The Transcription Factors Sp1, Sp3, and AP-2 Are Required for Constitutive Matrix Metalloproteinase-2 Gene Expression in Astroglioma Cells
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.29130
ISSN1083-351X
AutoresHongwei Qin, Yi Sun, Etty Benveniste,
Tópico(s)Cell Adhesion Molecules Research
ResumoMatrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that contribute to pathological conditions associated with angiogenesis and tumor invasion. MMP-2 is highly expressed in human astroglioma cells, and contributes to the invasiveness of these cells. The human MMP-2 promoter contains potential cis-acting regulatory elements including cAMP response element-binding protein, AP-1, AP-2, PEA3, C/EBP, and Sp1. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrates that the Sp1 site at −91 to −84 base pairs and the AP-2 site at −61 to −53 base pairs are critical for constitutive activity of this gene in invasive astroglioma cell lines. Electrophoretic gel shift analysis demonstrates binding of specific DNA-protein complexes to the Sp1 and AP-2 sites: Sp1 and Sp3 bind to the Sp1 site, while the AP-2 transcription factor binds the AP-2 element. Co-transfection expression experiments inDrosophilia SL2 cells lacking endogenous Sp factors demonstrate that Sp1 and Sp3 function as activators of the MMP-2 promoter and synergize for enhanced MMP-2 activation. Overexpression of AP-2 in AP-2-deficient HepG2 cells enhances MMP-2 promoter activation. These findings document the functional importance of Sp1, Sp3, and AP-2 in regulating constitutive expression of MMP-2. Delineation of MMP-2 regulation may have implications for development of new therapeutic strategies to arrest glioma invasion. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that contribute to pathological conditions associated with angiogenesis and tumor invasion. MMP-2 is highly expressed in human astroglioma cells, and contributes to the invasiveness of these cells. The human MMP-2 promoter contains potential cis-acting regulatory elements including cAMP response element-binding protein, AP-1, AP-2, PEA3, C/EBP, and Sp1. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrates that the Sp1 site at −91 to −84 base pairs and the AP-2 site at −61 to −53 base pairs are critical for constitutive activity of this gene in invasive astroglioma cell lines. Electrophoretic gel shift analysis demonstrates binding of specific DNA-protein complexes to the Sp1 and AP-2 sites: Sp1 and Sp3 bind to the Sp1 site, while the AP-2 transcription factor binds the AP-2 element. Co-transfection expression experiments inDrosophilia SL2 cells lacking endogenous Sp factors demonstrate that Sp1 and Sp3 function as activators of the MMP-2 promoter and synergize for enhanced MMP-2 activation. Overexpression of AP-2 in AP-2-deficient HepG2 cells enhances MMP-2 promoter activation. These findings document the functional importance of Sp1, Sp3, and AP-2 in regulating constitutive expression of MMP-2. 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Kuwano M. Clin. Exp. Metastasis. 1994; 12: 296-304Crossref PubMed Scopus (79) Google Scholar, 12Yamamoto M. Mohanam S. Sawaya R. Fuller G.N. Seiki M. Sato H. Gokaslan Z.L. Liotta L.A. Nicolson G.L. Rao J.S. Cancer Res. 1996; 56: 384-392PubMed Google Scholar, 14Uhm J.H. Dooley N.P. Villemure J.-G. Yong V.W. Clin. & Exp. Metastasis. 1996; 14: 421-433Crossref PubMed Scopus (128) Google Scholar, 15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar, 20Chintala S.K. Sawaya R. Gokaslan Z.L. Rao J.S. Cancer Lett. 1996; 103: 201-208Crossref PubMed Scopus (68) Google Scholar). A number of strategies have been utilized to modulate MMP-2 expression/activity, then assess subsequent changes in invasive potential. The MMP-2 proenzyme is activated by cell surface-associated membrane-type metalloproteinases (21Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2373) Google Scholar, 22Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). Transfection of U251.3 glioma cells with membrane-type metalloproteinases-1 leads to activation of the MMP-2 proenzyme and enhanced invasion as assessed by Matrigel assay (23Deryugina E.I. Luo G.