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• W2000529918 abstract "Pro-apoptotic Bax and Bak have been implicated in the regulation of p53-dependent apoptosis. We assessed the ability of primary baby mouse kidney (BMK) epithelial cells frombax−/−, bak−/−, andbax−/−bak−/− mice to be transformed by E1A alone or in conjunction with dominant-negative p53 (p53DD). Although E1A alone transformed BMK cells from p53-deficient mice, E1A alone did not transform BMK cells frombax−/−, bak−/−, orbax−/−bak−/− mice. Thus, the loss of both Bax and Bak was not sufficient to relieve p53-dependent suppression of transformation in epithelial cells. To test the requirement for Bax and Bak in other death signaling pathways, stable E1A plus p53DD-transformed BMK cell lines were derived from the bax−/−,bak−/−, and bax−/−bak−/− mice and characterized for their response to tumor necrosis factor-α (TNF-α)-mediated apoptosis. The loss of both Bax and Bak severely impaired TNF-α-mediated apoptosis, but the presence of either Bax or Bak alone was sufficient for cell death. Cytochrome c was released from mitochondria, and caspase-9 was activated in Bax- or Bak-deficient cells in response to TNF-α but not in cells deficient in both. Thus, either Bax or Bak is required for death signaling through mitochondria in response to TNF-α, but both are dispensable for p53-dependent transformation inhibition. Pro-apoptotic Bax and Bak have been implicated in the regulation of p53-dependent apoptosis. We assessed the ability of primary baby mouse kidney (BMK) epithelial cells frombax−/−, bak−/−, andbax−/−bak−/− mice to be transformed by E1A alone or in conjunction with dominant-negative p53 (p53DD). Although E1A alone transformed BMK cells from p53-deficient mice, E1A alone did not transform BMK cells frombax−/−, bak−/−, orbax−/−bak−/− mice. Thus, the loss of both Bax and Bak was not sufficient to relieve p53-dependent suppression of transformation in epithelial cells. To test the requirement for Bax and Bak in other death signaling pathways, stable E1A plus p53DD-transformed BMK cell lines were derived from the bax−/−,bak−/−, and bax−/−bak−/− mice and characterized for their response to tumor necrosis factor-α (TNF-α)-mediated apoptosis. The loss of both Bax and Bak severely impaired TNF-α-mediated apoptosis, but the presence of either Bax or Bak alone was sufficient for cell death. Cytochrome c was released from mitochondria, and caspase-9 was activated in Bax- or Bak-deficient cells in response to TNF-α but not in cells deficient in both. Thus, either Bax or Bak is required for death signaling through mitochondria in response to TNF-α, but both are dispensable for p53-dependent transformation inhibition. Apoptosis can be initiated in transformed cells by an intrinsic mechanism when deregulation of the cell cycle initiates an apoptotic response mediated by the tumor suppressor p53. Apoptosis can also be initiated by an extrinsic mechanism when TNF-α 1The abbreviations used are: TNF-αtumor necrosis factor-αCHAPSN,N-dimethyl-N-(3-sulfopropyl)-3-[[(3α,5β,7α,12α)-3,7,12 trihydroxy-24-oxocholan-24-yl]-amino]-1-propanaminium inner saltPBSphosphate-buffered salineFBSfetal bovine serumCHXcycloheximideDMEMDulbecco's modified Eagle's mediumBMKbaby mouse kidneyMEFmouse embryo fibroblastBRKbaby rat kidneyor Fas ligand initiates an apoptotic response mediated by death receptors. When the adenovirus E1A oncogene stimulates proliferation during transformation, the cellular response is apoptosis mediated by p53 (1.Debbas M. White E. Genes Dev. 1993; 7: 546-554Crossref PubMed Scopus (834) Google Scholar,2.White E. Oncogene. 2001; 20: 7836-7846Crossref PubMed Google Scholar). Activation of p53 results in altered transcription of a wide variety of genes that are involved in many facets of cell metabolism, cell cycle regulation, and apoptosis (3.Yu J. Zhang L. Hwang P.M. Rago C. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14517-14522Crossref PubMed Scopus (416) Google Scholar, 4.Zhao R. Gish K. Murphy M. Yin Y. Notterman D. Hoffman W.H. Tom E. Mack D.H. Levine A.J. Genes Dev. 2000; 14: 981-993Crossref PubMed Scopus (275) Google Scholar). Genes transcriptionally up-regulated by p53 that have been implicated in promoting apoptosis include the Bcl-2 family members bax,bak, puma, and noxa (5.Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar, 6.Nakano K. Vousden K. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1881) Google Scholar, 7.Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1707) Google Scholar, 8.Pearson A.S. Spitz F.R. Swisher S.G. Kataoka M. Sarkiss M.G. Meyn R.E. McDonnell T.J. Cristiano R.J. Roth J.A. Clin. Cancer Res. 2000; 6: 887-890PubMed Google Scholar). Evidence suggests that Bax and Bak function is required for the release of cytochrome c from the mitochondria to the cytosol during apoptosis (9.Jurgensmeier J.M. Zhihua X. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 10.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar). Cytochrome c release from the mitochondria occurs in many apoptotic signaling pathways including those implemented by p53 and TNF-α (11.Sundararajan R. White E. J. Virol. 2001; 75: 7506-7516Crossref PubMed Scopus (80) Google Scholar, 12.Henry, H., Thomas, A., Shen, Y., and White, E. (2002)Oncogene 748–760Google Scholar, 13.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In many cases, this event is pivotal in the regulation of apoptosis, because cytochromec in the cytosol complexes with APAF-1 and, in turn, promotes caspase-9 activation (14.Li P. Nijihawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar). This caspase activation initiates a caspase cascade that is required for p53-dependent apoptosis (12.Henry, H., Thomas, A., Shen, Y., and White, E. (2002)Oncogene 748–760Google Scholar, 15.Sabbatini P. Han J.H. Chiou S.-K. Nicholson D. White E. Cell Growth Differ. 1997; 8: 643-653PubMed Google Scholar) and results in DNA fragmentation, cleavage of cellular proteins such as poly(ADP-ribose) polymerase and nuclear lamins, and cell death by apoptosis (16.Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1161) Google Scholar). tumor necrosis factor-α N,N-dimethyl-N-(3-sulfopropyl)-3-[[(3α,5β,7α,12α)-3,7,12 trihydroxy-24-oxocholan-24-yl]-amino]-1-propanaminium inner salt phosphate-buffered saline fetal bovine serum cycloheximide Dulbecco's modified Eagle's medium baby mouse kidney mouse embryo fibroblast baby rat kidney Although there is up-regulation of bax, bak,puma, and noxa by p53, transcriptional up-regulation of at least Bax is not sufficient for p53-mediated apoptosis, because a mutant of p53 that up-regulates bax is not able to induce apoptosis (17.Sakamuro D. Sabbatini P. White E. Prendergast G.C. Oncogene. 1997; 15: 887-898Crossref PubMed Scopus (249) Google Scholar). Evidence suggests that Bax and Bak undergo changes in protein conformation that have been linked to their pro-apoptotic function (11.Sundararajan R. White E. J. Virol. 2001; 75: 7506-7516Crossref PubMed Scopus (80) Google Scholar, 13.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 18.Sundararajan R. Cuconati A. Nelson D. White E. J. Biol. Chem. 2001; 276: 45120-45127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19.Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar). Thus, merely an increase in the amount of Bax or Bak may not result in cell death. Furthermore, other Bcl-2 family members Bcl-2, Bcl-xL, and Bid, also regulate cytochrome c release, in some cases by modulating the function of Bax and/or Bak. Finally, caspase activation can also be regulated downstream of mitochondria by the inhibitors of apoptosis proteins (20.Goyal L. Cell. 2001; 104: 805-808Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Which of these events is essential for cell death in particular pathways has not always been clear. Cytokines such as TNF-α and Fas initiate apoptosis through separate, yet convergent, pathways. Upon receptor ligand interaction, the death receptor will recruit proteins into a death-inducing signaling complex (21.Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1787) Google Scholar). Among the proteins recruited to the receptor are FLICE-associated death domain-containing protein/caspase-8 heterodimers, and the complex formed will result in the activation of caspase-8 (22.Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2741) Google Scholar, 23.Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). Active caspase-8 cleaves Bid-generating tBid (24.Li H. Zhu H. Xu C.-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar) which promotes conformational changes in Bak (25.Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar) and Bax (13.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Evidence suggests that Bax and Bak oligomerize in mitochondrial membranes, the effect of which is to release cytochrome cfrom the inter-membrane space thereby inducing APAF-1-dependent caspase-9 activation (11.Sundararajan R. White E. J. Virol. 2001; 75: 7506-7516Crossref PubMed Scopus (80) Google Scholar, 14.Li P. Nijihawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar, 18.Sundararajan R. Cuconati A. Nelson D. White E. J. Biol. Chem. 2001; 276: 45120-45127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 25.Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar, 26.Korsmeyer S.J. Wei M.C. Saito M. Weiler S. Oh K.J. Schlesinger P.H. Cell Death Differ. 2000; 7: 1166-1173Crossref PubMed Scopus (845) Google Scholar, 27.Eskes R. Desagher S. Antonsson A. Martinou J. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1016) Google Scholar). Whether Bax and/or Bak function in this way in specific apoptotic pathways, and the mechanism by which they effect release of proteins from mitochondria, remains to be addressed. Recently, mice deficient for Bax, Bak, or both have been characterized (28.Knudson C.M. Tung K. Brown G. Korsmeyer S.J. Science. 1995; 270: 96-99Crossref PubMed Scopus (1311) Google Scholar, 29.Lindsten T. Ross A.J. King A. Zong W.-X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. Chen Y. Wei M. Eng V.M. Adelman D.M. Simon M.C. Ma A. Golden J.A. Evan G. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar). The bak−/− mice are developmentally normal, and the bax−/− mice have limited abnormalities including lymphoid hyperplasia and male sterility (28.Knudson C.M. Tung K. Brown G. Korsmeyer S.J. Science. 1995; 270: 96-99Crossref PubMed Scopus (1311) Google Scholar,29.Lindsten T. Ross A.J. King A. Zong W.-X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. Chen Y. Wei M. Eng V.M. Adelman D.M. Simon M.C. Ma A. Golden J.A. Evan G. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar). Mice deficient for both Bax and Bak die perinatally and have multiple developmental defects including webbing between the digits, imperforated vaginal canal, and accumulation of excess cells in the hematopoietic and central nervous systems (29.Lindsten T. Ross A.J. King A. Zong W.-X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. Chen Y. Wei M. Eng V.M. Adelman D.M. Simon M.C. Ma A. Golden J.A. Evan G. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar). Mouse embryo fibroblasts (MEF) from these mice show that Bax and Bak are necessary for apoptosis induced by staurosporin, UV radiation, etoposide, thapsigargin, and tunicamycin in the short term (30.Wei M. Zong W.-X. Cheng E. Lindsten T. Panoutsakopoulou V. Ross A. Roth K. MacGregor G. Thompson C. Korsmeyer S. Science. 2001; 292: 727-730Crossref PubMed Scopus (3354) Google Scholar). The release of cytochrome c in response to the overexpression of tBid is also inhibited in bax−/−bak−/− cells (30.Wei M. Zong W.-X. Cheng E. Lindsten T. Panoutsakopoulou V. Ross A. Roth K. MacGregor G. Thompson C. Korsmeyer S. Science. 