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• W2035510366 abstract "Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin αvβ3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to αvβ3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, αvβ3-negative melanoma cell lines MV3 and BLM, their β3-transfected αvβ3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription–polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both αvβ3-negative and αvβ3-positive cell lines. Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of αvβ3-negative cell lines and undetectable in the αvβ3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the αvβ3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in β3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the αvβ3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin αvβ3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and αvβ3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and αvβ3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and αvβ3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of αvβ3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation. Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin αvβ3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to αvβ3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, αvβ3-negative melanoma cell lines MV3 and BLM, their β3-transfected αvβ3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription–polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both αvβ3-negative and αvβ3-positive cell lines. Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of αvβ3-negative cell lines and undetectable in the αvβ3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the αvβ3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in β3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the αvβ3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin αvβ3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and αvβ3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and αvβ3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and αvβ3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of αvβ3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation. matrix metalloproteinase tissue inhibitors of metalloproteinases membrane-type MMP melanoma in situ ePM, early primary melanoma advanced primary melanoma melanoma metastasis serum-free culture medium Cellular invasion occurring in both physiologic and pathologic processes requires proteolytic degradation of the extracellular matrix (ECM), intercellular adhesion and detachment, and adhesion of cells to and detachment of cells from ECM components. During tumor progression, cells detach from the primary tumor, penetrate the basement membrane, and invade into lymph and blood vessels. These processes all depend on co-ordinated expression of proteolytic enzymes and adhesion molecules (Stetler Stevenson et al., 1993Stetler Stevenson W.G. Aznavoorian S. Liotta L.A. Tumor cell interactions with the extracellular matrix during invasion and metastasis.Annu Rev Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1489) Google Scholar;Brooks et al., 1996Brooks P.C. Stromblad S. Sanders L.C. et al.Localization of matrix metalloproteinase, MMP, -2 to the surface of invasive cells by interaction with integrin alpha v beta, 3.Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1399) Google Scholar). Matrix metalloproteinases (MMP) belong to a rapidly growing family of zinc-dependent endopeptidases that degrade many components of the ECM (Birkedal Hansen et al., 1993Birkedal Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal Hansen B. DeCarlo A. Engler J.A. Matrix metalloproteinases: a review.Crit Rev Oral Biol Med. 1993; 4: 197-250PubMed Google Scholar). They can be classified into different groups of closely related members including collagenases, gelatinases, stromelysins, elastases, the membrane-type MMP (MT-MMP) and colleagues (Woessner, 1991Woessner Jr, Jf Matrix metalloproteinases and their inhibitors in connective tissue remodeling.FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3007) Google Scholar;Sato et al., 1994Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells [see comments].Nature. 