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• W2090105093 abstract "In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1−/−) and heterozygous (Egr-1+/−) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis inEgr-1+/− cells (22.8%; p < 0.0001) when compared withEgr-1−/− cells (3.5%). Radiation elevated p53 protein in Egr-1+/− cells in 3–6 h. However, in Egr- 1−/− cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1+/− cells but not inEgr-1−/− cells. Interestingly, transient overexpression of EGR-1 in p53−/−MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53+/+ MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed inEgr-1 −/− MEF cells before and after radiation when compared with the levels observed inEgr-1 +/− cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed inEgr-1 −/− cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway inEgr-1 −/− MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53. In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1−/−) and heterozygous (Egr-1+/−) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis inEgr-1+/− cells (22.8%; p < 0.0001) when compared withEgr-1−/− cells (3.5%). Radiation elevated p53 protein in Egr-1+/− cells in 3–6 h. However, in Egr- 1−/− cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1+/− cells but not inEgr-1−/− cells. Interestingly, transient overexpression of EGR-1 in p53−/−MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53+/+ MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed inEgr-1 −/− MEF cells before and after radiation when compared with the levels observed inEgr-1 +/− cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed inEgr-1 −/− cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway inEgr-1 −/− MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53. early growth response early growth response-1 gene, CAT, chloramphenicol acetyltransferase, EBS, EGR-1-binding site, MEF, mouse embryonic fibroblast gray cytomegalovirus polymerase chain reaction reverse transcriptase-PCR terminal transferase-mediated dUTP-digoxigenin nick end labeling tumor necrosis factor-α platelet-derived growth factor retinoblastoma gene The apoptotic pathways consist of an early component that includes molecular events specific for an inducer or a group of inducers and of downstream effector components common to diverse apoptotic signals (1Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar). Apoptosis has also been reported in a variety of experimental tumor systems following exposure to radiation (2Meyn R.E. Stephens L.C. Ang K.K. Hunter N.R. Brock W.A. Milas L. Peters L.J. Int. J. Radiat. Biol. 1993; 64: 583-591Crossref PubMed Scopus (217) Google Scholar, 3Stephens L.C. Hunter N.R. Ang K.K. Milas L. Meyn R.E. Radiat. Res. 1993; 135: 75-80Crossref PubMed Scopus (130) Google Scholar). Ionizing radiation alters the expression of specific genes, the products of which may contribute to the events leading to apoptotic cell death. Ionizing radiation exposure is associated with activation of certain immediate-early genes that function as transcription factors (4Datta R. Taneja N. Sukhatme V.P. Qureshi S.A. Weichselbaum R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar). These include members of jun or fos and early growth response (EGR)1 gene families (5Sherman M.L. Datta R. Hallahan D.E. Weichselbaum R.R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5663-5666Crossref PubMed Scopus (277) Google Scholar, 6Hallahan D.E. Sukhatme V.P. Sherman M.L. Virudachalam S. Kufe D. Weichselbaum R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2156-2160Crossref PubMed Scopus (242) Google Scholar).The Egr gene family includes Egr-1 (7Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar),Egr-2 (8Chavrier P. Zerial M. Lemaire P. Almendral J. Bravo R. Charnay P. EMBO J. 1988; 7: 29-35Crossref PubMed Scopus (295) Google Scholar), Egr-3 (9Patwardhan S. Gashler A. Siegel M.G. Chang L.C. Joseph L.J. Shows T.B. Le Beau M.M. Sukhatme V.P. Oncogene. 1991; 6: 917-928PubMed Google Scholar), Egr-4 (10Crosby S.D. Puetz J.J. Simburger K.S. