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• W2103457557 abstract "Deregulated expression of c-Myc can sensitize cells to a variety of death stimuli, including loss of growth factors and oxygen. In this study, we examined whether rodent fibroblasts that conditionally express c-Myc undergo a similar mechanism of cell death in response to serum or oxygen deprivation. Our results demonstrate that murine embryonic fibroblasts from bax-/-bak-/- mice that conditionally express c-Myc did not die in response to either oxygen or serum deprivation. Fibroblasts from p53-/- mice that conditionally express c-Myc died in response to oxygen (but not serum) deprivation. The inability of p53 to regulate oxygen deprivation-induced cell death was due to the lack of induction of p53 target genes Puma, Noxa, and Pten. In contrast, serum deprivation transcriptionally induced Puma and Pten in cells that conditionally express c-Myc. The failure of p53 to regulate oxygen deprivation-induced cell death led us to hypothesize whether hypoxia-inducible factor (HIF) might be a critical regulator of cell death during oxygen deprivation. Fibroblasts from HIF-1β-/- cells that conditionally express c-Myc were not able to transcriptionally activate HIF during oxygen deprivation. These cells died in response to oxygen deprivation. Thus, oxygen deprivation-induced cell death in fibroblasts with deregulated expression of c-Myc is independent of p53 or HIF-1 status, but is dependent on the Bcl-2 family member Bax or Bak to initiate mitochondrial dependent cell death. Deregulated expression of c-Myc can sensitize cells to a variety of death stimuli, including loss of growth factors and oxygen. In this study, we examined whether rodent fibroblasts that conditionally express c-Myc undergo a similar mechanism of cell death in response to serum or oxygen deprivation. Our results demonstrate that murine embryonic fibroblasts from bax-/-bak-/- mice that conditionally express c-Myc did not die in response to either oxygen or serum deprivation. Fibroblasts from p53-/- mice that conditionally express c-Myc died in response to oxygen (but not serum) deprivation. The inability of p53 to regulate oxygen deprivation-induced cell death was due to the lack of induction of p53 target genes Puma, Noxa, and Pten. In contrast, serum deprivation transcriptionally induced Puma and Pten in cells that conditionally express c-Myc. The failure of p53 to regulate oxygen deprivation-induced cell death led us to hypothesize whether hypoxia-inducible factor (HIF) might be a critical regulator of cell death during oxygen deprivation. Fibroblasts from HIF-1β-/- cells that conditionally express c-Myc were not able to transcriptionally activate HIF during oxygen deprivation. These cells died in response to oxygen deprivation. Thus, oxygen deprivation-induced cell death in fibroblasts with deregulated expression of c-Myc is independent of p53 or HIF-1 status, but is dependent on the Bcl-2 family member Bax or Bak to initiate mitochondrial dependent cell death. The proto-oncogene c-Myc is a transcription factor that forms a heterodimer with Max and activates genes involved in proliferation (1Amati B. Brooks M.W. Levy N. Littlewood T.D. Evan G.I. Land H. Cell. 1993; 72: 233-245Abstract Full Text PDF PubMed Scopus (444) Google Scholar). However, cells expressing c-Myc rapidly undergo cell death under conditions in which survival factors are limiting (reviewed in Ref. 2Prendergast G.C. Oncogene. 1999; 18: 2967-2987Crossref PubMed Scopus (391) Google Scholar). The molecular machinery that regulates c-Myc-induced cell death is distinct and independent from proliferation because activation of the molecular machinery mediating cell cycle progression is not required for c-Myc-induced cell death (3Conzen S.D. Gottlob K. Kandel E.S. Khanduri P. Wagner A.J. O'Leary M. Hay N. Mol. Cell. Biol. 2000; 20: 6008-6018Crossref PubMed Scopus (94) Google Scholar, 4Wagner A.J. Kokontis J.M. Hay N. Genes Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (515) Google Scholar). c-Myc itself does not induce cell death, but acts to sensitize cells to other death stimuli. c-Myc expression has been shown to sensitize cells to a wide variety of death stimuli, including serum and growth factor deprivation (5Askew D. Ashmun R. Simmons B. Cleveland J. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar, 6Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar), glucose deprivation (7Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar), oxygen deprivation (8Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar, 9Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (198) Google Scholar), virus infection (10Cherney B.W. Bhatia K. Tosato G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12967-12971Crossref PubMed Scopus (42) Google Scholar), tumor necrosis factor (11Klefstrom J. Vastrik I. Saksela E. Valle J. Eilers M. Alitalo K. EMBO J. 1994; 13: 5442-5450Crossref PubMed Scopus (134) Google Scholar), interferons (12Bennett M.R. Evan G.I. Newby A.C. Circ. Res. 1994; 74: 525-536Crossref PubMed Scopus (185) Google Scholar), CD95/Fas (13Hueber A.O. Zornig M. Lyon D. Suda T. Nagata S. Evan G.I. Science. 1997; 278: 1305-1309Crossref PubMed Scopus (332) Google Scholar), and p53-dependent response to genotoxic damage (6Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar). The fact that c-Myc can sensitize to so many different triggers of cell death suggests an action at some common point in the death machinery. Previous studies have demonstrated that Myc-induced sensitization to death stimuli is mediated through changes in outer mitochondrial membrane permeabilization, resulting in the release of cytochrome c from the mitochondria to the cytosol (14Juin P. Hueber A.O. Littlewood T. Evan G. Genes Dev. 1999; 13: 1367-1381Crossref PubMed Scopus (303) Google Scholar, 15Kennedy S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (591) Google Scholar). Recent studies have indicated that key regulators of outer mitochondrial membrane permeabilization are the Bcl-2 family proteins (reviewed in Refs. 16Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar and 17Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2776) Google Scholar). Bcl-2 and Bcl-xL prevent outer mitochondrial membrane permeabilization in response to apoptotic signals, whereas the Bcl-2 family proteins Bax and Bak promote it (18Jurgensmeier J.M. Xie Z. 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, 19Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar, 20Kluck R.M. Esposti M.D. Perkins G. Renken C. Kuwana T. Bossy-Wetzel E. Goldberg M. Allen T. Barber M.J. Green D.R. Newmeyer D.D. J. Cell Biol. 1999; 147: 809-822Crossref PubMed Scopus (291) Google Scholar, 21Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1236) Google Scholar, 22Yang 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 (4410) Google Scholar). The overexpression of Bcl-2 and Bcl-xL can prevent Myc-overexpressing cells from serum or oxygen deprivation (8Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar, 23Bissonnette R. Echeverri F. Mahboubi A. Green D. Nature. 1992; 359: 552-554Crossref PubMed Scopus (923) Google Scholar, 24Fanidi A. Harrington E.A. Evan G.I. Nature. 1992; 359: 554-556Crossref PubMed Scopus (702) Google Scholar, 25Rupnow B.A. Alarcon R.M. Giaccia A.J. Knox S.J. Cell Death Differ. 