ATF3 Represses 72-kDa Type IV Collagenase (MMP-2) Expression by Antagonizing p53-dependent trans-Activation of the Collagenase Promoter

The murine homologue of the ATF3 transcription factor increases tumor metastases but, surprisingly, represses 72-kDa type IV metalloproteinase (MMP-2) expression. The current study describes a novel mechanism by which ATF3 regulates transcription. Progressive deletions of the MMP-2 promoter indicated a 38-base pair region (−1659/−1622) necessary for the ATF3-mediated repression. This region lacked CREB/AP-1 motifs but contained a consensus p53 motif shown previously to regulate MMP-2 expression. The activity of a p53 response element-driven luciferase reporter was reduced in ATF3-expressing HT1080 clones. Although MMP-2 promoter activity was not repressed by ATF3 in p53-deficient Saos-2 cells, p53 re-expression increased MMP-2 promoter activity and restored the sensitivity to ATF3. The activity of a GAL4-driven reporter in HT1080 cells co-expressing the full-length p53 sequence fused to the GAL4 DNA binding domain was diminished by ATF3. p53-ATF3 protein-protein interactions were demonstrated both in vivo and in vitro. Cell cycle analysis, performed as an independent assay of p53 function, revealed that γ-irradiation-induced slowed G2/M cell cycle progression (attributable to p53) was countered by ATF3. Thus, ATF3 represses MMP-2 expression by decreasing the trans-activation of this gene by p53. The murine homologue of the ATF3 transcription factor increases tumor metastases but, surprisingly, represses 72-kDa type IV metalloproteinase (MMP-2) expression. The current study describes a novel mechanism by which ATF3 regulates transcription. Progressive deletions of the MMP-2 promoter indicated a 38-base pair region (−1659/−1622) necessary for the ATF3-mediated repression. This region lacked CREB/AP-1 motifs but contained a consensus p53 motif shown previously to regulate MMP-2 expression. The activity of a p53 response element-driven luciferase reporter was reduced in ATF3-expressing HT1080 clones. Although MMP-2 promoter activity was not repressed by ATF3 in p53-deficient Saos-2 cells, p53 re-expression increased MMP-2 promoter activity and restored the sensitivity to ATF3. The activity of a GAL4-driven reporter in HT1080 cells co-expressing the full-length p53 sequence fused to the GAL4 DNA binding domain was diminished by ATF3. p53-ATF3 protein-protein interactions were demonstrated both in vivo and in vitro. Cell cycle analysis, performed as an independent assay of p53 function, revealed that γ-irradiation-induced slowed G2/M cell cycle progression (attributable to p53) was countered by ATF3. Thus, ATF3 represses MMP-2 expression by decreasing the trans-activation of this gene by p53. ATF3 (the human homologue of Ti241) is a member of the ATF/CREB subfamily of bZIP transcription factors (1.Liang G. Wolfgang C.D. Chen B.P.C. Chen T.-H. Hai T. J. Biol. Chem. 1996; 271: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and encoded by a four-exon gene spanning 15 kb. Transcription of this gene yields a 2-kb mRNA (1.Liang G. Wolfgang C.D. Chen B.P.C. Chen T.-H. Hai T. J. Biol. Chem. 1996; 271: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), or an alternatively spliced isoform ATF3ΔZip (2.Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 279: 15819-15826Abstract Full Text PDF Google Scholar), the former transcript encoding the full-length protein product (∼ 22 kDa) (2.Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 279: 15819-15826Abstract Full Text PDF Google Scholar). ATF3 homo- and heterodimers bind specifically to the ATF/CREB and AP-1 motifs (3.Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1114) Google Scholar, 4.Wolfgang C.D. Chen B.P.C. Martindale J.L. Holbrook N.J. Hai T. Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (143) Google Scholar) and regulate the expression of several genes (2.Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 279: 15819-15826Abstract Full Text PDF Google Scholar,4.Wolfgang C.D. Chen B.P.C. Martindale J.L. Holbrook N.J. Hai T. Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (143) Google Scholar, 5.Periz S. Vial E. van Damm H. Castallazzi M. Oncogene. 2001; 20: 1135-1141Crossref PubMed Scopus (53) Google Scholar, 6.Nawa T. Nawa M.T. Cai Y. Zhang C. Uchimura I. Narumi S. Numano F. Kitajima S. Biochem. Biophys. Res. Commun. 2000; 275: 406-411Crossref PubMed Scopus (57) Google Scholar) as well as gluconeogenic enzymes in transgenic mice as shown recently (7.Allen-Jennings A. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In a previous study comparing gene expression in metastatic and non-metastatic tumors, Ishiguro and co-workers (8.Ishiguro T. Nakajima M. Naito M. Muto T. Tsuruo T. Cancer Res. 1996; 56: 875-879PubMed Google Scholar) identified the murine homologue of ATF3 (Ti241) as overexpressed in metastatic melanoma and extended these studies to show a causal role for this gene in tumor dissemination. However, these authors did not determine the mechanism by which Ti241 induced this behavior. Considering the established role of type IV metalloproteinases in tumor metastases (9.Watson S.A. Morris T.M. Robinson G. Crimmin M.J. Brown P.D. Hardcastle J.D. Cancer Res. 1995; 55: 3629-3633PubMed Google Scholar, 10.Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Nature. 2000; 406: 532-535Crossref PubMed Scopus (1308) Google Scholar, 11.Kupferman M.E. Fini M.E. Muller W.J. Weber R. Cheng Y. Muschel R.J. Am. J. Pathol. 2000; 157: 1777-1783Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12.Bernhard E.J. Gruber S.B. Muschel R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4293-4297Crossref PubMed Scopus (389) Google Scholar, 13.Hua J. Muschel R.J. Cancer Res. 1996; 56: 5279-5284PubMed Google Scholar), we were interested in identifying which, if any, type IV collagenase(s) are regulated by ATF3 (the human homologue of Ti241). We report herein the unexpected finding that the 72-kDa type IV collagenase gene (MMP-2) 1The abbreviations used are: MMP-272-kDa Type IV collagenaseCMVcytomegalovirusDTTdithiothreitolEMSAelectrophoretic mobility shift assayGSTglutathione S-transferasePBSphosphate-buffered salinewtwild typeIRionizing radiationRLUrelative light unitsGAPDHglyceraldehyde-3-phosphate dehydrogenase is transcriptionally down-regulated by this transcription factor. More importantly, this transcriptional repression is achieved by ATF3 interfering with p53-dependent trans-activation of MMP-2 gene expression via a mechanism in which p53 transcriptional activity, but not DNA binding, is attenuated. Thus, our findings reveal a novel transcriptional mechanism in which ATF3 interferes with p53-dependent gene expression. 72-kDa Type IV collagenase cytomegalovirus dithiothreitol electrophoretic mobility shift assay glutathione S-transferase phosphate-buffered saline wild type ionizing radiation relative light units glyceraldehyde-3-phosphate dehydrogenase pGL2 reporters containing a luciferase reporter driven by full-length or 5′-deleted MMP-2 promoter fragments were as described elsewhere (14.Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar, 15.Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). p53-luc and pFR-luc were purchased from Stratagene (La Jolla, CA). p53-luc contains 15 tandem repeats of the p53 response element flanking a luciferase reporter whereas the pFR-luc comprises the luciferase coding sequence downstream of five tandem-repeated GAL4 binding sites. The plasmid encoding the chimeric GAL4 -p53 fusion protein has been described previously (16.Horikoshi M. Usheva A. Chen J. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Crossref PubMed Scopus (162) Google Scholar). The rabbit anti-human ATF3 antibody and mouse monoclonal antibody to human p53 (DO-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to human MMP-2 was supplied by Chemicon (Tamecula, CA). The pRc-p53 expression plasmid bearing the human p53 cDNA or its control vector (pRc-CMV) (17.Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (161) Google Scholar) has been described previously. Human fibrosarcoma HT1080 cells contain the wild-type p53 gene and were routinely cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and antibiotics. To obtain ATF3-overexpressing clones, pCG/ATF3, an expression plasmid encoding the full-length human ATF3 cDNA (18.Wolfgang C.D. Liang G. Okamoto Y. Allen A.E. Hai T. J. Biol. Chem. 2000; 275: 16865-16870Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) (kindly provided by Dr. T. Hai, The Ohio State University, Columbia, OH), was transfected into HT1080 cells using poly-l-ornithine as described previously (19.Nead M.A. McCance D.J. J. Invest. Dermatol. 1995; 105: 668-671Abstract Full Text PDF PubMed Scopus (31) Google Scholar). The transfectants were selected with 600 μg/ml G418, and resistant clones were isolated, expanded, and screened for ATF3 gene expression. Human osteosarcoma p53-deficient Saos-2 cells, or a derivative Saos-2/p53 made to express wild type p53 (20.Radinsky R. Fidler I.J. Price J.E. R. C. M. Oncogene. 1994; Google Scholar), were cultured in medium supplemented with were performed using the to the were and and luciferase activity was using the as of reporter was to for in indicated with the the full-length promoter a plasmid encoding wild type human p53 cDNA (17.Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (161) Google Scholar) or its control vector (pRc-CMV) were transfected into cells using the as by the from using was in a and to by using The was with to ATF3 and were performed with were by the with glyceraldehyde-3-phosphate dehydrogenase were as described by this elsewhere C. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). from cells were and in the of in transcription 10% was from with with and and using of was to to MMP-2 and glyceraldehyde-3-phosphate were performed as described by previously C. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). medium was in a containing The was for with in a containing and The was with and a light was as a in a was performed as described previously by C. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of and were as recently described by this C. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cell were by with of and of protein The were with of rabbit antibody were with of protein in a 15 and and by in p53 protein in the was by ATF3 protein was by the with a plasmid provided by Dr. T. Hai C.D. Chen B.P.C. Martindale J.L. Holbrook N.J. Hai T. Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (143) Google Scholar). To the was in the as by the was as we described previously C. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). were to the described by and co-workers (16.Horikoshi M. Usheva A. Chen J. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Crossref PubMed Scopus (162) Google Scholar) but with and fusion (16.Horikoshi M. Usheva A. Chen J. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Crossref PubMed Scopus (162) Google Scholar, J. M. R. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (95) Google Scholar) were in cells by with for cells were by and the was in and a the cell were for with The with or fusion were with bovine serum in the and ATF3 protein for with containing protein was from the by in in a and by were with using were with and in were using in containing μg/ml and μg/ml and for Cell cycle was performed a using CA). the previous study by Ishiguro (8.Ishiguro T. Nakajima M. Naito M. Muto T. Tsuruo T. Cancer Res. 1996; 56: 875-879PubMed Google Scholar) demonstrated the of Ti241 (the murine homologue of to tumor and the of type IV as of tumor we were interested in if ATF3 regulated expression of these this we transfected HT1080 cells with an expression encoding the full-length ATF3 ATF3 protein and mRNA in HT1080 cells bearing the vector were the of and In in independent clones transfected with the ATF3 expression the ATF3 transcript and protein were with a of the protein in the To determine the ATF3 regulated type IV collagenase medium from the vector and the ATF3 clones was and by that was in from the 72-kDa type IV collagenase (MMP-2) was in medium from the clones the vector In the of this was reduced in clones and shown to for ATF3 expression. The in MMP-2 the ATF3-expressing clones The were by in that reduced of this type IV collagenase were in clones transfected with ATF3 The MMP-2 in the medium was a of diminished transcription as by and and of the indicated a in the of MMP-2 mRNA in the ATF3-expressing clones. To the that reduced MMP-2 transcription achieved with ATF3 expression was a we HT1080 cells with a luciferase reporter regulated by the promoter of a collagenase gene type IV collagenase and of the ATF3 expression The ATF3 expression an in reporter activity in to the repression with the luciferase reporter the that the of ATF3 MMP-2 transcription is a To the role of ATF3 in MMP-2 we the of to ATF3 expression J. I. R. M. Oncogene. 2000; PubMed Scopus Google Scholar), the expression of this of HT1080 a of ATF3 protein as in The in ATF3 mRNA the in MMP-2 mRNA as shown in Thus, ATF3 transcript as as MMP-2 to using a MMP-2 luciferase reporter a in MMP-2 promoter activity in HT1080 cells the that ATF3 expression MMP-2 transcription. of the MMP-2 promoter (14.Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar, 15.Qin H. Sun Y. Benveniste E.N. J. Biol. Chem. 1999; 274: 29130-29137Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) revealed and AP-1 motifs and which bind ATF3 and its expression of this Thus, we the that the repression of MMP-2 expression was achieved these binding sites. this we ATF3 bind to these ATF3 was in and with an spanning the or AP-1 motifs in the MMP-2 a in ATF3 was with a consensus motif (Santa Cruz Santa Cruz, CA). the in ATF3 the consensus motif and was with an antibody In this transcription factor was not by spanning the MMP-2 or AP-1 motifs and from the ATF3-expressing HT1080 clones to show with these not the of ATF3 to bind to the and AP-1 the of flanking in the MMP-2 was that ATF3 was MMP-2 expression via its binding we a using MMP-2 promoter fragments to the of the this HT1080 cells the ATF3 expression or the vector were transfected with a luciferase reporter regulated by deletions of the MMP-2 promoter of luciferase activity was with the reporter regulated by of sequence However, of from the of the promoter reduced the activity of the reporter in the vector and the repression in the ATF3-expressing ATF3 luciferase reporter activity using MMP-2 promoter fragments from the that the of ATF3 to the activity of the MMP-2 promoter fragments and to a activity, these reporter relative light units as with a that repression of MMP-2 transcription by ATF3 is a promoter sequence and this region is of or AP-1 motifs S. Vial E. van Damm H. Castallazzi M. Oncogene. 2001; 20: 1135-1141Crossref PubMed Scopus (53) Google Scholar, J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar), contains a consensus p53 binding previously shown to MMP-2 expression in HT1080 cells (14.Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar). We were by the that ATF3 regulates MMP-2 expression by of interfering with the trans-activation of MMP-2 promoter by p53. HT1080 cells express wild type p53 A. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. M. A. A. G. Oncogene. 2001; 20: PubMed Scopus Google Scholar), which to its motif in the MMP-2 promoter (14.Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar), and our were with these previous To the that ATF3 down-regulated p53-dependent gene a luciferase reporter driven by 15 tandem repeats of the p53 response element was transfected into HT1080 cells the ATF3 expression or the vector were by with a luciferase the activity of the reporter driven by the tandem p53 repeats was down-regulated in the ATF3-expressing clones with HT1080 cells bearing the vector In the activity of a reporter was by ATF3 expression ATF3 represses MMP-2 expression by of trans-activation of the gene by that this activity in p53-deficient this Saos-2 cells, which p53 protein Y. J. PubMed Scopus (53) Google Scholar), were with the MMP-2 luciferase reporter and an expression vector bearing the coding ATF3 sequence The expression of the MMP-2 promoter in the p53-deficient cells was in the HT1080 cells, which the p53 More importantly, the expression of the ATF3 plasmid MMP-2 promoter activity in the Saos-2 cells that this of repression in Saos-2 cells not the of the luciferase In Saos-2 cells made to express p53 (20.Radinsky R. Fidler I.J. Price J.E. R. C. M. Oncogene. 1994; Google Scholar), demonstrated increased MMP-2 promoter activity with and repression of the MMP-2 these that ATF3 expression interferes with p53-dependent transcriptional of the MMP-2 ATF3 with trans-activation of gene expression by We the that ATF3 or interferes with DNA binding of p53. Hai and co-workers T. Wolfgang C.D. Allen A.E. U. 2001; Scholar) that is by ATF3. To this were for ATF3-expressing clones and clones bearing the plasmid and for p53 ATF3 did not the of p53 the that ATF3 is p53-dependent gene expression by p53 is that the DNA binding of p53 is reduced in response to ATF3 expression. To this was performed using from HT1080 clones the ATF3 or the vector and an spanning the p53 motif in the MMP-2 by an of a p53 consensus sequence not by a p53 was using from clones bearing the However, in the of this was the ATF3-expressing clones and clones bearing the vector that ATF3 not with of p53 or the binding of this transcription factor to its motif in the MMP-2 we that ATF3 was p53-dependent gene expression by of interfering with the DNA binding of the p53 transcription we the that the of p53 to an ATF3 To this cells were with an expression bearing the full-length p53 protein fused to the GAL4 DNA binding domain (16.Horikoshi M. Usheva A. Chen J. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Crossref PubMed Scopus (162) Google Scholar) and a luciferase reporter regulated by tandem GAL4 DNA binding sites. the activity of the luciferase reporter was reduced in the ATF3-expressing clones with the vector ATF3. To these the was using HT1080 cells with to ATF3 expression. trans-activation of the GAL4 promoter by the chimeric protein was reduced in cells induced for ATF3 expression. these that ATF3 represses the activity of p53. that ATF3 represses MMP-2 expression by of the activity of p53. This a of the To this independent were In the we p53 and ATF3 in HT1080 cells were transfected with a p53 expression and the of the was by for p53 The p53 