2001; 292: 727-730Crossref PubMed Scopus (3354) Google Scholar), suggesting that these proteins are essential regulators of apoptotic mitochondrial function in response multiple stimuli. Whether these findings can be extrapolated to other cell types and additional death stimuli was not known. Here we determined that Bax and/or Bak were not essential for p53 to suppress oncogenic transformation which is likely mediated in all or in part by apoptosis. E1A expression in primary kidney epithelial cells induces p53-dependent apoptosis that must be inhibited for transformation to occur (1.Debbas M. White E. Genes Dev. 1993; 7: 546-554Crossref PubMed Scopus (834) Google Scholar, 31.Sabbatini P. Lin J. Levine A.J. White E. Genes Dev. 1995; 9: 2184-2192Crossref PubMed Scopus (227) Google Scholar, 32.Chiou S.-K. Tseng C.C. Rao L. White E. J. Virol. 1994; 68: 6553-6566Crossref PubMed Google Scholar). We show that E1A alone transformed primary baby mouse kidney epithelial cells (BMK) fromp53−/− mice but not from wild-type,bax−/−, bak−/−, orbax−/−bak−/− mice. However, wild-type, bax−/−,bak−/−, andbax−/−bak−/− BMK cells were efficiently transformed by E1A and dominant-negative p53 (p53DD). Thus, the loss of Bax, Bak, or both did not abrogate the requirement for loss of p53 function during E1A-induced epithelial cell transformation. Stable E1A plus p53DD-transformed BMK cell lines were derived from the foci from bax−/−,bak−/−, and bax−/−bak−/− mice in the BMK cell transformation assay and were characterized for their ability to undergo apoptosis by death receptor signaling pathways. The loss of either Bax or Bak did not abrogate TNF-α-induced apoptosis; however, the loss of both Bax and Bak conferred resistance to apoptosis. The absence of either Bax or Bak did not affect the release of cytochrome c from mitochondria to the cytosol in response to TNF-α; however, the loss of both dramatically prevented the release of cytochrome cand caspase-9 activation. These findings indicate that Bax and Bak function redundantly to release cytochrome c from the mitochondria to implement apoptosis in response to death receptor signaling, but both are dispensable for p53-mediated suppression of oncogenic transformation. Three matings were performed as follows: mice were bred by crossing mice that werebax+/−bak−/− withbax+/−bak−/−;bax+/−bak+/− withbax+/−bak−/−; andbax+/−bak+/+ withbax+/−bak+/+. Newly born litters of pups (less than 48 h) were collected and numbered. Tail snips from each pup were collected and processed for DNA preparation, and PCR genotyping was performed as described for Bak (29.Lindsten T. Ross A.J. King A. Zong W.-X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. Chen Y. Wei M. Eng V.M. Adelman D.M. Simon M.C. Ma A. Golden J.A. Evan G. Korsmeyer S.J. MacGregor G.R. Thompson C.B. Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar) and Bax (33.Shindler K. Latham C. Roth K. J. Neurosci. 1997; 17: 3112-3119Crossref PubMed Google Scholar). Preparation of BMK cells from primary kidneys from murine pups was based on the BRK preparation protocol (34.Ruley H.E. Nature. 1983; 304: 602-606Crossref PubMed Scopus (741) Google Scholar) with the following modifications. Kidneys from each pup were removed and processed separately under sterile conditions. Each pair of kidneys was washed with PBS, placed into 10 ml of PBS containing 2.5 mg/ml dispase II (Roche Molecular Biochemicals) and 2.5 μg/ml collagenase A (Roche Molecular Biochemicals), mechanically disrupted, and then stirred at 37 °C for 30 min. Following the incubation, 5 ml of DMEM plus 5% FBS was added and mixed by pipetting, and clumps were allowed to settle for 5 min, and the BMK were collected from the supernatant by centrifugation at 400 × g for 5 min. The BMK cells were resuspended in 550 μl of DMEM plus 5% FBS, divided into two portions of 250 μl each, and electroporated in the presence of 10 μg of linearized pCMVE1A plasmid DNA (35.White E. Cipriani R. Sabbatini P. Denton A. J. Virol. 1991; 65: 2968-2978Crossref PubMed Google Scholar) and 110 μg of salmon sperm carrier DNA or 10 μg of linearized pCMVE1A plasmid DNA and 10 μg of linearized p53DD plasmid DNA (36.Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar) and 100 μg of salmon sperm carrier DNA. Post-transfection each condition was plated into three 6-cm tissue culture dishes. The culture medium was changed 1–2 times per week for 6 weeks to allow colonies to form. Colonies were ring-cloned from two plates to ensure that clones derived would be independent using standard tissue culture techniques. Giemsa stain (Sigma) was used according to the manufacturer's instructions to visualize colonies on culture plates. The following antibodies were used for indirect immunofluorescence, immunoprecipitation, and Western blotting analysis; the mouse monoclonal antibodies directed against the native and denatured forms of the cytochrome c protein were purchased from PharMingen; the rabbit N-20 polyclonal antibody directed against amino acids 11–30 of human Bax was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the rabbit polyclonal antibody (NT) against amino acids 1–21 of human Bax, which recognizes a conformation-specific form of Bax, and the rabbit polyclonal antibody (NT) raised against amino acids 23–37 of human Bak were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY); the mouse monoclonal antibodies directed against p53 (pAB421), the anti-adenovirus 2 E1A (M73), and actin were purchased from Oncogene Research Products (Boston, MA); the mouse monoclonal antibody directed against caspase-9 was purchased from StressGen Biotechnologies (Victoria, British Columbia, Canada); and the rhodamine-conjugated, goat-anti-mouse antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). BMK cells were prepared for immunoprecipitation as described previously (18.Sundararajan R. Cuconati A. Nelson D. White E. J. Biol. Chem. 2001; 276: 45120-45127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Briefly, the immunoprecipitation was carried out in 2% CHAPS lysis buffer (20 mm Tris (pH 7.4), 137 mm NaCl, 2 mmEDTA, 10% glycerol, and 2% CHAPS). The Sepharose was washed three times in a 0.5% CHAPS wash buffer (20 mm Tris (pH 7.4), 137 mm NaCl, 2 mm EDTA, 10% glycerol, and 0.5% CHAPS). Cell lysates and immunoprecipitates were resolved by SDS-PAGE and then analyzed by Western blotting with the Bax NT antibody. BMK cell lines were untreated, treated with CHX alone (0.05 μg/ml CHX) (Sigma), or treated with both TNF-α (100 units/ml mTNF-α) (Roche Molecular Biochemicals) and CHX (0.05 μg/ml). After 16 h of treatment cells were harvested by trypsinization, centrifuged, and resuspended in PBS. Cells were diluted 1:100 in 0.25% trypan blue solution (Invitrogen) and counted in a hemocytometer to assess the number of dead blue cells from the total number of cells counted. For fluorescence-activated cytometry BMK cells were treated with TNF/CHX or CHX alone for 16 h. After treatment cells were harvested by trypsinization, centrifuged, and resuspended in PBS. The cells were fixed with 70% ethanol and stained with propidium iodide (10 μg/ml) and RNase A (50 μg/ml) and incubated overnight. The cells were analyzed on a Becton Dickinson FACSCalibur system (San Jose, CA). Wild-type and Bax- and Bak-deficient BMK cells were harvested, counted, and plated at specific dilutions, as indicated in the figure. Twenty four hours after plating the cells were treated with or without TNF/CHX for 16 h, as described above. Following treatment with TNF/CHX, the plates were washed once with PBS and normal growth medium (DMEM + 5% FBS) was restored. After 4 days the plates were Giemsa-stained as described above, and colonies of cells were counted. Cell extracts were analyzed by SDS-PAGE and blotted semidry as described previously (13.Perez D. White E. Mol. Cell. 2000; 6: 53-63Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Proteins were detected by antibody as indicated and visualized by enhanced chemiluminescence according to the manufacturer's specifications (Amersham Biosciences). Cells were treated with TNF/CHX for 0 and 4 h. Subcellular fractionation was performed as described (37.Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Cells were harvested, washed in PBS, and resuspended in ice-cold lysis buffer (10 mm HEPES, pH 7.4, 42 mm KCl, 5 mm MgCl2, 2 mm EDTA) containing protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin A). Cells were incubated in lysis buffer for 20 min on ice. Sucrose (2 m in lysis buffer) was added to adjust to a final concentration of 0.32 m sucrose and passed through a 30-gauge syringed needle 10 times. The cell suspension was centrifuged at 1000 × g for 10 min to eliminate nuclei and unbroken cells. The supernatant was further centrifuged at 10,000 × g to remove the mitochondrial pellet and then at 100,000 × g to yield the cytosolic soluble fraction (S-100). The S-100 was subjected to Western blot analysis by probing with a monoclonal antibody that recognizes the denatured form of the cytochrome c. Cells were untreated or treated with TNF/CHX for 4 h. Indirect immunofluorescence was performed essentially as described (38.Perez D. White E. J. Cell Biol. 1998; 141: 1255-1266Crossref PubMed Scopus (103) Google Scholar). Cells were fixed at room temperature with 4.0% paraformaldehyde for 15 min and permeabilized with ice-cold PBS containing 0.2% Triton X-100 for 5 min. Cells were labeled with the antibodies that recognize native cytochrome c and visualized with rhodamine-conjugated goat anti-mouse antibody. Digital photography was performed using a Nikon FXA microscope equipped with epifluorescence optics (Nikon Inc., Garden City, NY). The p53 tumor suppressor protein is crucial in preventing E1A-mediated transformation in primary baby rat kidney (BRK) epithelial cells (1.Debbas M. White E. Genes Dev. 1993; 7: 546-554Crossref PubMed Scopus (834) Google Scholar,2.White E. Oncogene. 2001; 20: 7836-7846Crossref PubMed Google Scholar). BRK cells transfected with E1A alone fail to form foci due to the onset of apoptosis. However, BRK cells transfected with E1A and dominant-negative p53 efficiently form transformed foci and have been cloned into stable cell lines (1.Debbas M. White E. Genes Dev. 1993; 7: 546-554Crossref PubMed Scopus (834) Google Scholar, 31.Sabbatini P. Lin J. Levine A.J. White E. Genes Dev. 1995; 9: 2184-2192Crossref PubMed Scopus (227) Google Scholar, 32.Chiou S.-K. Tseng C.C. Rao L. White E. J. Virol. 1994; 68: 6553-6566Crossref PubMed Google Scholar). The utility of this strategy was expanded by adapting it to primary mouse cells where many apoptotic and tumor suppressor genes have been targeted for gene disruption. To explore the requirement of Bax and Bak in p53-mediated apoptosis, we transfected primary BMK cells with E1A or E1A plus p53DD and assayed for transformation. Wild-type BMK cells were not transformed by E1A alone (Fig. 1). Transformation by E1A requires inactivation of p53 with a dominant-negative p53 mutant in wild-type cells or the use of BMK cells from p53-deficient mice to abolish p53 function (Fig. 1). Expression of E1A alone does not promote transformation of BMK cells from mice lacking Bax, Bak, or both Bax and Bak, indicating that both Bax and Bak may be dispensable for p53-dependent apoptosis (Fig. 1). No transformants were obtained with E1A alone (Fig. 1), and all E1A plus p53DD-derived cell lines expressed E1A and high levels of the mutant p53 (see below). Interestingly, overexpression of anti-apoptotic Bcl-2 or the adenovirus Bcl-2 homologue E1B 19K inhibits p53-mediated apoptosis (39.Chiou S.-K. Rao L. White E. Mol. Cell. Biol. 1994; 14: 2556-2563Crossref PubMed Scopus (362) Google Scholar, 40.Han J. Sabbatini P. Perez D. Rao L. Modha D. White E. Genes Dev. 1996; 10: 461-477Crossref PubMed Scopus (315) Google Scholar, 41.Sabbatini P. Chiou S.