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• W1974341306 abstract "Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-xL). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochromec in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes. Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-xL). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochromec in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes. Bcl-2 homology dithiobis(succinimidyl propionate) bismaleimidohexane bis[2-(succinimidyloxycarbonyloxy)-ethyl] sulfone green fluorescence protein Apoptosis, also called programmed cell death, is a highly regulated process that plays an essential role in the development and maintenance of homeostasis within multicellular organisms (1Raff M.C. Barres B.A. Burne J.F. Coles H.S. Ishizaki Y. Jacobson M.D. Science. 1993; 262: 695-700Crossref PubMed Scopus (1353) Google Scholar, 2Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar). Dysregulation of apoptosis has been implicated in the development of cancer, autoimmune disorder, neurodegeneration, ischemic damage, and other devastating diseases (3Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar, 4Reed J.C. Nat. Rev. Drug Discov. 2002; 1: 111-121Crossref PubMed Scopus (603) Google Scholar, 5Green D.R. Evan G.I. Cancer Cell. 2002; 1: 19-30Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 6Cory S. Adams J.M. Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3338) Google Scholar). Whereas apoptosis regulation takes place at multiple levels, Bcl-2 family proteins are of paramount importance (7Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar, 8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 10Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar). Bcl-2 family proteins are defined by the presence of Bcl-2 homology (BH)1 domains (7Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar, 8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 10Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar, 11Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). They can be pro-apoptotic or anti-apoptotic. Specific function of individual members is determined by the presence and organization of the BH domains (8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar). For example, anti-apoptotic members, like Bcl-2 and Bcl-xL, contain four BH domains, whereas some pro-apoptotic molecules such as Bax and Bak contain three (BH1–3), and others contain only one, the BH3 domain (7Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar, 8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 10Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar, 11Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 12Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (629) Google Scholar). Bid is a unique BH3-only pro-apoptotic protein (13Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (807) Google Scholar). Unlike others, Bid activation depends on the proteolytic processing of intact Bid into truncated forms of tBid. tBid, thus generated, translocates to mitochondria and leads to disruption of the organelles and the release of apoptogenic molecules such as cytochrome c (14Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar, 15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar). Bid processing can be conducted by several proteases (16Alimonti J.B. Shi L. Baijal P.K. Greenberg A.H. J. Biol. Chem. 2001; 276: 6974-6982Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 17Stoka V. Turk B. Schendel S.L. Kim T.H. Cirman T. Snipas S.J. Ellerby L.M. Bredesen D. Freeze H. Abrahamson M. Bromme D. Krajewski S. Reed J.C. Yin X.M. Turk V. Salvesen G.S. J. Biol. Chem. 2001; 276: 3149-3157Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar); however, caspase-8 has been shown to be the major protease responsible for Bid cleavage during death receptor-mediated apoptosis (14Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar, 15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar). Caspase-8-mediated Bid processing therefore bridges the extrinsic death receptor-mediated pathway of apoptosis to the intrinsic mitochondrial pathway (14Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar, 15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar, 18Li S. Zhao Y. He X. Kim