-X. Reisfeld R.A. Bourdon M.A. Strongin A. Anticancer Res. 1997; 17: 3201-3210PubMed Google Scholar), as well as remodeling of the extracellular matrix in vitro (24Deryugina E. Bourdon M. Reisfeld R. Strongin A. Cancer Res. 1998; 58: 3743-3750PubMed Google Scholar). We have recently demonstrated that two cytokines, tumor necrosis factor-α and interferon-γ, inhibit MMP-2 expression in glioma cells, which results in decreased invasiveness of these cells (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). Collectively, these results highlight the important role of MMP-2 in the invasive potential of astroglioma cells. The activity of MMP-2 is regulated by several mechanisms, including gene expression, proenzyme activation by tissue inhibitor of metalloproteinase-2 and membrane-type metalloproteinases, and inhibition of enzyme activity by naturally occurring tissue inhibitor of metalloproteinases (for review, see Refs. 25Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar, 26Ries C. Petrides P.E. Biol. Chem. Hoppe-Seyler. 1995; 376: 345-355PubMed Google Scholar, 27Yu A.E. Hewitt R.E. Kleiner D.E. Stetler-Stevenson W.G. Biochem. Cell Biol. 1996; 74: 823-831Crossref PubMed Scopus (97) Google Scholar). Historically, the MMP-2 gene has been considered refractory to modulation, either inhibition or enhancement, due to a lack of well characterized regulatory elements in the MMP-2 promoter (for review, see Ref. 25Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar). However, sequence analysis of the MMP-2 promoter has revealed a number of potential cis-acting regulatory elements including p53, AP-1, Ets-1, C/EBP, CREB, PEA3, Sp1, and AP-2 that could be involved in regulation of MMP-2 expression (see Fig. 2). The human MMP-2 promoter lacks a typical TATA box but has a relatively GC-rich region in the proximal region. Limited functional analysis of the MMP-2 promoter has been performed. An enhancer sequence of 42 bp located at −1635 bp relative to the transcription initiation start site in the human MMP-2 promoter has been identified in HT1080 cells (human fibrosarcoma line) (28Frisch S.M. Morisaki J.H. Mol. Cell. Biol. 1990; 10: 6524-6532Crossref PubMed Scopus (109) Google Scholar). This enhancer region-2 (r2) was identified as an AP-2 binding sequence, allowing for several mismatches and gaps (28Frisch S.M. Morisaki J.H. Mol. Cell. Biol. 1990; 10: 6524-6532Crossref PubMed Scopus (109) Google Scholar). It has been suggested that AP-2 is an important transcription factor for activation of the MMP-2 promoter, and that adenovirus EIA represses MMP-2 gene expression by targeting AP-2 (29Somasundaram K. Jayaraman G. Williams T. Moran E. Frisch S. Thimmapaya B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3088-3093Crossref PubMed Scopus (69) Google Scholar). A silencer sequence at −1629 bp has also been functionally documented (28Frisch S.M. Morisaki J.H. Mol. Cell. Biol. 1990; 10: 6524-6532Crossref PubMed Scopus (109) Google Scholar). Recently, a p53-binding site at −1649 to −1630 bp (20 bp) has been identified which specifically binds the p53 protein and plays an important role in constitutive MMP-2 gene expression in human sarcoma cell lines (30Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar). Interestingly, the p53-binding site is localized within the enhancer region-2 (r2); however, Bian and Sun (30Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar) did not detect AP-2 binding in this region, only that of p53. No information is available regarding any other potential functional cis-acting elements in the MMP-2 promoter. The constitutive expression and regulation of the MMP-2 gene