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W2129127779 abstract "The urokinase type plasminogen activator (uPA), together with its receptor uPAR and the plasminogen activator inhibitor type-1 (PAI-1) plays a pivotal role during tumor invasion and metastasis. Integrins, via interaction with the extracellular matrix (ECM), control cell adhesion and motility. The two systems are functionally linked because uPAR and PAI-1 bind to the ECM component vitronectin (VN). Because integrin signaling alters gene expression patterns, we investigated whether the expression levels of uPA, uPAR, and PAI-1 are affected by ECM/integrin interactions. Expression of uPA, uPAR, and PAI-1 was significantly enhanced when human ovarian cancer cells (OV-MZ-6) were cultivated on fibronectin or collagen type IV. In contrast, VN induced down-regulation of uPA and uPAR while increasing PAI-1 by up to 4-fold. VN-dependent decrease of uPA protein was paralleled by a significant reduction of uPA promoter activity that was even more pronounced upon αvβ3overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon αvβ3-mediated cell adhesion to VN. The activity of the Rel-unresponsive PAI-1 promoter was up to 5-fold induced as a function of αvβ3/VN interaction. Thus, the balance between available concentrations of uPA, uPAR, PAI-1, and integrins in human ovarian cancer cells might provide a switch within the regulation of their invasive phenotype. The urokinase type plasminogen activator (uPA), together with its receptor uPAR and the plasminogen activator inhibitor type-1 (PAI-1) plays a pivotal role during tumor invasion and metastasis. Integrins, via interaction with the extracellular matrix (ECM), control cell adhesion and motility. The two systems are functionally linked because uPAR and PAI-1 bind to the ECM component vitronectin (VN). Because integrin signaling alters gene expression patterns, we investigated whether the expression levels of uPA, uPAR, and PAI-1 are affected by ECM/integrin interactions. Expression of uPA, uPAR, and PAI-1 was significantly enhanced when human ovarian cancer cells (OV-MZ-6) were cultivated on fibronectin or collagen type IV. In contrast, VN induced down-regulation of uPA and uPAR while increasing PAI-1 by up to 4-fold. VN-dependent decrease of uPA protein was paralleled by a significant reduction of uPA promoter activity that was even more pronounced upon αvβ3overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon αvβ3-mediated cell adhesion to VN. The activity of the Rel-unresponsive PAI-1 promoter was up to 5-fold induced as a function of αvβ3/VN interaction. Thus, the balance between available concentrations of uPA, uPAR, PAI-1, and integrins in human ovarian cancer cells might provide a switch within the regulation of their invasive phenotype. extracellular matrix vitronectin urokinase type plasminogen activator uPA receptor plasminogen activator inhibitor type-1 laminin fibronectin monoclonal antibody poly-l-lysin collagen type oligodeoxynucleotide(s) enzyme-linked immunosorbent assay phosphate-buffered saline bovine serum albumin polymerase chain reaction confocal laser scanning microscopy Tumor invasion into the surrounding tissue and metastasis require a finely tuned regulation of the formation and the loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM).1 These complex events depend on the concerted and controlled expression of adhesion molecules and proteolytic enzymes (1Reuning U. Magdolen V. Wilhelm O. Fischer K. Lutz V. Graeff H. Schmitt M. Int. J. Oncol. 