protein has a with the We if p53 was with ATF3 in these were were or an and the was to using the p53 protein was in from the ATF3-expressing with the antibody p53 protein was not by if the antibody was p53 was in the from the of The from the To the that p53 with were In ATF3 was with fusion the was for ATF3 protein by the fusion The of ATF3 in the protein of the ATF3 was by the full-length in to that of M. J. Levine A.J. 1999; PubMed Scopus Google indicated interactions the binding was with p53 spanning or In the p53 with ATF3 the of and The full-length p53 protein binding of ATF3 the of p53 for for the ATF3 these in and in vivo the that ATF3 and p53 our and to of p53-dependent gene by ATF3. To these we a p53 in response to ionizing radiation F. A. C. T. S. Brown PubMed Scopus Google Scholar, C.D. J.A. Mol. Cell. Biol. 2000; Scopus Google Scholar), we if p53 an ATF3 this cell cycle in the ATF3-expressing this HT1080 cells ATF3 or bearing the vector were to of ionizing radiation the cells were and to cell cycle of cells of both clones in G2/M to ionizing radiation with of cells from progression of G2/M was slowed in HT1080 cells bearing the and this was to radiation In the ATF3-expressing G2/M with of the cells this Thus, these the that ATF3 interferes with p53-dependent gene We report herein that ATF3 represses 72-kDa type IV collagenase (MMP-2) expression and that this is achieved by with the p53-dependent trans-activation of this To our this is the report to gene expression motifs the CREB/AP-1 binding ATF3 regulates the expression of several gluconeogenic and C.D. Chen B.P.C. Martindale J.L. Holbrook N.J. Hai T. Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (143) Google Scholar, 5.Periz S. Vial E. van Damm H. Castallazzi M. 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Biochem. 2000; PubMed Google Scholar), which a of a MMP-2 mRNA these is that diminished MMP-2 transcription by ATF3 the CREB/AP-1 motifs in the MMP-2 promoter for repression by ATF3 was not with 5′-deleted MMP-2 promoter containing the and AP-1 motifs the p53 we were to binding of ATF3 to the CREB/AP-1 motifs in the MMP-2 a of the that the of ATF3 with p53-dependent trans-activation of the MMP-2 of the p53 binding in the MMP-2 promoter the repression achieved with ATF3. diminished MMP-2 expression by ATF3 the p53 protein was not in p53-deficient Saos-2 ATF3 and p53 both in vivo and in vitro. trans-activation of a p53 response element-driven reporter is reduced in the ATF3-expressing clones. ATF3 as a protein to a of C. 1996; PubMed Scopus Google Scholar) and R. S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). More importantly, of p53 with transcription factors has been previously S. C. G. 1995; PubMed Scopus Google Scholar, F. J. Biol. Chem. 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However, this was not the as revealed by the p53 sequence of p53 was for the with the ATF3 Thus, that activity of p53 is interactions of ATF3 with of the former a has in that p53 represses the activity of the trans-activation domain J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of p53 its DNA binding Cell Biol. 2001; PubMed Scopus Google Scholar) the C. 1996; PubMed Scopus Google of the p53 DNA binding protein-protein interactions in repression of MMP-2 gene The that p53 protein T. T. E. M. G. M. Y. J. 1999; PubMed Scopus Google Scholar, M. A. S. J. G. J. Cell Sci. 2000; PubMed Google Scholar) is the ATF3-expressing clones contained of the p53 protein to the vector p53-ATF3 protein interactions in p53 or C. J. Pathol. 1999; PubMed Scopus Google Scholar, Chen J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google J. 1999; PubMed Scopus Google Scholar) reduced transcriptional activity M.G. S. T.R. J. Biol. 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Cancer Res. 1996; 56: 875-879PubMed Google Scholar) the in which Ti241 was in that the repression of MMP-2 expression in the HT1080 cells or is to as 2000; PubMed Scopus Google Scholar). Thus, in we demonstrated a novel transcriptional mechanism in which ATF3 represses MMP-2 expression by interfering with p53-dependent trans-activation of this collagenase the of ATF3 gene expression a reduced activity of p53 as to diminished DNA binding of the transcription factor. We Dr. Hai State University, for the ATF3 cDNA and in we express our to the for Dr. Shenk University, and fusion Dr. Benveniste of of MMP-2 Dr. Sun the full-length MMP-2 promoter We express our to Dr. for the Saos-2 cells transfected with p53 and Dr. Mukhopadhyay Cancer for the pRc-p53 and The provided the Dr. of fusion Dr. S. University, Dr. Cancer and Dr. A. of for the p53 fusion