-K. Rao L. White E. Mol. Cell. Biol. 1995; 15: 1060-1070Crossref PubMed Google Scholar) and enables transformation by E1A (42.Rao L. Debbas M. Sabbatini P. Hockenberry D. Korsmeyer S. White E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7742-7746Crossref PubMed Scopus (657) Google Scholar, 43.White E. Sabbatini P. Debbas M. Wold W.S.M. Kusher D.I. Gooding L. Mol. Cell. Biol. 1992; 12: 2570-2580Crossref PubMed Google Scholar). Thus, the loss of Bax and Bak function may not be equivalent to the gain of an anti-apoptotic Bcl-2 function. The formation of transformed foci by E1A plus p53DD in the wild-type, Bax, Bak, and Bax/Bak-deficient BMK cells allowed us to establish stable transformed cell lines, with defined deficiency in these apoptotic regulators. Although these cell lines are not suitable for studies on p53-mediated apoptosis due to the constitutive inactivation of endogenous p53, they can be used to define the roles of Bax and Bak in other apoptotic pathways. In order to study molecular events of apoptosis affected by Bax and Bak expression, stable BMK cell lines, transformed with E1A plus p53DD, were derived from the wild-type (W),bax−/− (X), bak−/−(K), and bax−/−bak−/− (D) mice. Individual foci from the transformation assay, as in Fig. 1, were cloned and grown into cell lines. Multiple independently cloned cell lines from each genotype were analyzed (W1–3, X1–3, K1–3, and D1–3), and expression of E1A, p53DD, Bax, and Bak was assessed by immunoblotting (Fig. 2A). The expression of E1A was similar in all of the cell lines tested, as were the levels of p53DD (Fig. 2A). Because all of the cell lines examined expressed both E1A and p53DD, this indicated that expression of both was required for transformation. The immunoblots for Bax and Bak matched genotyping of the parental mouse; however, the wild-type and Bak-deficient cell lines were derived from mice heterozygous for Bax (Bax−/+), and two Bax-deficient cell lines (X2, X3) were derived from mice heterozygous for Bak (Bak−/+) (Fig. 2B). The generation of these cell lines provides a renewable source of BMK epithelial cells with specific gene deficiencies inbax and/or bak. This strategy can be utilized to obtain epithelial cell lines deficient in nearly any gene for which a mouse with a targeted gene disruption has been developed and survives to birth. To test the requirement for Bax and Bak in extrinsic death signaling pathways, the Bax, Bak, and Bax plus Bak-deficient BMK cell lines were characterized for their apoptotic response induced by TNF-α. The BMK cell lines were untreated, treated with cycloheximide alone (CHX), or TNF-α and cycloheximide (TNF/CHX) for 16 h, photographed (Fig. 3A), and analyzed by trypan blue exclusion (Fig. 3B). Cell viability following cycloheximide treatment of all the cell lines was very similar to the untreated control (Fig. 3B). When treated with TNF/CHX the viability of the wild-type, bax−/−, andbak−/− BMK cells dropped dramatically (Fig. 3B), indicating that the presence of either Bax or Bak is sufficient for TNF/CHX to exert its apoptotic effect. In contrast, the absence of both Bax and Bak caused the BMK cells to be resistant to TNF/CHX-mediated killing (Fig. 3B). Viability was also assessed by fluorescence-activated cytometry for DNA content, and these results show the same trends as trypan blue exclusion with the exception that the relative resistance of D2 was slightly greater than D1 (data not shown). Taken together, these data indicate that the presence of either Bax or Bak is sufficient for TNF/CHX to mediate apoptosis. Because the absence of both Bax and Bak prevents TNF-α-mediated killing, this suggests that Bax and Bak are functionally redundant. To address whether the protection to TNF-α-mediated death signaling conferred by deficiency of both Bax and Bak in short term survival assays was sufficient to produce long term clonogenic survival, wild-type (bax+/−bak+/+; W3) and Bax plus Bak-deficient (bax−/−bak−/−; D3) BMK cell lines were untreated or treated with TNF/CHX for 16 h and assessed for colony formation ability 4–7 days later. When plated at a density of 5 × 105" @default.
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• W2000529918 title "Bax and Bak Independently Promote Cytochrome cRelease from Mitochondria" @default.
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