1998; 13: 893-906PubMed Google Scholar). Adhesive interactions of cells with the ECM are largely supported by adhesion receptors of the integrin superfamily. Integrins are transmembrane glycoproteins composed of noncovalently associated α and β chains that recognize peptide motifs, such as RGD, within adhesive ECM proteins (2Hynes R.O. Lander A.D. Cell. 1992; 68: 303-322Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Following ligand interaction, integrins generate intracellular signals that are propagated via their cytoplasmic domains, connecting the extracellular space with the cytoskeleton. Expression of ECM components and integrins is frequently modulated in response to changes within the tumor cell vicinity paralleling tumor cell progression. Certain integrins recognize multiple ECM ligands; precise biological responses, however, are achieved via differential integrin ligation with different ECM proteins (3Giancotti F.G. Rouslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar). Ovarian cancer cells bind to various ECM constituents, thereby facilitating invasion to the peritoneal epithelial layer. In ovarian cancer cell lines, the integrin repertoire very often encompasses β1 integrins in complex with the integrin subunits α1, α2, α3, α5, and α6, in addition to the integrin αvβ3, which associates with the ECM component vitronectin (VN) (4Carreiras F. Cruet S. Staedel C. Sichel F. Gaudochon P. Gynecol. Oncol. 1999; 72: 312-322Abstract Full Text PDF PubMed Scopus (45) Google Scholar). In fact, alteration of αvβ3 and VN expression influences tumor cell growth and survival necessary for cancer progression. The cellular mechanisms promoted via αvβ3 are still rather inconclusive (4Carreiras F. Cruet S. Staedel C. Sichel F. Gaudochon P. Gynecol. Oncol. 1999; 72: 312-322Abstract Full Text PDF PubMed Scopus (45) Google Scholar). It was noted that αvβ3 expression occurs at a significantly higher rate in advanced ovarian tumors than in ovarian tumors of low malignant potential (5Liapis H. Adler L. Wick M.R. Rader J.S. Human Pathol. 1996; 28: 443-449Crossref Scopus (103) Google Scholar). Compared with well differentiated ovarian cancers, in poorly differentiated ones, the β3 subunit is often absent, whereas the expression of αv is maintained. VN as a major ECM ligand of αvβ3 not only represents a primary adhesion substrate for established ovarian cancer cell lines but is also detectable in normal ovarian epithelium as well as in differentiated ovarian tumors (4Carreiras F. Cruet S. Staedel C. Sichel F. Gaudochon P. Gynecol. Oncol. 1999; 72: 312-322Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 6Carreiras F. Denoux Y. Staedel C. Lehmann M. Gaudochon P. Gynecol. Oncol. 1996; 62: 260-267Abstract Full Text PDF PubMed Scopus (99) Google Scholar). For invasive tumor cells a marked ability to degrade ECM molecules is well documented. There is a large body of experimental and clinical evidence for a key function of the serine protease uPA in pericellular proteolysis. By virtue of its ability to generate plasmin from plasminogen it promotes pro-invasive matrix degradation within the tumor vicinity (1Reuning U. Magdolen V. Wilhelm O. Fischer K. Lutz V. Graeff H. Schmitt M. Int. J. Oncol. 1998; 13: 893-906PubMed Google Scholar). On tumor cell surfaces, uPA interacts with a specific high affinity receptor, uPAR (CD87). Upon binding to uPAR, uPA enhances its catalytic activity (7Vassalli J.D. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (591) Google Scholar, 8Behrendt N. Rønne E. Danø K. Biol. Chem. Hoppe-Seyler. 1995; 376: 269-279PubMed Google Scholar), which is under the control of the plasminogen activator inhibitor type-1 (PAI-1) (9Blasi F. Immunol. Today. 1997; 18: 415-417Abstract Full Text PDF PubMed Scopus (240) Google Scholar). Because many tumor cells synthesize uPA, uPAR, and PAI-1, the underlying regulatory mechanisms have been studied at the protein and the transcriptional levels. As such, the promoters of uPA and PAI-1 have been characterized in detail (10Besser D. Verde P. Nagamine Y. Blasi F. Fibrinolysis. 1996; 10: 215-237Crossref Scopus (65) Google Scholar); the human uPA promoter contains, besides binding sites for other transcription factors (11Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar, 12Verde P. Boast S. Franze A. Robbiati F. Blasi F. Nucleic Acids Res. 1988; 16: 10699-10716Crossref PubMed Scopus (103) Google Scholar, 13Nerlov C. Rφrth P. Blasi F. Johnsen M. Oncogene. 