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1991; 11: 3835-3841Crossref PubMed Scopus (170) Google Scholar), and the tumor suppressor, Wilms' tumor gene product, WT1 (11Call K.M. Glaser T. Ito C.Y. Buckler A.J. Pelletier J. Haber D.A. Rose E.A. Kral A. Yeger H. Lewis W.H. Jones C. Housman D.E. Cell. 1990; 60: 509-520Abstract Full Text PDF PubMed Scopus (1654) Google Scholar, 12Rose E.A. Glaser T. Jones C. Smith C.L. Lewis W.H. Call K.M. Minden M. Champagne E. Bonetta L. Yeger H. Housman D.E. Cell. 1990; 60: 495-508Abstract Full Text PDF PubMed Scopus (211) Google Scholar). TheEgr family shows a high degree of homology in the amino acids constituting the zinc finger domain and binds to the same GC-rich consensus DNA sequence (13Christy B. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8737-8741Crossref PubMed Scopus (453) Google Scholar, 14Rauscher F.J.D. Morris J.F. Tournay O.E. Cook D.M. Curran T. Science. 1990; 250: 1259-1262Crossref PubMed Scopus (466) Google Scholar). The Egr-1 gene product, EGR-1, is a nuclear protein that contains three zinc fingers of the C2H2 subtype (15Cao X.M. Koski R.A. Gashler A. McKiernan M. Morris C.F. Gaffney R. Hay R.V. Sukhatme V.P. Mol. Cell. Biol. 1990; 10: 1931-1939Crossref PubMed Scopus (297) Google Scholar, 16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar). Structure-function mapping studies on EGR-1 protein suggest that the amino acids constituting the zinc finger motif confer DNA binding function, whereas the NH2- terminal amino acids confer transactivation function (16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar, 17Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (88) Google Scholar). More recent studies have found that sequences diverging from the consensus may also bind EGR-1 (18Molnar G. Crozat A. Pardee A.B. Mol. Cell. Biol. 1994; 14: 5242-5248Crossref PubMed Scopus (84) Google Scholar, 19Swirnoff A.H. Milbrandt J. Mol. Cell. Biol. 1995; 15: 2275-2287Crossref PubMed Scopus (300) Google Scholar), thus having a broader spectrum of potential target genes. It is interesting to note that within this family of transcription factors, EGR-1 was found to be a positive activator of transcription, whereas WT1 is a transcriptional repressor, both acting via binding to the same GC-rich consensus sequence in reporter constructs (20Drummond I.A. Madden S.L. Rohwer-Nutter P. Bell G.I. Sukhatme V.P. Rauscher F.J.D. Science. 1992; 257: 674-678Crossref PubMed Scopus (490) Google Scholar, 21Madden S.L. Cook D.M. Morris J.F. Gashler A. Sukhatme V.P. Rauscher III, F.J. Science. 1991; 253: 1550-1553Crossref PubMed Scopus (405) Google Scholar, 22Wang Z.Y. Madden S.L. Deuel T.F. Rauscher III, F.J. J. Biol. Chem. 1992; 267: 21999-22002Abstract Full Text PDF PubMed Google Scholar). Depending on the cell type, EGR-1 may behave as a positive or negative regulator of gene transcription (16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar, 23Ackerman S.L. Minden A.G. Williams G.T. Bobonis C. Yeung C.Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7523-7527Crossref PubMed Scopus (116) Google Scholar, 24Takimoto Y. Wang Z.Y. Kobler K. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1686-1690Crossref PubMed Scopus (39) Google Scholar). The EGR-1 GC-rich consensus target sequence, 5′-GCG(T/G)GGGCG-3′ or 5′-TCC(T/A)CCTCCTCC-3′ (25Nakagama H. Heinrich G. Pelletier J. Housman D.E. Mol. Cell. Biol. 1995; 15: 1489-1498Crossref PubMed Scopus (163) Google Scholar), has been identified in the promoter regions of the following: (a) transcription factors, such as MYC and NUR77; (b) growth factors or their receptors, such as transforming growth factor-β1, TNF-α, PDGF-A (26Wang Z.Y. Deuel T.F. Biochem. Biophys. Res. Commun. 1992; 188: 433-439Crossref PubMed Scopus (36) Google Scholar), PDGF-B (27Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (476) Google Scholar), insulin-like growth factor-II, fibroblast growth factor-β, or epidermal growth factor receptor (6Hallahan D.E. Sukhatme V.P. Sherman M.L. Virudachalam S. Kufe D. Weichselbaum R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2156-2160Crossref PubMed Scopus (242) Google Scholar,7Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar, 28Hu R.M. Levin E.R. J. Clin. Invest. 1994; 93: 1820-1827Crossref PubMed Scopus (50) Google Scholar, 29Sukhatme V.P. J. Am. Soc. Nephrol. 1990; 1: 859-866Crossref PubMed Google Scholar); (c) cell cycle regulators such as the retinoblastoma susceptibility gene Rb (30Day M.L. Wu S. Basler J.W. Cancer Res. 1993; 53: 5597-5599PubMed Google Scholar), cyclin D1 (31Philipp A. Schneider A. Vasrik I. Finke K. Xiong Y. Beach D. Alitalo K. Eilers M. Mol. Cell. Biol. 1994; 14: 4032-4043Crossref PubMed Scopus (246) Google Scholar), c-Ki-ras (26Wang Z.Y. Deuel T.F. Biochem. Biophys. Res. Commun. 