is cell- and stimulus-specific (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar, 30Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar, 31Harendza S. Pollock A.S. Mertens P.R. Lovett D.H. J. Biol. Chem. 1995; 270: 18786-18796Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Thus far, the molecular basis of MMP-2 gene expression in human astroglioma cells has not been addressed. In order to determine the sequence requirements for MMP-2 gene transcription, we utilized 5′-deletional MMP-2 reporter constructs in transcriptional activation studies. This approach, in conjunction with site-directed mutagenesis of the human MMP-2 promoter region, demonstrated that a Sp1 site spanning −91 to −84 bp and an AP-2 element at −61 to −53 bp are critical for constitutive expression of the MMP-2 gene in astroglioma cells. The p53/r2 elements identified as important for MMP-2 promoter activity in other cells are non-functional in astroglioma cells. We further defined DNA-protein complexes involved in MMP-2 transcription by electrophoretic mobility shift assays (EMSA). The transcription factors Sp1 and Sp3 both bind to the Sp1 element, while AP-2 binds to the AP-2 element. Furthermore, co-transfection experiments into mammalian cells lacking AP-2 and insect cells that lack Sp factors demonstrate that Sp1, Sp3, and AP-2 are all functionally important for constitutive expression of the MMP-2 gene. The U251-MG, U373-MG, and CH235-MG astroglioma cell lines were grown in Dulbecco's modified Eagle's/Ham's F-12 medium supplemented with 10 mm Hepes, 2 mml-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin, and 10% fetal bovine serum (FBS) as described previously (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar, 32Winkler M. Benveniste E.N. GLIA. 1998; 22: 171-179Crossref PubMed Scopus (42) Google Scholar). HeLa and HepG2 cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10 mm Hepes, 2 mml-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin, and 10% FBS as described previously (33Lee Y.-J. Han Y. Lu H.-T. Nguyen V. Qin H. Howe P.H. Hocevar B.A. Boss J.M. Ransohoff R.M. Benveniste E.N. J. Immunol. 1997; 158: 2065-2075PubMed Google Scholar, 34Wang D. Shin T.H. Kudlow J.E. J. Biol. Chem. 1997; 272: 14244-14250Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Schneider Drosophilia 2 (SL2) cells were grown in Schneider's medium (Sigma) supplemented with 100 units/ml penicillin, 10 μg/ml streptomycin, and 10% FBS at 25 °C. Polyclonal antisera against Sp1, Sp3, and AP-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Gelatin was purchased from Sigma. Mouse anti-human MMP-2 monoclonal antibody was the generous gift of Dr. J. Engler, The University of Alabama at Birmingham. The secondary peroxidase-conjugated antibodies and ECL reagents were from Amersham Pharmacia Biotech. Zymography was performed as described previously (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). In brief, astroglioma cell lines cells were incubated until ∼80% confluent, then the medium was aspirated and fresh serum-free medium added to the dish. Supernatants were collected after a 48-h incubation and concentrated. Concentrated supernatants (750 μl) were mixed with SDS sample buffer without reducing agent, and proteins subjected to SDS-polyacrylamide gel electrophoresis in 8% polyacrylamide gels that were copolymerized with 1 mg/ml gelatin. After electrophoresis, the gels were washed several times in 2.5% Triton X-100 for 1 h at room temperature to remove the SDS, and then incubated for 24–48 h at 37 °C in buffer containing 5 mm CaCl2 and 1 μmZnCl2. The gels were stained with Coomassie Blue (0.25%) for 30 min, then destained for 1 h in a solution of acetic acid and methanol. The proteolytic activity was evidenced as clear bands (zones of gelatin degradation) against the blue background of stained gelatin. The same supernatants were used in immunoblot analysis