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• W2090585013 abstract "The early growth response-1 (EGR-1) protein is an anti-proliferative signal for certain tumor cells and is required for apoptosis induced by stimuli that elevate intracellular Ca2+. We present evidence that EGR-1 transactivates the promoter of the p53 gene and up-regulates p53 RNA and protein levels. Inhibition of p53 function with dominant-negative p53 mutants abrogates EGR-1-dependent apoptosis. These findings establish a direct functional link between EGR-1 and the p53-mediated cell death pathway and suggest that mutant forms of p53 in tumor cells may provide resistance to the anti-proliferative effects of EGR-1. The early growth response-1 (EGR-1) protein is an anti-proliferative signal for certain tumor cells and is required for apoptosis induced by stimuli that elevate intracellular Ca2+. We present evidence that EGR-1 transactivates the promoter of the p53 gene and up-regulates p53 RNA and protein levels. Inhibition of p53 function with dominant-negative p53 mutants abrogates EGR-1-dependent apoptosis. These findings establish a direct functional link between EGR-1 and the p53-mediated cell death pathway and suggest that mutant forms of p53 in tumor cells may provide resistance to the anti-proliferative effects of EGR-1. Apoptosis, or programmed cell death, a genetic process of coordinated deletion of selective cells, is essential for metazoan development and homeostasis (1Wyllie A.H. Cancer Metastasis Rev. 1992; 11: 95-103Crossref PubMed Scopus (575) Google Scholar, 2Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2384) Google Scholar, 3Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2419) Google Scholar, 4Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (614) Google Scholar). The primordial forms of apoptosis in Caenorhabditis elegans and Drosophila have been recapitulated in mammalian cells, and striking similarities have been observed in the cell death programs of invertebrates and vertebrates (5Vaux D.L. Haecker G. Strasser A. Cell. 1994; 76: 777-779Abstract Full Text PDF PubMed Scopus (688) Google Scholar, 6White K. Tahaoglu E. Steller H. Science. 1996; 271: 805-807Crossref PubMed Scopus (335) Google Scholar, 7Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2229) Google Scholar). Apoptosis is characterized by cell membrane blebbing, chromatin condensation, changes in nuclear architecture, and oligonucleosome-length DNA fragmentation (8Williams G.T. Cell. 1991; 65: 1097-1098Abstract Full Text PDF PubMed Scopus (831) Google Scholar, 9Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6683) Google Scholar). The apoptotic pathways consist of an early component that includes molecular events that are specific for an inducer or a group of inducers and of downstream effector components that are common to diverse apoptotic signals (10Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar). The common components include a basal cell death machinery composed of initiator, amplifier, and effector proteases belonging to the interleukin-1 converting enzyme subfamily or an interleukin-1 converting enzyme-related family (10Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 11Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (797) Google Scholar, 12Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 13Kumar S. Trends Biochem. 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Prostate. 1988; 13: 119-130Crossref PubMed Scopus (124) Google Scholar, 16Hasbold J. Klaus G.G.B. Eur. J. Immunol. 1990; 20: 1685-1690Crossref PubMed Scopus (230) Google Scholar, 17Furuya Y. Lundmo P. Short A.D. Gill D.L. Isaacs J.T. Cancer Res. 1994; 54: 6167-6175PubMed Google Scholar, 18Nicotera P. Zhivotovsky B. Orrenius S. Cell Calcium. 1994; 16: 279-288Crossref PubMed Scopus (172) Google Scholar). Intracellular calcium levels become elevated after the activation of T lymphocytes by anti-CD3 or that of B lymphocytes by anti-IgM antibodies; after withdrawal of survival factors, such as testosterone in the prostate gland; or after exposure to certain exogenous stimuli, such as calcium ionophores or thapsigargin (TG), 1The abbreviations used are: TG, thapsigargin; EGR-1, early growth response-1; EBS, EGR-1-binding site(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; TUNEL, terminal transferase-mediated dUTP-nucleotide-end labeling; mEGR-1, mouse EGR-1. 1The abbreviations used are: TG, thapsigargin; EGR-1, early growth response-1; EBS, EGR-1-binding site(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; TUNEL, terminal transferase-mediated dUTP-nucleotide-end labeling; mEGR-1, mouse EGR-1. a potent inhibitor of the Ca2+-dependent ATPase in the endoplasmic reticulum (19Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2978) Google Scholar). Elevation of intracellular calcium causes induction of immediate-early genes that further trigger a cascade of downstream events leading ultimately to cell death. Although several immediate-early genes such as early growth response-1 (Egr-1; also referred to as zif268, NGF-IA, TIS8, and Krox-24), nur77, and par-4 have been functionally linked to apoptosis caused by intracellular calcium elevation (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 21Sells S.F. Wood Jr., D.P. Joshi-Barve S.S Muthukkumar S. Jacob R.J. Crist S.A. Humphreys S. Rangnekar V.M. Cell Growth & Differ. 