1992; 188: 433-439Crossref PubMed Scopus (36) Google Scholar), and p53 (32Nair P. Muthukkumar S. Sells S.F. Han S.S. Sukhatme V.P. Rangnekar V.M. J. Biol. Chem. 1997; 272: 20131-20138Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar); and (d) thymidine kinase, an enzyme crucial in DNA biosynthesis (18Molnar G. Crozat A. Pardee A.B. Mol. Cell. Biol. 1994; 14: 5242-5248Crossref PubMed Scopus (84) Google Scholar) and MDR-1 (33McCoy C. Smith D.E. Cornwell M.M. Mol. Cell. Biol. 1995; 15: 6100-6108Crossref PubMed Scopus (81) Google Scholar).It has also been speculated that x-ray induction of PDGF, transforming growth factor-β1, and TNF-α may be regulated by Egr-1 and c-jun (6Hallahan D.E. Sukhatme V.P. Sherman M.L. Virudachalam S. Kufe D. Weichselbaum R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2156-2160Crossref PubMed Scopus (242) Google Scholar). Apart from being a potential transcriptional regulator, Egr-1 has a radiation-inducible promoter. Through these distinct induction pathways, Egr-1 has been linked to signaling events initiating cell phenotypic response to radiation injury.On the other hand, wild type p53 has been shown to be functionally necessary for growth inhibition and apoptosis following exposure to ionizing radiation, and p53 mutations have been reported to increase resistance to apoptosis (34Lee J.M. Bernstein A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5742-5746Crossref PubMed Scopus (569) Google Scholar). In our previous report, using melanoma cells, which contain wild type p53, a dose-dependent increase in EGR-1 expression with dose-dependent growth inhibition was observed when exposed to ionizing radiation (35Ahmed M.M. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Wood Jr., D.P. Sells S.F. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1996; 271: 29231-29237Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Transfectant melanoma cells stably expressing the dominant-negative mutant protein of EGR-1 showed significantly reduced (<50%) sensitivity to radiation-inducible growth inhibition, and this resistance was found to be dose-dependent. These observations suggest that the EGR-1 induction is involved in the regulation of radiation-inducible apoptosis despite the presence of wild type p53. Recently, we used a p53 null prostate cancer cell line (PC-3), which was found to be moderately resistant to ionizing radiation-inducible apoptosis (36Ahmed M.M. Sells S.F. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Harp C. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1997; 272: 33056-33061Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Western blot analysis and immunocytochemistry studies indicate that EGR-1 is induced in the PC-3 cells by ionizing radiation. Experiments with the Egr-1 dominant-negative mutant or Egr-1 overexpression suggest that Egr-1 function is required for the radiation-inducible apoptosis. Despite the absence of wild type functional p53 protein, the transfected cells expressing the dominant-negative mutant of EGR-1 were resistant to ionizing radiation, and cells overexpressing EGR-1 protein were sensitive to ionizing radiation. Our findings strongly suggested that the radiation-induced apoptotic response in PC-3 cells is elicited through up-regulation of TNF-α protein via EGR-1-mediated transactivation. Thus, EGR-1 is an important mediator of radiation responsiveness irrespective of p53 functional status. However, in a recent report, it was found that EGR-1 protein transactivates the promoter of p53 gene and up-regulates p53 mRNA and protein levels in response to apoptotic stimuli (32Nair P. Muthukkumar S. Sells S.F. Han S.S. Sukhatme V.P. Rangnekar V.M. J. Biol. Chem. 1997; 272: 20131-20138Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). This prompted us to investigate further the interactive role of Egr-1 with p53 during the process of apoptosis. We sought to investigate this mechanism in a normal cell background using isogenic normal primary culture cells derived from mouse embryonic fibroblasts (MEF) with varied genomic status for Egr-1 gene (cells with both intact Egr-1 alleles,Egr-1 +/+; cells with homozygous deletion ofEgr-1 alleles,Egr-1 −/−; and heterozygous deletion of one Egr-1 allele,Egr-1 +/−). Based on findings from these isogenic normal cells with varied genomic status of Egr-1, we suggest that EGR-1 function is necessary for enhanced sensitivity to radiation-induced apoptosis and that the radiation-induced proapoptotic function of Egr-1 is directly mediated by the target genesp53 and Rb.DISCUSSIONExposure to ionizing radiation is associated with the formation of reactive oxygen intermediates causing direct damage to DNA (44Ward J.F. Prog. Nucleic Acids Res. Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1170) Google Scholar). These reactive oxygen intermediates target the sequence CC(A/T)6GG to mediate the activation of EGR-1 (4Datta R. Taneja N. Sukhatme V.P. Qureshi S.A. Weichselbaum R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar). Previous studies from our laboratory (35Ahmed M.M. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Wood Jr., D.P. Sells S.F. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1996; 271: 29231-29237Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) have suggested that despite the presence of wild type p53 background, inhibition of the expression or function of EGR-1 causes a diminution of radiation-induced growth inhibition in melanoma cells. In the absence of p53, radiation-induced apoptosis of prostate cancer cells was found to be mediated by EGR-1 via TNF-α transactivation (36Ahmed M.M. Sells S.F. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Harp C. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1997; 272: 33056-33061Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These results suggest thatEgr-1 induction is involved in the radiation-induced signaling of the cascades of apoptosis pathway.In this study, in contrast to Egr-1+/− MEF cells, Egr-1−/− MEF cells were significantly resistant to radiation-inducible apoptosis and showed no elevation of p53 protein after radiation. These observations indicate that radiation-induced EGR-1-mediated transactivation of downstream genes is essential for radiation sensitivity. Thus, in support of previous reports, the present study demonstrates that EGR-1 is the upstream mediator for the initiation of the radiation-induced signaling cascade leading to cell death.The tumor suppressor gene p53 is a central mediator of apoptotic pathways in diverse model systems (45Huang R.P. Liu C. Fan Y. Mercola D. Adamson E.D. Cancer Res. 1995; 55: 5054-5062PubMed Google Scholar, 46Buckbinder L. Talbott R. Velasco-Miguel S. Takenaka I. Faha B. Seizinger B.R. Kley N. Nature. 1995; 377: 646-649Crossref PubMed Scopus (804) Google Scholar, 47Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 48Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.W. Kruzel E. Radinsky R. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (690) Google Scholar). The p53 protein can cause transcriptional up-regulation of a number of downstream genes, such as mdm-2, p21waf1/cip1, bax, fas/apo1, insulin-like growth factor-binding protein-3, which are implicated in growth inhibition and apoptotic cell death (46Buckbinder L. Talbott R. Velasco-Miguel S. Takenaka I. Faha B. Seizinger B.R. Kley N. Nature. 1995; 377: 646-649Crossref PubMed Scopus (804) Google Scholar, 47Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (302) Google Scholar, 48Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.W. Kruzel E. Radinsky R. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (690) Google Scholar). In this study, it was found that mRNA levels ofp53, p21waf1/cip1, mdm-2, andbax were elevated after irradiation inEgr-1+/− cells but not inEgr-1−/− cells. In addition, the basal levels of these mRNAs were high inEgr-1−/− MEF cells when compared with Egr-1+/− cells. Loss of radiation-induced elevation of p53 may be attributed to the loss of Egr-1 mediated transregulation of p53 inEgr-1−/− MEF cells, and this may have led to the loss of up-regulation of p53 target genes, p21waf1/cip1, mdm-2, and bax.Radiation caused degradation of p53 protein inEgr-1−/− cells, and this led to enhanced resistance to radiation-inducible apoptosis. Transient overexpression of EGR-1 protein inEgr-1−/− cells restored radiation sensitivity and stabilized the p53 protein levels. Thus, this observation suggests that EGR-1 protein is necessary for the up-regulation and the stability of p53 protein and radiation sensitivity. Moreover, radiation elevated the p53-CAT reporter activity in Egr-1+/− cells but not inEgr-1−/− cells. This observation is supported by a recent study that EGR-1 can directly bind with thep53 promoter at two consensus EGR-1-binding sites and induce the p53 mRNA and protein (32Nair P. Muthukkumar S. Sells S.F. Han