. M. M. Boise L.H. Thompson C.B. M. G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) presented an to the hypothesis that Bcl-2 family members function primarily to regulate cytochromec we that with cycloheximide plus either TNF or anti-Fas a of apoptosis over an in MCF7F cells (17Tewari M. Dixit V.M. J. Biol. Chem. 1995; 270: 3255-3260Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, H. A.M. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and that this was inhibited by expression of However, microinjection of cytochrome at levels to be at least the cytochrome c of the cell to induce apoptosis in MCF7F or MCF7F cells. these cells were insensitive to these results suggested that the were to be due to cytochrome that anti-Fas or TNF have cellular that cytochrome in MCF7F cells. Bcl-xL function by cytochrome c release in these death MCF7F cells cytochrome c this we cytochrome c microinjection overcome the inhibition by Bcl-xL of death. shown in Bcl-xL transfected MCF7F cells were insensitive to cycloheximide plus anti-Fas with or without cytochromec In contrast, cycloheximide plus anti-Fas the control MCF7F and this death was by microinjection of cytochrome c. The that microinjection of cytochrome c not overcome the Bcl-xL inhibition of apoptosis that the Bcl-xL in this was not due to cytochrome c release from mitochondria. We have used microinjection techniques to the effects of cytochrome c in intact cells. cytochrome c is localized in mitochondria but is released into the cytoplasm during we used microinjection to this we cell all of which can undergo caspase-dependent apoptosis in response to but that in their response to microinjection of cytochrome c. microinjection was sufficient to induce cell death in 293 MCF7F cells were HeLa cells sensitivity. The ability of cytochrome c to induce apoptosis in a cell with ability to induce caspase in from that cell Furthermore, induction of apoptosis by cytochrome c was in the insensitive MCF7F cells by transfection of pro-caspase 3 but not by other the the of cytochromec into cells with the of cytochrome c These were on the of cell to cytochrome c Although the were from human cells the were from this was to have the cytochromec The used in the was cytochrome c and shown to with a that is between and human R. E. J. PubMed Scopus Google Scholar, D. E. of and The Scholar). In demonstrated that the with to and cytochrome they at in the R. E. J. PubMed Scopus Google Scholar). we that an of cytochrome in 293 and MCF7 cells. Our are lower from mitochondrial in cytochromec may be Cell W. Scholar). However, at the used in experiments, we of cytochrome c to present in the we that MCF7F cells not undergo apoptosis with of cytochrome c. that the cytochrome c in these experiments was from Although we cannot the that microinjection of human cytochrome c have demonstrated and human cytochrome are in of D. E. of and The Scholar). Our results support a in which cytochrome c can promote and apoptosis in cells expressing pro-caspase 3, but they on the hypothesis (13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar) that cytochromec release is for apoptosis to in in does not pro-caspase 3 as by a response to apoptotic in from caspase 3 K. S. Yang D. H. P. Nature. 1996; PubMed Scopus Google Scholar). In with the of the have a in caspase 3 that apoptosis in cell is not on caspase 3 K. S. Yang D. H. P. Nature. 1996; PubMed Scopus Google Scholar). Furthermore, MCF7F cells lack pro-caspase they are of undergoing apoptosis in response to cytokines plus cycloheximide and Although we not analyze all pro-caspases, data that cytochrome c has specificity for pro-caspase 3 as with and or the caspases in MCF7F cells H. A.M. Dixit V.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and The specificity of cytochromec for pro-caspase 3 with the that apoptosis can of this caspase that cytochrome c release is not for the induction of apoptosis. However, we have that cytochrome in released from mitochondria in MCF7F cells Our data the that this mitochondrial release is a of apoptotic a of caspase experiments with and cells have suggested that cytochrome c release is not by caspase R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 16Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (371) Google Scholar). experiments in which cytochromec is or be to or not this mitochondrial protein a causative role in apoptosis. c is in cells from and specifically into the it in the between the and mitochondrial membranes W. J. Google Scholar). Bcl-2 and Bcl-xL are localized to intracellular membranes including the mitochondrial (11Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google M. Pérez-Ballestero R. Ding L. Duan L. Boise L.H. Thompson C.B. Núñez G. Development. 1994; 120: 3033-3042Crossref PubMed Google Scholar), and it has been that these exert their antiapoptotic effects by preventing cytochrome c from released into the cytoplasm (14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 16Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (371) Google Scholar). In support of this Bcl-2 expression does inhibit cytochrome c release from mitochondria in cells and from isolated (14Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, 15Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 16Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (371) Google Scholar). However, from these experiments it is to between a of Bcl-2 on cytochrome c release and an Bcl-2 inhibits other proapoptotic thus preventing mitochondrial Furthermore, the Bcl-2 is a of apoptosis but is from the mitochondrial E. S. Cell Biol. 4: PubMed Scopus Google Scholar, E. R. Proc. Natl. Acad. Sci. U. S. A. 86: PubMed Scopus Google Scholar), the of that are not to mitochondria. Bcl-2 to intracellular membranes in of apoptosis W. A. J. 1996; PubMed Scopus Google Scholar). 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