for MMP-2 protein. Proteins were detected using anti-MMP-2 antibody (5 μg/ml) as described previously (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). Blots were washed four times in TBS with 0.01% Tween 20, and subsequently incubated in sheep anti-mouse peroxidase-conjugated antibody (1:3000) in antibody dilution buffer. ECL reagents were used for development. Total RNA was isolated from confluent monolayers of astroglioma cell lines that had been incubated in serum-free medium for 24 h (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). Human MMP-2 cDNA (gift of Dr. W. G. Stetler-Stevenson, National Cancer Institute, Bethesda, MD) was digested with SacI/PstI, and a 324-bp fragment corresponding to 1500–1824 nucleotides was subcloned into the SacI/PstI polylinker site of the pGEM4Z vector (Promega, Madison, WI). The construct was linearized byEcoRI and used to generate a radiolabeled antisense RNA probe of 354 nucleotides with T7 RNA polymerase. A pAMP-1 vector containing a fragment of the human glyceraldehyde-3-phosphate dehydrogenase cDNA (corresponding to 43–531 nucleotides) was linearized with NcoI, and used to generate a radiolabeled antisense RNA probe of 290 nucleotides with T7 polymerase. Fifteen μg of total RNA was hybridized with MMP-2 (50 × 103 cpm) and glyceraldehyde-3-phosphate dehydrogenase (25 × 103 cpm) riboprobes at 42 °C overnight. The hybridized mixture was then treated with RNase A/T1 (1:200) at room temperature for 1 h, analyzed by 5% denaturing (8 m urea) polyacrylamide gel electrophoresis, and the gels were exposed to x-ray film. The protected fragments of the MMP-2 and glyceraldehyde-3-phosphate dehydrogenase riboprobes are 324 and 230 bp in length. A luciferase reporter plasmid driven by 1659 bp of the human MMP-2 promoter was used in this study, and is referred to as wild type (WT) (30Bian J. Sun Y. Mol. Cell. Biol. 1997; 17: 6330-6338Crossref PubMed Scopus (249) Google Scholar). Serial deletion mutants were synthesized using polymerase chain reaction byPfu DNA polymerase to delete out potential regulatory elements in the MMP-2 promoter, including one p53 site, two silencers, one AP-1 element, four PEA3 elements, two Sp1 elements, and one AP-2 site (see Fig. 2). They are named as follows: D1, which lacks the p53 site; D2, lacking the first silencer element (S1); D3, lacking both silencer elements (S1 and S2); D4, which lacks the AP-1 element; D5, lacking the Ets-1 and c-Myc/c-Myb sites; D6, lacking one PEA3 site and the C/EBP element; D7, lacking the second PEA3 site; D8, which lacks the third PEA3 site, the CREB site, and GCN-His region; D9, lacking the fourth PEA3 site; D10, lacking the first Sp1 element; D11, lacking both Sp1 elements; and D12, which lacks the AP-2 site (see Fig. 3,A and B). The polymerase chain reaction amplification protocol consisted of an initial 1-min melting step at 94 °C, followed by 30 cycles with 40 s melting at 92 °C, 40 s annealing at 60 °C, and 1 min 30 s extension at 75 °C, except for the last cycle which contained a 5-min extension step. The restriction enzyme EcoRI was used to check positive clones and AvaI to verify the correct orientation. The deletion mutants were inserted into the pGL2-Basic vector, which contains the gene for luciferase as reporter, using theKpnI/XhoI restriction site. Site-directed mutagenesis was utilized to mutate potential transcription elements in the proximal sequence (−139 to −7) of the human MMP-2 promoter. 