1994; 370: 61-65Crossref PubMed Scopus (2317) Google Scholar). Most of the MMP are secreted in latent forms (proMMP). A specific multistep activation process is required to convert proMMP to proteolytic active forms (Nagase, 1998Nagase H. Cell surface activation of progelatinase A (proMMP-2) and cell migration.Cell Res. 1998; 8: 179-186Crossref PubMed Scopus (167) Google Scholar;Morgunova et al., 1999Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed.Science. 1999; 284: 1667-1670Crossref PubMed Scopus (465) Google Scholar). Activation of proMMP, and localization of functionally active MMP on the cell surface are essential and tightly regulated elements during tumor cell invasion (Murphy and Gavrilovic, 1999Murphy G. Gavrilovic J. Proteolysis and cell migration: creating a path?.Curr Opin Cell Biol. 1999; 11: 614-621Crossref PubMed Scopus (340) Google Scholar). The presence of activated MMP-2 correlates strongly with tumor cell invasion and metastasis in a variety of cancers (Nomura et al., 1995Nomura H. Sato H. Seiki M. Mai M. Okada Y. Expression of membrane-type matrix metalloproteinase in human gastric carcinomas.Cancer Res. 1995; 55: 3263-3266PubMed Google Scholar;Tokuraku et al., 1995Tokuraku M. Sato H. Murakami S. Okada Y. Watanabe Y. Seiki M. Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis.Int J Cancer. 1995; 64: 355-359Crossref PubMed Scopus (264) Google Scholar;Yamamoto et al., 1996Yamamoto M. Mohanam S. Sawaya R. et al.Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro.Cancer Res. 1996; 56: 384-392PubMed Google Scholar;Kanayama et al., 1998Kanayama H. Yokota K. Kurokawa Y. Murakami Y. Nishitani M. Kagawa S. Prognostic values of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression in bladder cancer.Cancer. 1998; 82: 1359-1366Crossref PubMed Scopus (164) Google Scholar;Nakamura et al., 1999Nakamura H. Ueno H. Yamashita K. et al.Enhanced production and activation of progelatinase A mediated by membrane-type 1 matrix metalloproteinase in human papillary thyroid carcinomas.Cancer Res. 1999; 59: 467-473PubMed Google Scholar;Hofmann et al., 2000Hofmann U.B. Westphal J.R. Zendman A.J. Becker J.C. Ruiter D.J. van Muijen G. Expression and activation of matrix metalloproteinase-2 (MMP-2) and its co-localization with membrane-type matrix metalloproteinase 1 (MT1-MMP) correlate with melanoma progression.J Pathol. 2000; 191: 295-296Crossref Scopus (144) Google Scholar). Previously it has been demonstrated that increased expression of MT1-MMP results in activation of MMP-2 on the cell surface of human melanoma cells, inducing invasive behavior (Nakahara et al., 1997Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion.Proc Natl Acad Sci USA. 1997; 94: 7959-7964Crossref PubMed Scopus (351) Google Scholar). MMP-2 activation is further regulated by formation of a tissue inhibitor of metalloproteinase (TIMP) -2/MT1-MMP complex, which functions as a receptor for MMP-2 at the cell surface (Strongin et al., 1995Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease.J Biol Chem. 1995; 270: 5331-5338Crossref PubMed Scopus (1399) Google Scholar;Butler et al., 1998Butler G.S. Butler M.J. Atkinson S.J. et al.The TIMP2 membrane type 1 metalloproteinase “receptor” regulates the concentration and efficient activation of progelatinase A. A kinetic study.J Biol Chem. 1998; 273: 871-880Crossref PubMed Scopus (530) Google Scholar;Zucker et al., 1998Zucker S. Drews M. Conner C. et al.Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).J Biol Chem. 1998; 273: 1216-1222Crossref PubMed Scopus (252) Google Scholar). Other mechanisms of localizing MMP-2 in a functionally active form on the cell surface seem to involve the integrin αvβ3 (Brooks et al., 1996Brooks P.C. Stromblad S. Sanders L.C. et al.Localization of matrix metalloproteinase, MMP, -2 to the surface of invasive cells by interaction with integrin alpha v beta, 3.Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1399) Google Scholar;Deryugina et al., 1997Deryugina E.I. Bourdon M.A. Luo G.X. Reisfeld R.A. Strongin A. Matrix metalloproteinase-2 activation modulates glioma cell migration.J Cell Sci. 1997; 110: 2473-2482Crossref PubMed Google Scholar). Integrin αvβ3 belongs to a family of heterodimeric transmembrane receptors composed of noncovalently linked α and β subunits that mediate adhesion of cells to other cells, and to the ECM (Hynes, 1992Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8809) Google Scholar;Seftor, 1998Seftor R.E. Role of the beta3 integrin subunit in human primary melanoma progression: multifunctional activities associated with alpha (v) beta3 integrin expression [comment].Am J Pathol. 1998; 153: 1347-1351Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Both MMP-2 (Ray and Stetler Stevenson, 1995Ray J.M. Stetler Stevenson W.G. Gelatinase A activity directly modulates melanoma cell adhesion and spreading.EMBO J. 1995; 14: 908-917Crossref PubMed Scopus (170) Google Scholar;Vaisanen et al., 1998Vaisanen A. Kallioinen M. Taskinen P.J. Turpeenniemi-Hujanen T. Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma.J Pathol. 1998; 186: 51-58Crossref PubMed Scopus (130) Google Scholar;Hofmann et al., 1999Hofmann U.B. Westphal J.R. Waas E.T. Zendman A.J.W. Cornelissen I.M.H.A. Ruiter D.J. van Muijen G.N.P. Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression.Br J Cancer. 1999; 81: 774-782Crossref PubMed Scopus (128) Google Scholar) and αvβ3 (Filardo et al., 1995Filardo E.J. Brooks P.C. Deming S.L. Damsky C. Cheresh D.A. Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo.J Cell Biol. 1995; 130: 441-450Crossref PubMed Scopus (181) Google Scholar;Albelda et al., 1990Albelda S.M. Mette S.A. Elder D.E. Stewart R. Damjanovich L. Herlyn M. Buck C.A. Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression.Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar;Nip et al., 1992Nip J. Shibata H. Loskutoff D.J. Cheresh D.A. Brodt P. Human melanoma cells derived from lymphatic metastases use integrin alpha v beta 3 to adhere to lymph node vitronectin.J Clin Invest. 1992; 90: 1406-1413Crossref PubMed Scopus (144) Google Scholar;Danen et al., 1995Danen E.H. Jansen K.F. van-Kraats A.A. Cornelissen I.M. Ruiter D.J. Van-Muijen G.N. Alpha v-integrins in human melanoma: gain of alpha v beta 3 and loss of alpha v beta 5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice.Int J Cancer. 1995; 61 ([published erratum appears in Int J Cancer 62 (3): 365, 1995].): 491-496Crossref PubMed Scopus (59) Google Scholar;Nip and Brodt, 1995Nip J. Brodt P. The role of the integrin vitronectin receptor, alpha v beta 3 in melanoma metastasis.Cancer Metastasis Rev. 1995; 14: 241-252Crossref PubMed Scopus (50) Google Scholar;Petitclerc et al., 1999Petitclerc E. Stromblad S. von Schalscha T.L. et al.Integrin αvβ3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.Cancer Res. 1999; 59: 2724-2730PubMed Google Scholar) have been implicated in melanoma progression in vitro and in vivo; however, there are no reports of a functional colocalization of these two molecules in human melanoma lesions, or of the possible relevance of this association for human melanoma progression. In this study, we determined the expression and activation status of MMP-2 in a previously described melanoma xenograft model (Danen et al., 1996Danen E.H. van-Kraats A.A. Cornelissen I.M. Ruiter D.J. Van-Muijen G.N. Integrin beta 3 cDNA transfection into a highly metastatic alpha v beta 3-negative human melanoma cell line inhibits invasion and experimental metastasis.Biochem Biophys Res Commun. 1996; 226: 75-81Crossref PubMed Scopus (32) Google Scholar). The model consists of two highly metastatic αvβ3-negative human melanoma cell lines MV3 and BLM, their β3-transfected counterparts, and their corresponding xenografts. Transfection of the cell lines with β3-cDNA resulted in surface expression of αvβ3. In order to evaluate the clinical relevance from this model system, we characterized the expression and colocalization of MMP-2 and αvβ3 in 46 human cutaneous melanoma lesions, comprising melanoma in situ, primary melanoma and melanoma metastasis. We show that the expression of membrane-bound activated MMP-2 correlates with expression of αvβ3 in the xenograft model and in human melanoma lesions. Our findings provide strong evidence that a cooperative expression of MMP-2 and αvβ3 may be involved in melanoma cell invasion. The highly metastatic, αv + /β3– human melanoma cell lines MV3 and BLM have been described before (van Muijen et al., 1991van Muijen G.N.P. Cornelissen L.M. Jansen C.F. Figdor C.G. Johnson J.P. Brocker E.B. Ruiter D.J. Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice.Clin Exp Metastasis. 1991; 9: 259-272Crossref PubMed Scopus (114) Google Scholar, van Muijen et al., 1991van Muijen G.N.P. Jansen K.F. Cornelissen I.M. Smeets D.F. Beck J.L. Ruiter D.J. Establishment and characterization of a human melanoma cell line (MV3) which is highly metastatic in nude mice.Int J Cancer. 1991; 48: 85-91Crossref PubMed Scopus (132) Google Scholar). MV3 and BLM cells were either untransfected, or transfected with plasmid pBJl containing only the neomycin resistance gene (pBJlneo) (MV3-neo and BLM-neo), or with pBJlneo, including the full-length cDNA for the β3 subunit (MV3-β3 and BLM-β3) as described previously (Danen et al., 1996Danen E.H. van-Kraats A.A. Cornelissen I.M. Ruiter D.J. Van-Muijen G.N. Integrin beta 3 cDNA transfection into a highly metastatic alpha v beta 3-negative human melanoma cell line inhibits invasion and experimental metastasis.Biochem Biophys Res Commun. 1996; 226: 75-81Crossref PubMed Scopus (32) Google Scholar). As both parental cell lines express the αv gene product endogenously, transfection of β3-cDNA resulted in cell surface expression of αvβ3. Expression of αvβ3 was confirmed by immunocytochemistry with monoclonal antibody (MoAb) LM609, which recognizes an epitope formed by a combination of the α and the β chains. Cells were cultured in Dulbecco's minimal essential medium (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum (Integro, Zaandam, The Netherlands) and gentamycin (40 μg per ml, Gibco/BRL, Gaithersburg, MD) in an atmosphere of 95% humidified air and 5% CO2 at 37°C. Transfected cells were cultured in medium containing 1 mg per ml neomycin (G418, Life Technologies, Gaithersburg, MD). Human melanoma cell lines were xenografted in BALB/C nu/nu mice kept under specific pathogen-free conditions according to NIH guidelines. Cells (3–5 × 106) of cell lines MV3, MV3-neo, MV3-β3, BLM, BLM-neo, and BLM-β3 were injected subcutaneously into the flanks of nude mice. The animals were killed when the tumor had a diameter of approximately 1 cm. Parts of the tumors were snap-frozen and stored in liquid nitrogen. Representative tissue samples from 46 patients suffering from all stages of melanoma tumor progression were obtained from patients at the University Hospital, Nijmegen, The Netherlands and from patients at the Department of Dermatology, University Hospital, Würzburg, Germany. Clinical data of patients and tumor specimens are summarized in Table 1. Samples were snap-frozen in liquid nitrogen and stored at - 80°C until sectioning. Based on histopathologic examination of hematoxylin and eosin-stained paraffin sections, lesions were classified into four groups: melanoma in situ (MIS, n = 10), early primary melanoma (tumor thickness < 1.5 mm) (ePM, n = 10), advanced primary melanoma (aPM, tumor thickness ≥ 1.5 mm) (n = 6), and melanoma metastasis (MM, n = 20).Table IClinical data and experimental data on coexpression of MMP-2 and integrin αvβ3 in melanoma patients and tumor specimens aSSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanomaPatient characteristicsnNo. of specimens with ≥ 15% MMP-2/ αvβ3-positive tumor cells vs total number of specimensTumor subtype MIS100/10 SSM121/12 NM32/3 LMM10/1 Metastases2015/20Tumor thickness < 1.5 mm100/10 ≥ 1.5 mm63/6Level of invasion I100/10 II50/5 III40/4 ≥ IV73/7Patient age (y) < 200 20–3972/7 40–59167/16 60–79176/17 ≥ 8063/6Patient sex Female187/18 Male2812/28Tumor localization of primary melanomas Head/neck21/2 Trunk91/9 Extremities51/5Metastases Yes42/4 No122/12a SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma Open table in a new tab For RNA extraction from tissue samples at least five frozen sections of 10 μm thickness were homogenized