S.S. Sukhatme V.P. Rangnekar V.M. J. Biol. Chem. 1997; 272: 20131-20138Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Thus, Egr-1 is an important transregulator of p53. However, at this point we cannot rule out the possibility that other genes that are also regulated byEgr-1 may play a role in the stability of p53 protein.A marginal induction of radiation-induced apoptosis observed in p53−/−/CMV-EGR-1 MEF transfectants when compared with p53+/+/CMV-EGR-1 MEF cells suggests that p53 played an important downstream role in regulation ofEgr-1-mediated radiation-induced apoptosis. It also suggests that the absence of p53 may not contribute toward complete abrogation of EGR-1-mediated radiation-induced apoptosis. This is supported by our previous data that in p53 null prostate cancer cell line PC3, EGR-1 overexpression caused super induction of radiosensitivity (36Ahmed M.M. Sells S.F. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Harp C. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1997; 272: 33056-33061Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The degree of induction of apoptosis was much higher in p53 null PC3 cells when compared with p53−/−/CMV-EGR-1 MEF transfectant cells in this study. The difference may be due to the tumor cell background versus the normal cell background. Thus, in the absence of p53, EGR-1 may mediate the proapoptotic action of radiation via TNF-α (36Ahmed M.M. Sells S.F. Venkatasubbarao K. Fruitwala S.M. Muthukkumar S. Harp C. Mohiuddin M. Rangnekar V.M. J. Biol. Chem. 1997; 272: 33056-33061Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) or other downstream cell-death effector genes.p53 can bind to the promoter region of MDM2 and activate its transcription, forming an autoregulation loop between the expression and function of p53 and MDM2 (49Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2282) Google Scholar). It is also reported that MDM2-p53 interaction can target p53 for degradation (43Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Rb can regulate the apoptotic function of p53 through binding to MDM2, thus preventing MDM2-mediated degradation of p53 (42Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Rb can also prevent MDM2 from inhibiting p53-mediated apoptosis. In addition, Rb can protect p53 from MDM2-mediated degradation by forming a trimeric complex with p53 via binding to MDM2 (42Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). To understand further the mechanism of p53 degradation in irradiated Egr-1−/−MEF cells, we investigated the expression and functional interaction of Rb with p53 and MDM2 in Egr-1+/− andEgr-1−/− MEF cells. The rationale for analyzing the Rb function in this normal isogenic cell system is that (a) Rb regulates the apoptotic function of p53 by mitigating MDM2 mediated degradation (42Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and (b) the Rb gene promoter contains EGR-1-binding sites that conform to the GC-rich consensus (30Day M.L. Wu S. Basler J.W. Cancer Res. 1993; 53: 5597-5599PubMed Google Scholar). Low expression levels of hypophosphorylated forms of Rb and decreased Rb-CAT reporter activity were found inEgr-1−/− MEF cells before and after irradiation when compared with Egr-1+/−MEF cells. Relatively higher levels of Rb-MDM2-bound complex and lower levels of p53-MDM2-bound complex were observed in irradiatedEgr-1+/− MEF cells. In contrast, higher amounts of p53-MDM2 complex and low bound forms of the Rb-MDM2 complex were observed in Egr-1−/− cells. Lower amounts of the Rb-MDM2 complex along with higher amounts of p53-MDM2 inEgr-1−/− MEF cells might have contributed to p53 degradation after radiation. Thus, apoptosis caused by ionizing radiation requires the induction of EGR-1 protein, which then transregulates the expression of p53 protein and also indirectly regulates the stability of p53 via Rb. The apoptotic pathways consist of an early component that includes molecular events specific for an inducer or a group of inducers and of downstream effector components common to diverse apoptotic signals (1Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar). Apoptosis has also been reported in a variety of experimental tumor systems following exposure to radiation (2Meyn R.E. Stephens L.C. Ang K.K. Hunter N.R. Brock W.A. Milas L. Peters L.J. Int. J. Radiat. Biol. 1993; 64: 583-591Crossref PubMed Scopus (217) Google Scholar, 3Stephens L.C. Hunter N.R. Ang