1998; 5: 141-147Crossref PubMed Scopus (33) Google Scholar, 26Wagner A.J. Small M.B. Hay N. Mol. Cell. Biol. 1993; 13: 2432-2440Crossref PubMed Scopus (207) Google Scholar). Primary cells isolated from mice lacking both Bax and Bak are resistant to cell death signals that cause outer mitochondrial membrane permeabilization, such as staurosporine, UV radiation, etoposide, thapsigargin, and serum and oxygen deprivation (27Lindsten 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, 28McClintock D.S. Santore M.T. Lee V.Y. Brunelle J. Budinger G.R. Zong W.X. Thompson C.B. Hay N. Chandel N.S. Mol. Cell. Biol. 2002; 22: 94-104Crossref PubMed Scopus (151) Google Scholar, 29Wei 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). Bcl-2 family proteins are also key downstream targets of the transcription factor p53 or survival-signaling pathways such as that initiated by insulin-like growth factor-1. Insulin-like growth factor-1 or loss of p53 can inhibit growth factor or serum deprivation-induced death in cells with deregulated c-Myc (4Wagner A.J. Kokontis J.M. Hay N. Genes Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (515) Google Scholar, 30Harrington E.A. Bennett M.R. Fanidi A. Evan G.I. EMBO J. 1994; 13: 3286-3295Crossref PubMed Scopus (736) Google Scholar, 31Hermeking H. Eick D. Science. 1994; 265: 2091-2093Crossref PubMed Scopus (708) Google Scholar). The ability of Myc to concurrently induce proliferation and cell death has led to the proposal that death due to inadequate survival factors provides a mechanism to guard against the appearance of neoplastic clones that can lead to unrestrained cell growth. Tumors with deregulated Myc expression require a continuous supply of survival factors or the acquisition of mutations such as in the p53 pathway or overexpression of Bcl-2 or Bcl-xL (32Alarcon R.M Rupnow B.A. Graeber T.G. Knox S.J. Giaccia A.J. Cancer Res. 1996; 56: 4315-4319PubMed Google Scholar, 33Pelengaris S. Khan M. Evan G.I. Cell. 2002; 109: 321-334Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 34Schmitt C.A. McCurrach M.E. de Stanchina E. Wallace-Brodeur R.R. Lowe S.W. Genes Dev. 1999; 13: 2670-2677Crossref PubMed Scopus (407) Google Scholar). Two survival factors that are likely to be limiting to induce cell death in tumors with deregulated c-Myc are growth factors and oxygen. Presently, it remains unknown whether loss of either oxygen or growth factors elicits a similar death pathway in cells with deregulated c-Myc expression. In this study, we investigated the role of p53 and Bcl-2 proteins in regulating oxygen deprivation-induced cell death in fibroblasts that express a conditionally active c-Myc and compared these genetic manipulations in the same cells exposed to serum deprivation. Cell Culture—Rat1a fibroblasts and murine embryonic fibroblasts (MEFs) 1The abbreviations used are: MEFsmurine embryonic fibroblastsOHT4-hydroxytamoxifenHIF-1hypoxia-inducible factor-1HREhypoxic response elementTKthymidine kinaseFBSfetal bovine serumDNp53dominant-negative p53WTwild-type. were cultured to 30-40% confluence in Dulbecco's modified essential medium supplemented with 25 mm HEPES, 1 mm pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen). Primary MEFs were generated from wild-type, p53-/-, or bax-/-bak-/- embryos. Rat1a fibroblasts or MEFs stably expressing a cDNA encoding human c-Myc fused at its C terminus to the hormone binding domain of 4-hydroxytamoxifen (4-OHT)-responsive mutant murine estrogen receptor (MycER) were established by retroviral infection as previously described (35Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar). Rat1a fibroblasts containing MycER and expressing Bcl-xL or dominant-negative p53 (GSE56) were established by retroviral infection as described previously (36Gottlob K. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (769) Google Scholar, 37Ossovskaya V.S. Mazo I.A. Chernov M.V. Chernova O.B. Strezoska Z. Kondratov R. Stark G.R. Chumakov P.M. Gudkov A.