T.H. Kuharsky D.K. Rabinowich H. Chen J. Du C. Yin X.M. J. Biol. Chem. 2002; 277: 26912-26920Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This provides a mechanism to amplify the execution signal and exacerbate the pace of cell demise. Interactions among Bcl-2 family proteins have been documented (7Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar, 8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 10Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar, 11Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 12Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (629) Google Scholar). Functionally, expression of anti-apoptotic Bcl-2 or Bcl-xLsuppresses cell death initiated or mediated by pro-apoptotic members. Recent studies (19Wei 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 (3354) Google Scholar) have further suggested a sequence of Bcl-2 family protein activation during apoptosis, where Bcl-2 is positioned to block apoptosis at two separate steps, through inhibition of Bax/Bak and BH3-only proteins including Bid. Despite these important observations, it remains unclear how Bcl-2 antagonizes the pro-apoptotic action of Bid or tBid (20Esposti M.D. Apoptosis. 2002; 7: 433-440Crossref PubMed Scopus (185) Google Scholar, 21Yin X.M. Cell Res. 2000; 10: 161-167Crossref PubMed Scopus (274) Google Scholar). Here, by using an expression model, we have systematically analyzed Bid-induced apoptosis and its inhibition by Bcl-2. Our results show that Bcl-2 inhibited Bid-induced cytochrome c release and apoptosis, without suppressing Bid processing into tBid and subsequent tBid translocation to mitochondria. At the mitochondria, three critical events that were blocked by Bcl-2 have been identified. First, Bcl-2 suppressed tBid insertion into mitochondrial membranes. Second, Bcl-2 inhibited Bid-induced Bax translocation from the cytosol to mitochondria. Third, Bcl-2 diminished Bid-induced Bax/Bak oligomerization in the mitochondrial membranes. The results suggest that Bcl-2 may suppress Bid-induced apoptosis at the mitochondrial level by multiple mechanisms. PcDNA-Bid was prepared as described previously (13Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (807) Google Scholar). pऔactBcl-2 was a gift from Dr. Junyin Yuan at Harvard Medical School. pEGFP was purchased from Clontech. Transfection reagents were purchased from Invitrogen. Chemical cross-linkers dithiobis(succinimidyl propionate (DSP), bismaleimidohexane (BMH), and bis[2-(succinimidyloxycarbonyloxy)-ethyl] sulfone (BSOCOES) were purchased from Pierce. Antibodies used in this study were from the following sources: rabbit polyclonal anti-Bax (N-20), mouse monoclonal anti-Bcl-2 (C-2), rabbit polyclonal anti-Bcl-2 (ΔC21), and goat polyclonal anti-lamin B from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-Bak from Upstate Biotechnology, Inc. (Lake Placid, NY); mouse monoclonal antibodies against native (6H2.B4) and denatured (7H8.2C12) cytochrome c from Pharmingen; mouse monoclonal 20E8 anti-cytochrome oxidase IV from Molecular Probes (Eugene, OR); rabbit polyclonal anti-murine Bid was prepared as described previously (13Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (807) Google Scholar). All secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). HeLa cells were maintained in minimum essential medium supplemented with 107 fetal bovine serum, 0.1 mm non-essential amino acids, 2 mml-glutamine, and 17 antibiotics. The cells were plated at 3.0 × 105/35-mm dish to reach ∼507 confluence by the next day for transfection. Cells in each dish were transfected with 0.25 ॖg of empty pcDNA vectors, 0.25 ॖg of pcDNA-Bid, or 0.25 ॖg of pcDNA-Bid along with 0.25 ॖg of pऔactBcl-2. To identify the transfectants, the same dishes were co-transfected with pEGFP-C1, which led to the expression of green fluorescence protein in transfected cells. Transfection was facilitated with LipofectAMINE PLUS (Invitrogen), according to the manufacturer's instructions. Transfection efficacy was usually over 707. After transfection, cells were incubated in serum-free medium for 4–5 h and then transferred to full growth medium. Morphological and biochemical analyses were conducted at ∼17 h post-transfection. Apoptotic cells were identified by their morphology as described in our previous studies (22Dong Z. Venkatachalam M.A. Wang J. Patel Y. Saikumar P. Semenza G.L. Force T. Nishiyama J. J. Biol. Chem. 2001; 276: 18702-18709Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Typical apoptotic morphology evaluated included cellular shrinkage and the formation of apoptotic bodies. To verify apoptosis, nucleus was stained with Hoechst 33342 to reveal nuclear condensation and fragmentation. For each condition, apoptosis was monitored in five fields with ∼60 cells per field. The experiments were repeated for at least four times with duplicate dishes for each condition in every experiment. To analyze the distributions of various proteins, cells were fractionated into cytosolic and membrane-bound organellar fractions with low concentrations of digitonin (23Dong Z. Saikumar P. Patel Y. Weinberg J.M. Venkatachalam M.A. Biochem. J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar, 24Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (260) Google Scholar, 25Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Selective permeabilization of plasma membranes was monitored by microscopy. Digitonin permeabilization has been used to study protein redistributions within cells during apoptosis (24Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (260) Google Scholar, 25Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 26Waterhouse N.J. Goldstein J.C. von Ahsen O. Schuler M. Newmeyer D.D. Green D.R. J. Cell Biol. 2001; 153: 319-328Crossref PubMed Scopus (374) Google Scholar, 27Gottlieb R.A. Granville D.J. Methods. 2002; 26: 341-347Crossref PubMed Scopus (69) Google Scholar). Briefly, cells were exposed to 0.057 digitonin in isotonic sucrose buffer (in mm: 250 sucrose, 10 Hepes, 10 KCl, 1.5 MgCl2, 1 EDTA, and 1 EGTA; pH 7.1) for 2 min at room temperature to collect the soluble fraction as cytosolic extracts. Digitonin-insoluble fraction was washed with isotonic sucrose buffer and further dissolved in 27 SDS buffer to collect the membrane-bound organellar fraction. Because apoptotic redistribution of cytochromec and Bcl-2 family proteins including Bid and Bax mainly takes place between the cytosol and mitochondria, immunoblot analysis of the membrane-bound part is expected to reveal mitochondrial content of the molecules. Cross-linking was conducted by procedures modified from our previous work (25Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). All cross-linking chemicals (DSP, BMH, and BSOCOES) were dissolved in Me2SO at concentrations of 100 mm prior to experiments and further diluted in phosphate-buffered saline to 10 mmbefore using. DSP was added to the cells for 30 min of incubation at room temperature under constant mixing. The cells were subsequently fractionated by digitonin as described above. For BMH and BSOCOES, cells were first fractionated to collect the membrane fraction for cross-linking. Cross-linked samples were subjected to electrophoresis under non-reducing conditions for immunoblot analysis. Cytochromec immunofluorescence was examined as described in our previous publications (23Dong Z. Saikumar P. Patel Y. Weinberg J.M. Venkatachalam M.A. Biochem. J. 2000; 347: 669-677Crossref PubMed Scopus (72) Google Scholar, 24Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (260) Google Scholar). Briefly, cells were grown on collagen-coated glass coverslips and subjected to transfection. The cells were fixed in a modified Zamboni's fixative containing 47 paraformaldehyde and 0.197 picric acid and permeabilized with 0.17 SDS prior to blocking and primary antibody (mouse anti-native cytochrome c) exposure. Finally, antigenic sites within the cells were revealed by staining with CY-3-conjugated goat anti-mouse antibodies. To examine the nucleus, Hoechst 33342 (10 ॖg/ml) was added to cells in phosphate-buffered saline and stained for 5 min at room temperature. After digitonin permeabilization, the membrane-bound organellar fraction was collected and washed once with phosphate-buffered saline. The fraction was incubated on ice in 0.1m Na2CO3 at pH 11.5 for 30 min and then subjected to 1 h of centrifugation at 100,000 ×g at 4 °C to collect the supernatant and pellet. The supernatant contained the proteins released during alkaline incubation, whereas the pellet contained the proteins that were resistant to the treatment (28Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar). Cells were extracted directly with RIPA buffer to collect whole cell lysate, or subjected to sequential extractions with 0.057 digitonin and RIPA buffer to collect cytosolic and membrane-bound organellar extracts, respectively. Isotonic digitonin buffer was described above under “Subcellular Fractionation.” The composition of RIPA buffer was (in mm): 150 NaCl, 1 MgCl2, 1 EGTA, 10 औ-mercaptoethanol, 15 Tris-HCl, pH 7.4, containing 0.57 sodium deoxycholate, 0.27 SDS, and 17 Triton X-100. Immunoprecipitation was conducted according to our previous work (24Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (260) Google Scholar, 25Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Extracts (whole cell extracts by RIPA, cytosolic extracts by digitonin, post-digitonin extracts by RIPA) from ∼1 × 106 cells were pre-cleared by incubation with 1 ॖg of normal serum and 30 ॖl of agarose protein A/G (Santa Cruz Biotechnology). The pre-cleared lysates were subsequently incubated for 2 h with 2 ॖg of immunoprecipitation antibody and 30 ॖl of agarose protein A/G. Immunoprecipitates were collected by centrifugation and dissolved in SDS sample buffer for immunoblot analysis. To detect Bcl-2 and Bid/tBid interaction, a mouse monoclonal anti-Bcl-2 was used for immunoprecipitation, and the resultant precipitates were subjected to immunoblot analysis of Bid/tBid. We initially determined whether transfection of full-length Bid induced apoptosis in HeLa cells and whether Bcl-2 co-transfection was able to suppress it. To identify the transfected cells, a vector containing green fluorescence protein (GFP) was co-transfected. The results are shown in Fig. 1A. The control group that was transfected with empty vectors had an apoptosis rate of 22.87. Bid transfection led to a dramatic increase in apoptosis, to 81.37. Bid-induced apoptosis was blocked by Bcl-2 co-transfection. As a result, cells co-transfected with Bid + Bcl-2 showed an apoptosis rate of 33.77 (Fig. 1A). The morphological observations were confirmed by biochemical analyses. As shown in Fig. 1B, Bid transfection led to the cleavage of lamin B into an apoptosis-indicative fragment of 46 kDa (lane 2). Co-transfection of Bcl-2 blocked lamin B cleavage (lane 3). Data not shown also demonstrated apoptotic cleavage of poly(ADP-ribose) polymerase in Bid-only transfected cells, which was again suppressed by Bcl-2 co-transfection. Together, these experiments have demonstrated Bcl-2 inhibition of Bid-induced apoptosis in HeLa cells. A major cellular site targeted by Bcl-2 family proteins is the mitochondrion (7Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar, 8Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4811) Google Scholar, 9Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 10Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (940) Google Scholar, 11Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 12Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (629) Google Scholar). Disruption of this organelle results in the release of apoptogenic molecules including cytochromec and may therefore underlie the pro-apoptotic actions of Bid (20Esposti M.D. Apoptosis. 2002; 7: 433-440Crossref PubMed Scopus (185) Google Scholar, 21Yin X.M. Cell Res. 2000; 10: 161-167Crossref PubMed Scopus (274) Google Scholar). Thus, to pursue the mechanisms responsible for Bcl-2 inhibition of Bid-induced apoptosis, we examined cellular distributions of cytochrome c. The results are shown in Fig.2. By immunoblot analyses, the majority of cytochrome c was detected in the mitochondrial fraction of the control cells (Fig. 2A, lane 1). Bid transfection led to an increase of cytochrome c in the cytosol, accompanied by loss of the molecule from the membrane-bound organellar fraction (lane 2), indicating cytochromec release from mitochondria in these cells. Significantly, Bcl-2 co-transfection blocked Bid-induced cytochrome crelease (lane 3). The immunoblot results were confirmed by immunofluorescence staining of cytochrome c within these cells. As shown in Fig. 2B, Bid-transfected cells (a, green) exhibited typical morphology of apoptosis, assuming a round-up and fragmented configuration. In the same cells, cytochrome c (Fig. 2B, b,red) was released from mitochondria, resulting in whole cell