15-bp lengths of DNA in the D3 and D9 reporter constructs were serially mutated to the EcoRV andXbaI restriction enzyme sequence by polymerase chain reaction using QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) (see Fig. 4). The introduction of mutations was verified by the restriction enzymes EcoRV andXbaI and DNA sequencing. After 18 cycles of polymerase chain reaction by Pfu polymerase at 95 °C for 30 s, 55 °C for 1 min, and 68 °C for 20 min, DpnI was used to digest the parental supercoiled double-stranded DNA. Transformations were performed in Escherichia coli supercompetent cells using DpnI-treated DNA. A total of three site-directed mutants were generated using the D3 and D9 promoter constructs as templates (Fig. 4), and were confirmed by sequencing. They are mSp1A, mSp1B, and mAP-2. As well, constructs containing mutations in both the Sp1A and AP-2 elements were created. Ten μg of the MMP-2 promoter constructs (both MMP-2 deletion and mutation constructs) were co-transfected into human astroglioma cells with 1 μg of the pCMV-β-galactosidase construct into 3 × 106 cells by electroporation with a Bio-Rad Gene Pulser set at 250 V, 960 microfarads, as described previously (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). After transfection, cells were allowed to recover for 18 h and cultured in 1% FBS/Dulbecco's modified Eagle's/F-12 medium for 24 h. Cells were washed with phosphate-buffered saline and lysed with 180 μl of lysis buffer containing 25 mm trisphosphate (pH 7.8), 2 mm DTT, 2 mm diaminocyclohexane tetraacetic acid, 10% glycerol, and 1% Triton X-100. Extracts were assayed in triplicate for luciferase activity in a volume of 130 μl containing 30 μl of cell extract, 20 mm Tricine, 0.1 mmEDTA, 1 mm magnesium carbonate, 2.67 MgSO4, 33.3 mm DTT, 0.27 mm coenzyme A, 0.47 mm luciferin, and 0.53 mm ATP, and light intensity was measured using a luminometer (Promega, Madison, WI). Luciferase activity was integrated over a 10-s time period. Extracts were also assayed in triplicate for β-galactosidase enzyme activity as described previously (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). The luciferase activity of each sample was normalized to β-galactosidase activity to calculate relative luciferase activity (RLA) before calculating the fold activation value. For transfection with HepG2 cells, 3 × 106 cells were electroporated with 10 μg of the indicated MMP-2 luciferase promoter constructs, 1 μg of the pCMV-β-galactosidase construct, and 1.0 μg of either the pSX-AP-2 expression vector or pSX control vector (34Wang D. Shin T.H. Kudlow J.E. J. Biol. Chem. 1997; 272: 14244-14250Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). After transfection, cells were allowed to recover for 18 h, changed to serum-free medium for 24 h, and luciferase and β-galactosidase activities assayed as described above. For transfection of SL2 cells, 1 day prior to transfection, cells were plated onto 60-mm2 dishes at a density of 4 × 106 cells/plate. Cells were transfected by the calcium phosphate method as described previously (35Hagen G. Dennig J. Preiß A. Beato M. Suske G. J. Biol. Chem. 1995; 270: 24989-24994Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Each plate received up to 20 μg of DNA including 10 μg of the indicated MMP-2 promoter constructs, and variable amounts of the expression plasmids pPacSp1 and/or pPacUSp3 (36Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar). Variable amounts of the expression plasmids were adjusted with the control plasmid pPac. The medium was changed 24 h after addition of DNA, and 24 h later the cells were harvested for luciferase activity. Luciferase values were normalized against total protein concentrations determined by the Bio-Rad protein assay. Nuclear extracts were prepared as described previously (37Panek R.B. Lee Y.-J. Lindstrom-Itoh Y. Ting J.P.-Y. Benveniste E.N. J. Immunol. 