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10309-10314Crossref PubMed Scopus (160) Google Scholar). GSE56 in pLXSN was a gift of Andrei Gudkov. Primary MEFs were generated from HIF-1β+/+ and HIF-1β-/- embryos and subsequently immortalized with a plasmid containing the large T antigen. Oxygen deprivation conditions (0% O2, 85% N2, 10% H2, and 5% CO2) were achieved in a humidified anaerobic workstation (BugBox, Ruskinn Technologies) at 37 °C. An anaerobic color indicator (Oxoid Limited) confirms the anaerobicity of the chamber. murine embryonic fibroblasts 4-hydroxytamoxifen hypoxia-inducible factor-1 hypoxic response element thymidine kinase fetal bovine serum dominant-negative p53 wild-type. Measurement of Cell Death—Cell death was assayed by measuring lactate dehydrogenase activity in culture supernatants from cells plated on 60-mm culture plates using the cytotoxicity detection kit from Roche Applied Science according to the manufacturer's protocol. Briefly, 500 μl of medium was removed, and the remaining cells were lysed by adding the same volume of 0.5% Triton X-100. After 30 min, 500 μl of the lysate was removed. The samples were incubated for 30 min with buffer containing NAD+, lactate, and tetrazolium. Lactate dehydrogenase converts lactate to pyruvate, generating NADH. The NADH then reduces tetrazolium (yellow) to formazan (red), which was detected at 490 mm. Lactate dehydrogenase release is expressed as the percentage of the lactate dehydrogenase measured in the medium over the total lactate dehydrogenase released after treating with Triton X-100. Measurement of Cytochrome c Release and Caspase-9 Activation— Rat1a/MycER cells were plated on 60-mm culture dishes at 30-40% confluence and exposed to experimental conditions. Both adherent and non-adherent cells were collected and centrifuged for 5 min at 200 × g. The resulting pellet was then washed with phosphate-buffered saline and centrifuged again for 5 min at 200 × g. The cells were subsequently resuspended in 100 μl of phosphate-buffered saline and centrifuged on microscope glass plates at 200 × g for 5 min (Cytospin®-3 Cytocentrifuge, Shandon). The cells were then incubated at 20 °C for 5 min in methanol/acetone (1:1) and blocked for 45 min in phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma). Following blocking, the cells were removed, air-dried, and incubated for 2 h with a 1:500 dilution of anti-cytochrome c monoclonal antibody 7H8.2C12 (Pharmingen) at 37 °C in a humidified environment. The cells were then washed for 4 × 15 min in phosphate-buffered saline containing 0.1% bovine serum albumin, air-dried, and incubated for 1 h with a 1:50 dilution of a rhodamine-conjugated secondary antibody (Chemicon International, Inc.). Subsequently, the cells were washed as before, air-dried, and stained with 4′,6-diamidino-2-phenylindole/1,4-diazabicyclo[2.2.2]octane. The cells were then visualized via fluorescence microscopy (15Kennedy S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (591) Google Scholar). Caspase-9 enzymatic activity was determined in cytosolic extracts by LEHD-cleaving activity using a fluorometric assay kit (R&D Systems). Data were normalized using total protein concentration as determined by the Bio-Rad protein assay (Bio-Rad). Reporter Assays—Transfections of HIF-1β+/+ and HIF-1β-/- MEFs were carried out on cells plated on 60-mm Petri dishes at 50% confluence using TransIT-LT1 (Mirus) according to the manufacturer's protocol. A typical transfection was performed using 2.0 μg of a firefly luciferase reporter driven by a trimer of a hypoxic response element (HRE-luciferase) and 50 ng of Renilla luciferase driven by the herpes simplex virus thymidine kinase (TK) promoter (TK-luciferase; Promega). HRE-luciferase reporter constructs consisted of a trimerized 24-mer containing 18 bp of sequence from the phosphoglycerate kinase promoter. Luciferase assays were performed using the Dual Luciferase assay system (Promega). Values are reported as a ratio of HRE-luciferase to TK-luciferase. RNA Isolation and Real-time Reverse Transcription-PCR—Total RNA was isolated from cells grown in monolayers using a modified guanidine isothiocyanate protocol (TRIzol reagent, Invitrogen) according to the manufacturer's protocol. The expression of p21, Puma, Noxa, and