K.K. Milas L. Meyn R.E. Radiat. Res. 1993; 135: 75-80Crossref PubMed Scopus (130) Google Scholar). Ionizing radiation alters the expression of specific genes, the products of which may contribute to the events leading to apoptotic cell death. Ionizing radiation exposure is associated with activation of certain immediate-early genes that function as transcription factors (4Datta R. Taneja N. Sukhatme V.P. Qureshi S.A. Weichselbaum R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2419-2422Crossref PubMed Scopus (165) Google Scholar). These include members of jun or fos and early growth response (EGR)1 gene families (5Sherman M.L. Datta R. Hallahan D.E. Weichselbaum R.R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5663-5666Crossref PubMed Scopus (277) Google Scholar, 6Hallahan D.E. Sukhatme V.P. Sherman M.L. Virudachalam S. Kufe D. Weichselbaum R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2156-2160Crossref PubMed Scopus (242) Google Scholar). The Egr gene family includes Egr-1 (7Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (552) Google Scholar),Egr-2 (8Chavrier P. Zerial M. Lemaire P. Almendral J. Bravo R. Charnay P. EMBO J. 1988; 7: 29-35Crossref PubMed Scopus (295) Google Scholar), Egr-3 (9Patwardhan S. Gashler A. Siegel M.G. Chang L.C. Joseph L.J. Shows T.B. Le Beau M.M. Sukhatme V.P. Oncogene. 1991; 6: 917-928PubMed Google Scholar), Egr-4 (10Crosby S.D. Puetz J.J. Simburger K.S. Fahrner T.J. Milbrandt J. Mol. Cell. Biol. 1991; 11: 3835-3841Crossref PubMed Scopus (170) Google Scholar), and the tumor suppressor, Wilms' tumor gene product, WT1 (11Call K.M. Glaser T. Ito C.Y. Buckler A.J. Pelletier J. Haber D.A. Rose E.A. Kral A. Yeger H. Lewis W.H. Jones C. Housman D.E. Cell. 1990; 60: 509-520Abstract Full Text PDF PubMed Scopus (1654) Google Scholar, 12Rose E.A. Glaser T. Jones C. Smith C.L. Lewis W.H. Call K.M. Minden M. Champagne E. Bonetta L. Yeger H. Housman D.E. Cell. 1990; 60: 495-508Abstract Full Text PDF PubMed Scopus (211) Google Scholar). TheEgr family shows a high degree of homology in the amino acids constituting the zinc finger domain and binds to the same GC-rich consensus DNA sequence (13Christy B. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8737-8741Crossref PubMed Scopus (453) Google Scholar, 14Rauscher F.J.D. Morris J.F. Tournay O.E. Cook D.M. Curran T. Science. 1990; 250: 1259-1262Crossref PubMed Scopus (466) Google Scholar). The Egr-1 gene product, EGR-1, is a nuclear protein that contains three zinc fingers of the C2H2 subtype (15Cao X.M. Koski R.A. Gashler A. McKiernan M. Morris C.F. Gaffney R. Hay R.V. Sukhatme V.P. Mol. Cell. Biol. 1990; 10: 1931-1939Crossref PubMed Scopus (297) Google Scholar, 16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar). Structure-function mapping studies on EGR-1 protein suggest that the amino acids constituting the zinc finger motif confer DNA binding function, whereas the NH2- terminal amino acids confer transactivation function (16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar, 17Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (88) Google Scholar). More recent studies have found that sequences diverging from the consensus may also bind EGR-1 (18Molnar G. Crozat A. Pardee A.B. Mol. Cell. Biol. 1994; 14: 5242-5248Crossref PubMed Scopus (84) Google Scholar, 19Swirnoff A.H. Milbrandt J. Mol. Cell. Biol. 1995; 15: 2275-2287Crossref PubMed Scopus (300) Google Scholar), thus having a broader spectrum of potential target genes. It is interesting to note that within this family of transcription factors, EGR-1 was found to be a positive activator of transcription, whereas WT1 is a transcriptional repressor, both acting via binding to the same GC-rich consensus sequence in reporter constructs (20Drummond I.A. Madden S.L. Rohwer-Nutter P. Bell G.I. Sukhatme V.P. Rauscher F.J.D. Science. 1992; 257: 674-678Crossref PubMed Scopus (490) Google Scholar, 21Madden S.L. Cook D.M. Morris J.F. Gashler A. Sukhatme V.P. Rauscher III, F.J. Science. 1991; 253: 1550-1553Crossref PubMed Scopus (405) Google Scholar, 22Wang Z.Y. Madden S.L. Deuel T.F. Rauscher III, F.J. J. Biol. Chem. 1992; 267: 21999-22002Abstract Full Text PDF PubMed Google Scholar). Depending on the cell type, EGR-1 may behave as a positive or negative regulator of gene transcription (16Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar, 23Ackerman S.L. Minden A.G. Wil" @default.
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