staining. In sharp contrast, cells co-transfected with Bid + Bcl-2 displayed a much healthier morphology (Fig. 2B,c). These cells maintained cytochrome c in the mitochondria, showing a punctated perinuclear staining (Fig.2B, d). The results suggest that Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochromec leakage from mitochondria. The pro-apoptotic activity of Bid depends on its processing into the active forms of tBid (14Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar, 15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar). Thus, to identify further the mechanisms responsible for Bcl-2 inhibition, we examined Bid processing in transfected cells and the effects of Bcl-2 co-transfection by using whole cell lysates. The results are shown in Fig. 3. In Bid-transfected cells, intact Bid of 22 kDa was expressed at high levels (lane 2), compared with control transfection (lane 1). Moreover, tBid of 15 and 13 kDa was detected, indicating Bid processing in these cells (lane 2). Bcl-2 co-transfection did not attenuate either Bid expression or it processing into tBid (lane 3). The results suggest that Bcl-2 inhibited Bid-induced apoptosis without significantly suppressing Bid expression or processing. An important event for Bid activation and toxicity is the targeting of mitochondria by tBid (14Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar, 15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar). Thus, our subsequent experiments tested the possibility that Bcl-2 might inhibit Bid-induced apoptosis by blocking tBid translocation. For this purpose, transfected cells were fractionated into cytosolic and membrane-bound organellar fractions for immunoblot analysis of Bid/tBid. The results are shown in Fig. 4. In Bid-only transfected cells (lanes 1 and 3), intact Bid of 22 kDa was detected mainly in the cytosol, whereas tBid of 15 kDa showed both cytosolic and organellar distributions, and p13 was detected only in the membrane-bound organellar fraction. Bcl-2 co-transfection did not change the cellular localization of these molecules (Fig. 4,lanes 2 and 4). The results, together with those shown in Fig. 3, indicate that Bcl-2 inhibited Bid-induced cytochromec leakage and apoptosis without blocking Bid processing and translocation. Bcl-2 did not affect Bid processing into tBid or tBid translocation (Figs. 3 and4). However, it inhibited Bid-induced mitochondrial disruption including the release of cytochrome c (Fig. 2). These observations promoted us to examine the status of tBid association with mitochondrial membranes. Specifically, we asked the following: does Bcl-2 prevent tBid integration or insertion into the membranes? To address this question, we utilized a classical method of alkaline treatment to examine the integration status of tBid (28Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar). Alkaline incubation of cellular membranes at pH 11.5 leads to the dissociation of loosely attached proteins, whereas integrated proteins remain. This approach was successfully employed to demonstrate Bax insertion into mitochondria (29Eskes R. Desagher S. Antonsson B. Martinou J.C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1016) Google Scholar, 30Goping I.S. Gross A. Lavoie J.N. Nguyen M. Jemmerson R. Roth K. Korsmeyer S.J. Shore G.C. J. Cell Biol. 1998; 143: 207-215Crossref PubMed Scopus (549) Google Scholar). In our experiments, cells were transfected with Bid alone or Bid + Bcl-2. Membrane-bound organellar fractions were collected for incubation with 0.1 m NaHCO3 at pH 11.5. Proteins stripped into the incubation solution were collected and analyzed for Bid/tBid, along with the proteins that were resistant to alkaline incubation. The results are shown in Fig.5. In Bid-only transfected cells, alkaline incubation stripped off intact Bid of 22 kDa from the organellar membranes into the supernatant (lane S1). However, tBid of 15 and 13 kDa was rather resistant to the treatment and thus remained associated with the membranes after alkaline exposure (lane P1), suggesting that tBid and not intact Bid had integrated into the mitochondrial membranes in these cells. In sharp contrast, for Bid + Bcl-2-co-transfected cells, alkaline treatment led to the release of not only intact Bid but also a significant portion of 15-kDa tBid (lane