1994; 5: 457-466PubMed Google Scholar, 22Liu Z.-G. Smith S.W. McLaughlin K.A. Schwartz L.M. Osborne B.A. Nature. 1994; 367: 281-284Crossref PubMed Scopus (494) Google Scholar, 23Woronicz J.D. Calnan B. Ngo V. Winoto A. Nature. 1994; 367: 277-281Crossref PubMed Scopus (504) Google Scholar, 24Sells S.F. Han S.-S. Muthukkumar S. Maddiwar N. Johnstone R. Boghaert E. Gillis D. Liu G. Nair P. Monning S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S. Wood Jr., D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (179) Google Scholar), the precise downstream events that are important for successful apoptosis via this pathway have not been delineated. Egr-1 was first identified as an immediate-early gene induced by mitogenic stimulation and during membrane depolarization and seizure (25Sukhatme V.P. Cao X. Chang L.C. Tsai-Morris C.-H. Stemenkovich D. Ferreira P.C.P. Cohen R. Edwards S.A. Shows T.B. Curran T. Le Beau M.M. Adamson E.D. Cell. 1988; 53: 37-43Abstract Full Text PDF PubMed Scopus (1016) Google Scholar, 26Milbrandt J. Science. 1988; 238: 797-799Crossref Scopus (929) Google Scholar). Subsequently, EGR-1 was shown to be induced by diverse exogenous stimuli (27Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. 1995; 50: 191-224Crossref PubMed Scopus (551) Google Scholar). EGR-1 is a nuclear protein that contains three zinc finger motifs of the C2H2subtype that bind to a GC-rich consensus DNA sequence TGCG(T/g)(G/A)GG(C/a/t)G(G/T) (where lowercase letters indicate bases of relatively lower binding affinity) or to a (TCC) n motif (28Wang Z.-Y. Qiu Q.-Q. Enger K.T. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8896-8900Crossref PubMed Scopus (145) Google Scholar,29Swirnoff A.H. Milbrandt J. Mol. Cell. Biol. 1995; 15: 2275-2287Crossref PubMed Scopus (298) Google Scholar). Structure-function mapping studies suggest that the NH2 terminus of EGR-1 confers transactivation function to the protein (27Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. 1995; 50: 191-224Crossref PubMed Scopus (551) Google Scholar, 30Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (87) Google Scholar). The Egr-1 gene was localized to human chromosome 5q31.1, a region known to be often deleted from patients suffering from therapy-induced acute myeloid leukemia (31Le Beau M.M. Espinosa III, R. Neuman W.L. Stock W. Roulston D. Larson R.A. Keinanen M. Westbrook C.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 5484-5488Crossref Scopus (232) Google Scholar). Moreover, studies using diverse tumor cells suggest that endogenous levels of EGR-1 act to impede proliferation (32Huang R.-P. Darland T. Okamura D. Mercola D. Adamson E.D. Oncogene. 1994; 9: 1367-1377PubMed Google Scholar, 33Huang R.-P. Liu C. Fan Y. Mercola D. Adamson E.D. Cancer Res. 1995; 55: 5054-5062PubMed Google Scholar). Consistent with an anti-tumor role for EGR-1, apoptosis-inducing stimuli, such as TG and ionizing radiation, up-regulate EGR-1 expression (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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). Furthermore, our previous studies, which used antisense oligomers to block EGR-1 expression or a dominant-negative EGR-1 mutant to inhibit EGR-1 function, confirmed that EGR-1 is essential for apoptosis induced by TG or by ionizing radiation (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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). Because EGR-1 is induced very early in the apoptotic process (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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), it is expected to mediate the activation of downstream genes that play crucial roles in growth control. EGR-1-binding sites (EBS) that conform to the GC-rich consensus sequence have been identified in the promoters of genes such as thymidine kinase, an enzyme integral to DNA biosynthesis; cell cycle regulators such as cyclin D1 and the retinoblastoma susceptibility gene Rb; and (TCC) n motifs have been identified in the promoter regions of genes encoding growth factors such as platelet-derived growth factor and basic fibroblast growth factor; growth factor receptors such as epidermal growth factor-receptor and the insulin-like growth factor-receptor; and protooncogenes c-Ki-ras and c-myc (cited in Refs.20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar and 27Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. 1995; 50: 191-224Crossref PubMed Scopus (551) Google Scholar). However, none of these genes has been shown to be directly involved in apoptosis as a consequence of EGR-1 induction. 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Zhang W.-W. Kruzel E. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (687) Google Scholar). These regulatory effects require binding of p53 protein to consensus binding sites in the promoter region of target genes (35Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4402) Google Scholar, 46Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-830Crossref PubMed Scopus (886) Google Scholar, 47El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1728) Google Scholar). The p53-dependent downstream induction or activation of the Fas/Apo1 pathway leads to the activation of a cascade of downstream effector proteases resulting in apoptotic death (12Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 45Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. 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Halachmi S. Bronson R.T. Weinberg R.A. Curr. Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar, 57Malkin D. Li F.P. Strong L.C. Fraumeni Jr., J.F. Nelson C.E. Kim D.R. Kassel J. Gryka M.A. Bischoff F.Z. Tainsky M.A. Friend S.H. Science. 1990; 250: 1233-1238Crossref PubMed Scopus (3013) Google Scholar, 58Shrivastava S. Zou Z. Pirollo K. Blattner W. Chang E.H. Nature. 1990; 348: 747-749Crossref PubMed Scopus (1019) Google Scholar). Most tumor types either contain no p53 protein owing to loss of the p53 alleles or of chromosome 17 wherep53 is located or contain point mutations in thep53 gene that result in mutant protein products that are functionally inactive (54Hollstein M. Rice K. Greenblatt M.S. Soussi T. Fuchs R. Sorlie T. Hovig E. Smith-Sorensen B. Montesano R. Harris C.C. Nucleic Acids Res. 1994; 22: 3551-3555PubMed Google Scholar). Most of the commonly occurring mutations inp53 are located in the DNA-binding or transactivation domains of the protein and render p53 inactive in the transcription of downstream proapoptotic genes such as bax (39Friedlander P. Haupt Y. Prives C. Oren M. Mol. Cell. Biol. 1996; 16: 4961-4971Crossref PubMed Scopus (268) Google Scholar, 40Ludwig R.L. Bates S. Vousden K.H. Mol. Cell. Biol. 1996; 16: 4952-4960Crossref PubMed Scopus (252) Google Scholar, 54Hollstein M. Rice K. Greenblatt M.S. Soussi T. Fuchs R. Sorlie T. Hovig E. Smith-Sorensen B. Montesano R. Harris C.C. Nucleic Acids Res. 1994; 22: 3551-3555PubMed Google Scholar, 59Hollstein M. Sidransky D. Vogelstein B. Harris C. Science. 1991; 253: 49-52Crossref PubMed Scopus (7400) Google Scholar). Some mutant forms of p53 protein can oligomerize with wild-type p53 protein and abrogate its growth-inhibitory functions and thus act as dominant-negative inhibitors of wild-type p53 (46Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. 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Genes & Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (512) Google Scholar), and c-Fos and c-MYC have been shown to require wild-type p53 for apoptosis (70Preston G.A. Lyon T.T. Yin Y. Lang J.E. Solomon G. Annab L. Srinivasan D.G. Alcorta D.A. Barrett J.C. Mol. Cell. Biol. 1996; 16: 211-218Crossref PubMed Scopus (205) Google Scholar, 72Hemerking H. Eick D. Science. 1994; 265: 2091-2093Crossref PubMed Scopus (699) Google Scholar,73Wagner A.J. Kokontis J.M. Hay N. Genes & Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (512) Google Scholar). It is not known, however, whether the apoptotic action of the other immediate-early genes is dependent on wild-type p53 function. Studies directed at identifying whether p53 is required or not for the transduction of an apoptotic signal from immediate-early proapoptotic genes encoding transcription factors, which couple the early events in the plasma membrane to long term cellular phenotypic changes in the cell, should provide valuable insights into the downstream targets of the transcription factors and help identify a link with components of the cell death pathways. In the course of our studies aimed at understanding the mechanism of EGR-1-mediated apoptosis, we have identified p53 as a direct transcriptional target of EGR-1. Our data suggest that the proapoptotic action of EGR-1 is mediated via wild-type p53. Human melanoma cells A375-C6 and transfected cell lines that expressed pCMV-mEGR1 or vector were cultured as described previously (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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). Pools of approximately 200 transfected clones were maintained as cell lines. Expression constructs m143 and m175 that encode mutant p53 proteins (46Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-830Crossref PubMed Scopus (886) Google Scholar) were kindly provided by Bert Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD). The constructs pCMV-ΔTA, pCMV-ΔRM, pCMV-ΔZF, and pCMV-ΔTA/RM that encode mutant EGR-1 proteins have been previously described (27Gashler A. Sukhatme V.P. Prog. Nucleic Acid Res. 1995; 50: 191-224Crossref PubMed Scopus (551) Google Scholar, 74Gashler A.L. Swaminathan S. Sukhatme V.P. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (211) Google Scholar). Reporter construct p53(2.2+1.6)-CAT, which contains about 2.2 kb of sequence upstream of the human p53 cap site and about 1.6 kb of sequence downstream of the p53 cap site placed downstream of the chloramphenicol acetyltransferase (CAT) cDNA (75Reisman D. Greenberg M. Rotter V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5146-5150Crossref PubMed Scopus (70) Google Scholar), was kindly provided by David Reisman (University of South Carolina, Columbia, SC). Polymerase chain reaction was employed to synthesize a 0.8-kb p53 promoter fragment from p53(2.2+1.6)-CAT DNA template. The reactions used a downstream primer (5′-TTTTTGGCTCTAGACTTTTGAGAA-3′) complementary to the region +11 to −9 (underlined) and an upstream primer (5′-TTTTTCTAGAGAAGCGCCTACGCTCCC-3′) corresponding to the region −758 to −774 (underlined) in the p53 promoter. The polymerase chain reaction product was subcloned in the XbaI site upstream of the CAT sequence in pGCAT-C vector and the construct, p53(0.8)-CAT, thus obtained was confirmed for orientation. Whole-cell protein extracts were prepared and analyzed by using Western blot analysis as described previously (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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). EGR-1 (sc-110) and p53 (DO-1) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the actin antibody was purchased from Sigma. Cells were quantified for apoptosis by terminal transferase-mediated dUTP-nucleotide-end labeling (TUNEL) as described previously (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar, 34Ahmed 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). Transfections were performed by the calcium phosphate coprecipitation method, and stable transfectants were selected in 300 μg/ml G418-sulfate as described previously (20Muthukkumar S. Nair P. Sells S.F. Maddiwar N.G. Jacob R.J. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 6262-6272Crossref PubMed Scopus (105) Google Scholar). Pools of about 200 stably transfected clones were maintained as cell lines. CAT assays were performed by using thin layer chromatography as described previously (76Joshi-Barve S.S. Rangnekar V.V. Sells S.F. Rangnekar V.M. J. Biol. Chem. 1993; 268: 18018-18029Abstract Full Text PDF PubMed Google Scholar, 77Sells S.F. Muthukkumar S. Sukhatme V.P. Crist S.A. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 682-692Crossref PubMed Google Scholar). Total RNA extraction and Northern blot analysis were performed as described previously (77Sells S.F. Muthukkumar S. Sukhatme V.P. Crist S.A. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 682-692Crossref PubMed Google Scholar). The cDNA probe for Egr-1 has been described previously (77Sells S.F. Muthukkumar S. Sukhatme V.P. Crist S.A. Rangnekar V.M. Mol. Cell. Biol. 1995; 15: 682-692Crossref PubMed Google Scholar). The cDNA for human p53 was purchased from the American Type Culture Collection (Rockville, MD). Preparation of nuclear extracts from transfected cells and electrophoretic mobility shift assay were preformed as described previously (76Joshi-Barve S.S. Rangnekar V.V. Sells S.F. Rangnekar V.M. J. Biol. Chem. 1993; 268: 18018-18029Abstract Full Text PDF PubMed Google Scholar). Two complementary primers, 5′-GCGCCTACGCTC-3′ and 5′-GAGCGTAGGCGC-3′, corresponding to the upper and lower strands of EBS-1 (−770 to −762) in the upstream region of the p53 promoter were synthesized at the Macromolecular Synthesis Facility, University of Kentucky. The primers were annealed, end-labeled with [γ-32P]ATP and used as probe. The reaction mixture consisting of nuclear extract, 5 × reaction buffer (1m Tris, pH 7.5, 2.5 m potassium chloride, 0.5m EDTA, 1 m dithiothreitol, glycerol, bovine serum albumin), and 1 μm poly(dI-dC)·poly(dI-dC) was incubated on ice for 15 min. Probe was added, and the mixture was further incubated at room temperature for 20 min. The reaction mixture for the supershift assay consisted of all of the reagents described above for electrophoretic mobility shift assay plus 2 μl of EGR-1 antibody (sc-110X from Santa Cruz Biotechnology, Inc.) or preimmune antibody. The reaction mixtures were preincubated for 20 min on ice, then incubated with the probe for 40 min, and subjected to electrophoresis on polyacrylamide gels to separate the bound complexes from the free probe. To examine ectopically overexpressed EGR-1 as a cause or enhancer of apoptosis, human A375-C6 cells were stably transfected with pCMV-mEGR1, an expression construct for mouse EGR-1 (mEGR1), or with vector for a control. For each of the two constructs, we examined three transfected cell lines for EGR-1 expression by Western blot analysis. Data representative of the transfected cell lines shown in Fig.1 A confirmed overexpression of EGR-1 in the pCMV-mEGR1-transfected cell line as compared with the vector-transfected cell line. These transfected cell lines we" @default.
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• W2090585013 title "Early Growth Response-1-dependent Apoptosis Is Mediated by p53" @default.
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