1994; 153: 4555-4564PubMed Google Scholar). Cells were grown in 100-mm dishes, allowed to adhere overnight, and then were incubated in serum-free medium for 24 h. Cells were then washed with cold phosphate-buffered saline, harvested by scraping, and pelleted. Cells were resuspended in 1 ml of buffer A (10 mm KCl, 20 mm HEPES, 1 mmMgCl2, 1 mm DTT, 0.4 mmphenylmethylsulfonyl fluoride, 1 mm sodium fluoride, 1 mm Na3VO4), incubated on ice for 10 min, and pelleted at 1000 × g for 10 min. Pellets were resuspended in 0.5 ml of buffer A plus 0.1% Nonidet P-40, incubated on ice for 10 min, and centrifuged at 3,000 × g for 10 min. The nuclear pellet was resuspended in 1 ml of buffer B (10 mm HEPES, 400 mm NaCl, 0.1 mm EDTA, 1 mm MgCl2, 1 mm DTT, 0.4 mm phenylmethylsulfonyl fluoride, 15% glycerol, 1 mm sodium fluoride, 1 mmNa3VO4) and incubated for 30 min at 4 °C with constant gentle mixing. Nuclei were then pelleted at 40,000 × g for 30 min, and extracts were dialyzed for 2 h at 4 °C against 1 liter of buffer C (20 mm HEPES, 200 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 1 mm DTT, 0.4 mmphenylmethylsulfonyl fluoride, 15% glycerol, 1 mm sodium fluoride, 1 mm Na3VO4). Extracts were cleared by centrifugation at 14,000 × g for 15 min at 4 °C. Protein concentrations were determined using a Bio-Rad protein assay. EMSA was performed using the following oligonucleotides as probes and/or competitors: the oligonucleotide Sp1A has the sequence 5′-CAGAGAGGGGCGGGCCCGAGTG-3′, corresponding to the human MMP-2 promoter sequence −98 to −76, and the AP-2 oligonucleotide has the sequence 5′-CCCCAGCCCCGCTCTGCCAGCT-3′, and corresponds to the human MMP-2 promoter sequence −66 to −44. The mutant Sp1 oligonucleotide (mSp1A) has the sequence 5′-CAGAtAtctagatGatatcGTG-3′, and the mutant AP-2 oligonucleotide (mAP-2) is 5′-CCgatatCatctagaatCAGCT-3′. Mutations are indicated by lowercase letters. 0.2 ng of 32P-labeled oligonucleotide (20,000 cpm) were incubated for 30 min at room temperature with 10 μg of nuclear extract in a volume of 20 μl containing 50 mm KCl, 2.5 mm MgCl2,1 mm EDTA, 1 mm DTT, 10 mm Tris-Cl (pH 7.5), 10% glycerol, 1 μg of salmon sperm DNA, and 1 μg of poly(dI-dC). For supershift analysis, 1 μl of antibody was incubated with the nuclear extracts at 4 °C for 30 min in binding buffer, followed by an additional incubation for 30 min at room temperature with labeled oligonucleotide. For competitions, unlabeled DNA was incubated with the nuclear extracts at 4 °C for 20 min before addition of labeled probe. Bound and free DNA were resolved by electrophoresis through a 6% polyacrylamide gel at 250 V in 0.25 × TBE (50 mm Tris-Cl, 2 mm EDTA). Dried gels were exposed to Kodak XAR-5 film at −70 °C with intensifying screens. Four different preparations of nuclear extracts were tested by EMSA. Levels of significance for comparisons between samples were determined using Student's t test distribution. High levels of MMP-2 expression correlate with malignant progression of astrogliomasin vivo (11Nakano A. Tani E. Miyazaki K. Yamamoto Y. Furuyama J.-I. J. Neurosurg. 1995; 83: 298-307Crossref PubMed Scopus (224) Google Scholar, 12Yamamoto M. Mohanam S. Sawaya R. Fuller G.N. Seiki M. Sato H. Gokaslan Z.L. Liotta L.A. Nicolson G.L. Rao J.S. Cancer Res. 1996; 56: 384-392PubMed Google Scholar, 14Uhm J.H. Dooley N.P. Villemure J.-G. Yong V.W. Clin. & Exp. Metastasis. 1996; 14: 421-433Crossref PubMed Scopus (128) Google Scholar, 16Nakagawa T. Kubota T. Kabuto M. Sato K. Kawano H. Hayakawa T. Okada Y. J. Neurosurg. 1994; 81: 69-77Crossref PubMed Scopus (206) Google Scholar, 18Sawaya R.E. Yamamoto M. Gokaslan Z.L. Wang S.W. Mohanam S. Fuller G.N. McCutcheon I.E. Stetler-Stevenson W.G. Nicolson G.L. Rao J.S. Clin. & Exp. Metastasis. 1996; 14: 35-42Crossref PubMed Scopus (169) Google Scholar). We have recently determined that a variety of human astroglioma cell lines constitutively express high levels of MMP-2, which correlates with the in vitroinvasive capacity of these cells (15Qin H. Moellinger J.D. Wells A. Windsor L.J. Sun Y. Benveniste E.N. J. Immunol. 1998; 161: 6664-6673PubMed Google Scholar). As illustrated in Fig.1, the MMP-2 gene is constitutively expressed in U251-MG astroglioma cells. The 72-kDa MMP-2 protein can be detected by zymography (lane 1) and i
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