S2). To estimate the percentage of 15-kDa tBid that was sensitive to alkaline treatment, blots from 4 separate experiments were analyzed by densitometry (Fig. 5B). In cells co-transfected with Bcl-2, over 307 of 15-kDa tBid was released during alkaline incubation; by sharp contrast, less than 27 was released from cells transfected with Bid only. As a control, cytochrome oxidase IV, an integral mitochondrial membrane protein, was not stripped off from the membranes under these experimental conditions, regardless of the presence or absence of Bcl-2 (Fig. 5A,lower panel). The results suggest that Bcl-2 may suppress Bid-induced mitochondrial disruption partly by prevention of tBid insertion into the organellar membranes. Our results suggested that Bcl-2 inhibited tBid insertion into mitochondrial membranes (Fig. 5). However, the underlying mechanism was unclear. One hypothesis was that Bcl-2 might directly interact with tBid, resulting in conformational changes in this molecule that prevented its integration into organellar membranes. To test this possibility, we examined the interactions between Bcl-2 and Bid/tBid by co-immunoprecipitation. In this experiment, cells were extracted either directly with RIPA buffer to collect whole cell lysate or sequentially with digitonin and RIPA buffer to collect the cytosolic fraction and the membrane-bound organellar fraction including mitochondria. The extracts were subjected to immunoprecipitation with Bcl-2 antibodies. The immunoprecipitates were analyzed for the presence of Bid and tBid. As shown in Fig. 6A, intact Bid was detected in all Bcl-2 immunoprecipitates, irrespective of the extracts utilized for immunoprecipitation. On the contrary, tBid was not shown in any of the Bcl-2 immunoprecipitates (Fig. 6A). To demonstrate that the extracts prior to immunoprecipitation did contain tBid," @default.
• W1974341306 created "2016-06-24" @default.
• W1974341306 creator A5023791678 @default.
• W1974341306 creator A5050155862 @default.
• W1974341306 creator A5058297699 @default.
• W1974341306 date "2003-05-01" @default.
• W1974341306 modified "2023-10-02" @default.
• W1974341306 title "Inhibition of Bid-induced Apoptosis by Bcl-2" @default.
• W1974341306 cites W1527449265 @default.
• W1974341306 cites W1554372630 @default.
• W1974341306 cites W1963564407 @default.
• W1974341306 cites W1964617786 @default.
• W1974341306 cites W1978813721 @default.
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• W1974341306 cites W1982784171 @default.
• W1974341306 cites W1984547206 @default.
• W1974341306 cites W1986388339 @default.
• W1974341306 cites W1991865867 @default.
• W1974341306 cites W1994611236 @default.
• W1974341306 cites W2000093730 @default.
• W1974341306 cites W2000175408 @default.
• W1974341306 cites W2003380824 @default.
• W1974341306 cites W2012173926 @default.
• W1974341306 cites W2012825099 @default.
• W1974341306 cites W2014586657 @default.
• W1974341306 cites W2018077387 @default.
• W1974341306 cites W2019572526 @default.
• W1974341306 cites W2033847504 @default.
• W1974341306 cites W2036529420 @default.
• W1974341306 cites W2036822121 @default.
• W1974341306 cites W2043051754 @default.
• W1974341306 cites W2050477129 @default.
• W1974341306 cites W2062807784 @default.
• W1974341306 cites W2062853648 @default.
• W1974341306 cites W2063362926 @default.
• W1974341306 cites W2064127449 @default.
• W1974341306 cites W2064727849 @default.
• W1974341306 cites W2073028327 @default.
• W1974341306 cites W2075477970 @default.
• W1974341306 cites W2084533871 @default.
• W1974341306 cites W2087877138 @default.
• W1974341306 cites W2102584757 @default.
• W1974341306 cites W2105253445 @default.
• W1974341306 cites W2112998068 @default.
• W1974341306 cites W2118731822 @default.
• W1974341306 cites W2132411546 @default.
• W1974341306 cites W2136269288 @default.
• W1974341306 cites W2142120946 @default.
• W1974341306 cites W2143067729 @default.
• W1974341306 cites W2152247516 @default.
• W1974341306 cites W2160107586 @default.
• W1974341306 cites W2160169717 @default.
• W1974341306 cites W2165546509 @default.
• W1974341306 cites W2166219468 @default.
• W1974341306 cites W2166601968 @default.
• W1974341306 cites W2750896950 @default.
• W1974341306 cites W4243035579 @default.
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