in 1 ml RNAzolB (Campro, Veenendaal, The Netherlands) using a pestle. Melanin and DNA were removed from RNA samples using the RNeasy kit. RNA isolation from cell lines was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was reverse-transcribed with the First Strand cDNA Synthesis Kit for reverse transcription–PCR (Boehringer Mannheim, Penzberg, Germany) using random hexadeoxynucleotide primers according to the manufacturer's instructions. One microliter aliquots of cDNA were subjected to PCR, using PCR buffer IV (20 mM (NH4)2SO4, 75 mM Tris/HCl pH 9, 0.1% Tween), 0.2 M dNTP, 2 pmol of each primer, 1.5 mM MgCl2, and 0.15 units of Thermoperfectplus DNA polymerase (Integro, Zaandam, The Netherlands). Water was added to a final volume of 25 μl. Each PCR was performed for 30 cycles (45 s at 94°C, 1 min at 59°C, 1 min 30 s at 72°C), preceded by a denaturation step at 94°C for 3 min, and terminated with an elongation step at 72°C for 5 min. PCR products were visualized on 2% agarose gels containing ethidium bromide. The following human specific MMP-2 primers were used: 5′-GTGCTGAAGGACACACTAAA- GAAGA-3′ (sense) and 5′-TTGCCATCCTTCTCAAAGTTGTAGG-3′ (anti-sense), yielding a 580 bp product (Grant et al., 1996Grant G.M. Cobb J.K. Castillo B. Klebe R.J. Regulation of matrix metalloproteinases following cellular transformation.J Cell Physiol. 1996; 167: 177-183Crossref PubMed Scopus (34) Google Scholar). As positive controls for the reverse transcription–PCR procedure we used phorphobilinogen-deaminase primers: 5′-CTGGTAACGGCAATGC- GGCT-3′ (sense) and 5′-GCAGATGGCTCCGATGGTGA-3′ (anti-sense), yielding a 339 bp product. Serum-free conditioned medium (SFCM) of parental and (mock) transfected MV3 and BLM cell lines was prepared from 1 × 107 cells by washing cells three times with phosphate-buffered saline, followed by incubation overnight in serum-free medium. Cell lysates were prepared as described previously (Brooks et al., 1996Brooks P.C. Stromblad S. Sanders L.C. et al.Localization of matrix metalloproteinase, MMP, -2 to the surface of invasive cells by interaction with integrin alpha v beta, 3.Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1399) Google Scholar) by suspending cultured tumor cells in lysis buffer containing 1% Triton X-100, 50 mM Tris, 300 mM NaCl, pH 8.0. Lysates were incubated for 15 min on ice with occasional vortexing. Lysates from xenograft tumors and human melanoma metastases were prepared by homogenizing four serial sections (10 μm) in 4 × sample-buffer (62.5 mM Tris/HCl (pH 6.8), 8.8% glycerol, 2% (wt/vol) sodium dodecyl sulfate (SDS), 0.05% bromophenol blue), followed by centrifugation for 1 min at 10,000 × g, incubation at 37°C for 30 min, and 1:1 dilution with distilled water. Protein content of supernatants was measured using BCA Protein Assay Reagent (Pierce, Rockford, IL). Gelatinolytic activity of MMP-2 was determined using SDS–polyacrylamide gel zymography as described previously with minor alterations (Seifert et al., 1996Seifert W.F. Wobbes T. Hendriks T. Divergent patterns of matrix metalloproteinase activity during wound healing in ileum and colon of rats.Gut. 1996; 39: 114-119Crossref PubMed Scopus (43) Google Scholar). Briefly, after heating at 50°C for 20 min, total protein of SFCM (10 μg) from each cell line or tissue extracts (20 μg) were electrophoresed at 4°C on a 7.5% SDS–polyacrylamide gel containing 50 mg per ml gelatin. After electrophoresis, gels were washed three times in 2.5% Triton X-100 for 10 min to remove SDS. After rinsing twice in substrate buffer (50 mM Tris–HCl, pH 7.8, containing 5 mM CaCl2 and 0.1% Triton X-100) gels were incubated at 37°C for 18 h in the same buffer under gentle agitation. Gels were stained for 45 min in 40% methanol/10% glacial acetic acid containing 0.1% (wt/vol) Coomassie Brilliant Blue R 250 and destained in the same solution without Coomassie Brilliant Blue. ProMMP-2 (Mr 66,000) and activated MMP-2 (Mr 62,000) were identified by control samples containing MMP-2 protein. MMP-2 activity was quantified by densitometric scanning of zymographic bands. Zymography was performed at least twice for each sample. For