Pten was determined by real-time quantitative PCR using the ABI 7700 sequence detector system (PDAR, PerkinElmer Life Sciences) and the TaqMan gold reverse transcription-PCR kit (Applied Biosystems). Murine primers and probes were selected based on nucleotide sequences downloaded from the NCBI Data Bank and designed by Primer Express Software 1.0 (Applied Biosystems): Puma sense, 5′-TGCACTGATGGAGATACGGACTT-3′; Puma antisense, 5′-ACCATGAGTCCTTCAGCCCTC-3′; Puma TaqMan probe, 5′-CCTGGCCTCCCGAAAGCCAGG-3′; Pten sense, 5′-GCCACAGGCTCCCAGACAT-3′; Pten antisense, 5′-TCCATCCTCTTGATATCTCCTTTTG-3′; Pten TaqMan probe, 5′-ACAGCCATCATCAAAGAGATCGTTAGCAGAA-3′; p21 sense, 5′-CTGTTCCGCACAGGAGCAA-3′; p21 antisense, 5′-CGCAACTGCTCACTGTCCA-3′; p21 TaqMan probe, 5′-TGTGCCGTTGTCTCTTCGGTCCC-3′; Noxa sense, 5′-CTGGGAAGTCGCAAAAGAGC-3′; Noxa antisense, 5′-CCTTCAAGTCTGCTGGCACC-3′; and Noxa TaqMan probe 5′-ATGAGGAGCCCAAGCCCAACCC-3′. The TaqMan probe consists of an oligonucleotide with a 5′-reporter dye (6-carboxyfluorescein) and a 3′-quencher dye (6-carboxytetramethylrhodamine). The reaction mixture was prepared according to the manufacturer's protocol (Applied Biosystems). All samples were read in duplicate, and values were normalized for base-line expression and for expression of glyceraldehyde-3-phosphate dehydrogenase (internal reference). Immunoblotting—The primary antibodies used for immunoblotting were anti-p53 monoclonal antibody (Ab-1, Oncogene Research) at a concentration of 4.0 μg/ml and anti-α-tubulin clone B-5-1-2 (Sigma) at a concentration of 0.5 μg/ml. The secondary antibody used was horseradish peroxidase-linked anti-mouse IgG (H+L) antibody (Cell Signaling) at a concentration of 1 μg/ml. Cell lysates were prepared using cell lysis buffer (New England Biolabs Inc.) supplemented with 1 mm phenylmethylsulfonyl fluoride. c-Myc Sensitizes Rat1a Fibroblasts to Serum or Oxygen Deprivation-induced Cell Death—Deregulated c-Myc expression in Rat1a fibroblasts without serum or oxygen has been demonstrated to cause death (6Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar, 8Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar, 9Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (198) Google Scholar). Rat1a fibroblasts were stably transfected with a fusion protein containing a modified hormone-binding domain of the murine estrogen receptor and the full-length human c-Myc protein (Rat1a/MycER cells) (35Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar). This c-Myc chimeric protein can be activated by the addition of (100 nm) to cell cultures. Rat1a/MycER cells were cultured at low confluence (30-40%) under normal oxygen conditions (21% O2) in medium containing 10% fetal bovine serum (FBS) and 25 mm HEPES to buffer any changes in pH. Rat1a/MycER cells were incubated with or without OHT for 16 h. Subsequently, Rat1a/MycER cells were deprived of either serum or oxygen for 16 and 24 h. Total cell death was measured by lactate dehydrogenase release. The addition of OHT accelerated cell death in these cells when deprived of either serum or oxygen (Fig. 1, A and B). In accordance with previous studies, our present data demonstrate that expression of c-Myc accelerates cell death following oxygen or serum deprivation. c-Myc Sensitization to Serum (but Not Oxygen) Deprivation-induced Cell Death Requires p53—The cell death activity of p53 has been suggested to be responsible for suppression of Myc-mediated tumorigenesis (38Schmitt C.A Fridman J.S. Yang M. Baranov E. Hoffman R.M. Lowe S.W. Cancer Cells. 2002; 1: 289-298Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Studies have also demonstrated that c-Myc-mediated cell death is dependent on p53 in rodent fibroblasts (4Wagner A.J. Kokontis J.M. Hay N. Genes Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (515) Google Scholar). We examined the role of p53 in our Rat1a/MycER cells by stably expressing a dominant-negative mutant of p53 (DNp53). In control experiments, Rat1a/MycER cells containing DNp53 were markedly protected against daunorubicin-induced cell death (Fig. 2A). Rat1a/MycER cells containing DNp53 and incubated with OHT were protected from serum (but not oxygen) deprivation-induced cell death (Fig. 2, B and C). We further confirmed our findings by expressing the conditional OHT-dependent c-Myc protein in MEFs from wild-type (p53+/+/MycER MEFs) and p53-null (p53-/-/MycER MEFs) animals. Both p53+/+/MycER and p53-/-/MycER cells were incubated with OHT for 16 h and subsequently deprived of either serum or oxygen for 24 h. p53-/-/MycER cells incubated with OHT were protected from serum deprivation-induced cell death (Fig. 3). In contrast, p53-/-/MycER cells incubated with OHT were not protected from oxygen deprivation-induced cell death. Collectively these results indicate that p53 is required for serum (but not oxygen) deprivation-induced cell death in rodent fibroblasts that conditionally express c-Myc.Fig. 3Wild-type or p53-/- MEFs that express a conditional OHT-dependent c-Myc protein were incubated under normal oxygen conditions with 10% FBS in the presence of OHT (100 nm) for 16 h. Subsequently, cell death was measured in cells deprived of either oxygen or serum for 24 h. Data are expressed as means ± S.E. from four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bcl-2 Proteins Regulate c-Myc Sensitization to Serum or Oxygen Deprivation-induced Cell Death—Previous studies have shown that Bcl-xL prevents either serum or oxygen deprivation-induced cell death in Rat1a/MycER cells (8Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar, 23Bissonnette R. Echeverri F. Mahboubi A. Green D. Nature. 1992; 359: 552-554Crossref PubMed Scopus (923) Google Scholar, 24Fanidi A. Harrington E.A. Evan G.I. Nature. 1992; 359: 554-556Crossref PubMed Scopus (702) Google Scholar, 25Rupnow B.A. Alarcon R.M. Giaccia A.J. Knox S.J. Cell Death Differ. 1998; 5: 141-147Crossref PubMed Scopus (33) Google Scholar, 26Wagner A.J. Small M.B. Hay N. Mol. Cell. Biol. 1993; 13: 2432-2440Crossref PubMed Scopus (207) Google Scholar). In accordance with these previous reports, stable overexpression of Bcl-xL prevented cell death in Rat1a/MycER cells incubated with OHT for 16 h and subsequently deprived of either serum or oxygen for 16 and 24 h (Fig. 4, A and B). Recent studies have indicated that the ability of Bcl-2 or Bcl-xL to inhibit cell death is through prevention of Bax or Bak from causing outer mitochondrial membrane permeabilization (reviewed in Ref. 39Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (848) Google Scholar). To test whether Bax or Bak is required for oxygen or serum deprivation-induced cell death in cells overexpressing c-Myc, the conditional OHT-dependent c-Myc protein was expressed in MEFs from wild-type (WT/MycER MEFs) and Baxand Baknull animals (bax-/-bak-/-/MycER MEFs). Both WT/MycER and bax-/-bak-/-/MycER MEFs were incubated with OHT for 16 h and subsequently deprived of either serum or oxygen for 24 h. The bax-/-bak-/-/MycER cells incubated with OHT were protected from both serum and oxygen deprivation-induced cell death (Fig. 5). These results indicate that both serum and oxygen deprivation-induced cell death pathways converge on Bax or Bak to cause outer mitochondrial membrane permeabilization.Fig. 5Wild-type or bax-/-bak-/- fibroblasts that express a conditional OHT-dependent c-Myc protein were incubated under normal oxygen conditions with 10% FBS in the presence of OHT (100 nm) for 16 h. Subsequently, cell death was measured in cells deprived of either oxygen or serum for 24 h. Data are expressed as means ± S.E. from four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bcl-xL(but Not p53) Regulates Oxygen Deprivation-induced Cytochrome c Release and Caspase-9 Activation—We further investigated the role of p53 and Bcl-xL in regulating cell death by examining cytochrome c release and caspase-9 activation. Cells release cytochrome c early in the course of cell death due to loss of outer mitochondrial membrane integrity. Cytochrome c in the cytosol binds to Apaf-1 (apoptotic protease-activating factor-1), which then undergoes a conformational change that allows the cleavage and activation of caspase-9 (40Li P. Nijhawan 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, 41Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 42Yoshida H. Kong Y.Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar, 43Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar). Rat1a/MycER cells were incubated with OHT for 16 h, subsequently deprived of either serum or oxygen for 16 h, and analyzed for cytochrome c release and caspase-9 activation. It is important to note that there was minimal cell death at 16 h in response to either death stimulus (see Fig. 1). The addition of OHT resulted in increased levels of cytochrome c release and caspase-9 activation in Rat1a/MycER cells following oxygen or serum deprivation compared with cells that did not receive OHT (Fig. 6). Rat1a/MycER cells overexpressing Bcl-xL did not release cytochrome c or activate caspase-9 upon deprivation of serum or oxygen in the presence of OHT (Fig. 6). In contrast, Rat1a/MycER cells containing DNp53 in the presence of OHT released cytochrome c and activated caspase-9 in response to oxygen (but not serum) deprivation (Fig. 6). Thus, Bcl-xL prevents both the cytochrome c release and caspase-9 activation in Rat1a cells conditionally expressing c-Myc upon serum or oxygen deprivation. The loss of p53 function prevents only serum deprivation-induced cell death in Rat1a fibroblasts conditionally expressing c-Myc at a level upstream of cytochrome c release and caspase-9 activation. Oxygen Deprivation Fails to Activate p53 Target Genes in Cells with Deregulated c-Myc Expression—To further understand why p53 is required only under conditions of serum (but not oxygen) deprivation, we examined p53 protein levels and p53 transcriptionally regulated genes p21, Puma, Noxa, and Pten in WT/MycER MEFs in the absence of either serum or oxygen. Puma and Noxa are BH3 proteins that can activate Bax or Bak and have been implicated in p53-dependent cell death (44Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2728) Google Scholar). Pten is a negative regulator of the phosphatidylinositol 3-kinase/Akt-dependent cellular survival signal and has been shown to be required for p53-mediated cell death in immortalized MEFs (45Stambolic V. MacPherson D. Sas D. Lin Y. Snow B. Jang Y. Benchimol S. Mak T.W. Mol. Cell. 2001; 8: 317-325Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar). It has been proposed that c-Myc favors the initiation of cell death by inhibiting the expression of the cyclin-dependent kinase inhibitor p21 and allowing the p53-dependent expression of cell death genes such as Puma (46Seoane J. Le H.V. Massagué J. Nature. 2002; 419: 729-734Crossref PubMed Scopus (568) Google Scholar). WT/MycER MEFs were incubated with OHT for 16 h and deprived of either serum or oxygen for 6 h (prior to any cell death), and the levels of p53 protein and p53 target genes were assessed. WT/MycER MEFs treated with OHT stabilized p53 protein levels under normal oxygen conditions as well as serum or oxygen deprivation (Fig. 7A). However, the p53 target genes Puma and Pten were expressed only under normal oxygen conditions or serum deprivation in the presence of OHT (Fig. 7B). Noxa mRNA levels" @default.
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• W2103457557 date "2004-02-01" @default.
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• W2103457557 title "c-Myc Sensitization to Oxygen Deprivation-induced Cell Death Is Dependent on Bax/Bak, but Is Independent of p53 and Hypoxia-inducible Factor-1" @default.
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