immunohistochemistry mouse MoAb MAB903 (4 μg per ml) (R&D Systems Europe Ltd, Abingdon, U.K.) and for double staining rabbit polyclonal antibody AB809 (100 ng per ml) (Chemicon International Inc., Temecula, CA) against human MMP-2 were used. For western blot analysis mouse MoAb (Oncogene Research Products, Cambridge, MA) against MMP-2 (IM33L) (1 μg per ml), TIMP-2 (IM11L) (5 μg per ml), and rabbit polyclonal antibody AB815 (100 ng per ml) (Chemicon International Inc.) directed against human MT1-MMP were used. For integrin αvβ3 staining MoAb LM609 (2 ng per ml) (Merck KgaA, Darmstadt, Germany) was used. For western blot analysis cell lysates were prepared by suspending cultured tumor cells in lysis buffer containing 1% SDS, 5 mM ethylenediamine tetraacetic acid, 10 μg per ml leupeptin, 200 μg per ml aminoethylbenzenesulfonyl fluoride (AEBSF), and 10 μg per ml chymostatin. Lysates were incubated for 45 min at 37°C with occasional vortexing. SFCM was prepared as described followed by 10 × concentration (Centricon 10 microconcentrator; Amicon, Beverly, MA). Analysis was performed according to standard protocols. Briefly, equal protein amounts (20 μg) from cell and tissue lysates, or SFCM were loaded on a 10% polyacrylamide gel. Gels were run under reducing conditions. After electrophoresis, samples were electroblotted on to nitrocellulose (Schleicher & Schuell, Dassel, Germany). Blots were blocked for 30 min in blocking solution (Boehringer Mannheim, Germany) at room temperature and then incubated overnight with primary antibody in blocking solution. After washing with phosphate-buffered saline/0.05% Tween-20, membranes were incubated for 2 h at room temperature with a rabbit anti-mouse or swine anti-rabbit peroxidase-labeled (1:2000) (Dako, Glastrup, Denmark) secondary antibody in blocking solution. After additional washes, binding of the peroxidase-labeled antibody was visualized by chemiluminescence (Boehringer Mannheim). Analysis was performed at least twice for each sample. To investigate the expression of MMP-2 and αvβ3 at the cellular level, cell lines were cultured on microscope slides for 24 h. Slides were washed three times in serum-free Dulbecco's minimal essential medium and fixed for 10 min at room temperature in acetone with 0.15% H2O2. Slides were incubated overnight with primary antibody in a humidified chamber at 4°C. Subsequent steps were performed at room temperature. Prior to incubation with a biotinylated goat anti-rabbit or horse anti-mouse secondary antibody (1:200) (Vector Laboratories, Burlingame, CA) for 30 min, the avidin–biotin blocking kit (Vector Laboratories) was used according to the manufacturer's suggestions. Forty-five minutes after addition of the avidin–biotin–peroxidase complex (Vectastain Elitekit, Vector Laboratories), slides were developed with amino-ethyl carbazol, and counterstained with Meyer's hematoxylin. As negative control the first antibody was omitted. To study the expression of MMP-2 and αvβ3 in tissue, 4 μm cryostat serial sections were used. At least three different xenografts per cell line, and three consecutive sections of melanoma lesions were stained. Procedures used for immunohistochemistry were identical as described for cultured cells. For double staining, sections were first subjected to a polyclonal anti-MMP-2 antibody and developed with Fast Blue. Subsequently, the section was stained with a MoAb against integrin αvβ3 as described above without counterstaining. For each section the percentage of single and double MMP-2/αvβ3-positive melanoma cells was estimated by counting 500 consecutive malignant cells. One per cent of stained tumor cells was taken as threshold for positivity. Positively stained endothel" @default.
• W2035510366 created "2016-06-24" @default.
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• W2035510366 date "2000-10-01" @default.
• W2035510366 modified "2023-10-12" @default.
• W2035510366 title "Coexpression of Integrin αvβ3 and Matrix Metalloproteinase-2 (MMP-2) Coincides with MMP-2 Activation: Correlation with Melanoma Progression" @default.
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