1991; 6: 1583-1592PubMed Google Scholar, 14Nerlov C. De Cesare D. Pergola F. Caracciolo A. Blasi F. Johnsen M. Verde P. EMBO J. 1992; 11: 4573-4582Crossref PubMed Scopus (142) Google Scholar, 15Lengyel E. Gum R. Stepp E. Juarez J. Wang H. Boyd D. J. Cell. Biochem. 1996; 61: 430-443Crossref PubMed Scopus (48) Google Scholar, 16Ried S. Jäger C. Jeffers M. Van de Woude G.F. Graeff H. Schmitt M. Lengyel E. J. Biol. Chem. 1999; 274: 16377-16386Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), κB consensus motifs for Rel transcription factors. Two Rel-like binding sites are located in tandem repeat at nucleotide positions −1865 and −1835, respectively (11Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar,17Novak U. Cocks B.G. Hamilton J.A. Nucleic Acids Res. 1991; 19: 3389-3393Crossref PubMed Scopus (82) Google Scholar); another has been identified in the 5′-flanking region of the uPA gene between nucleotide positions −1592 and −1572 and was termed Rel-related binding element (18Hansen S.K. Nerlov C. Zabel U. Verde M. Johnson M. Baeuerle P.A. Blasi F. EMBO J. 1992; 11: 205-231Crossref PubMed Scopus (135) Google Scholar). In fact, Rel proteins are involved in the development and progression of cancer by altering the adhesive and proteolytic properties of tumor cells (19Higgins K.A. Perez J.R. Coleman T.A. Dorshkind K. McComas W.A. Sarmiento U.M. Rosen C.A. Narayanan R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9901-9905Crossref PubMed Scopus (272) Google Scholar, 20Sokoloski J.A. Sartorelli A.C. Rosen C.A. Narayanan R. Blood. 1993; 82: 625-632Crossref PubMed Google Scholar). Members of the Rel transcription factor family, such as RelA (p65) and NFKB1 (p50), as heterodimers rapidly transmit signals from the cytoplasm to the nucleus after dissociation from their cytoplasmic inhibitor IκB, resulting in immediate changes in gene transcription (21Grilli M. Chiu J.J. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (883) Google Scholar). In earlier studies we have demonstrated that Rel proteins substantially contribute to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis (22Reuning U. Wilhelm O. Nishiguchi T. Blasi F. Graeff H. Schmitt M. Nucleic Acids Res. 1995; 23: 3889-3893Crossref Scopus (48) Google Scholar, 23Reuning U. Guerrini L. Nishiguchi T. Page S. Seibold H. Magdolen V. Graeff H. Schmitt M. Eur. J. Biochem. 1999; 259: 143-148Crossref PubMed Scopus (20) Google Scholar). The human PAI-1 promoter contains recognition sites for transcription factors including PEA3, AP1, AP2, and Sp1, whereas perfect consensus motifs for the binding of Rel proteins are lacking (24Irigoyen J.P. Munoz-Canoves P. Montero L. Koziczak M. Nagamine Y. Cell. Mol. Life Sci. 1999; 56: 104-136Crossref PubMed Scopus (343) Google Scholar, 25Riccio A. Lund L.R. Sartorio R. Lania A. Andreasen P.A. Danφ K. Blasi F. Nucleic Acids Res. 1988; 16: 2805-2824Crossref PubMed Scopus (60) Google Scholar). Recently, important links have been identified between the uPA and the integrin system. Physical association of uPAR with β integrins modulates their interaction with certain ECM proteins (26Xue W. Mizukami I. Todd III, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 27Bohuslav J. Horejsi V. Hansmann C. Stockl J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar, 28Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar, 29Carriero M.V. Del Vecchio S. Capozzoli M. Franco P. Fontana L. Zannetti A. Botti G. D'Aiuto G. Salvatore M. Stoppelli M.P. Cancer Res. 1999; 59: 5307-5314PubMed Google Scholar), whereas a functional interdependence has been elucidated by virtue of the ability of uPAR (28Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar, 30Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 31Kanse S. Kost C. Wilhelm O. Andreasen P.A. Preissner K.T. Exp. Cell Res. 1996; 224: 344-353Crossref PubMed Scopus (232) Google Scholar) and PAI-1 (32Deng G. Royle G. Wang S. Crain K. Loskutoff D.J. J. Biol. Chem. 1996; 271: 12716-12723Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Loskutoff D.J. Curriden S.A. Hu G. Deng G. APMIS. 1999; 107: 54-61Crossref PubMed Scopus (144) Google Scholar, 34Lauffenburger D.A. Nature. 1996; 383: 390-391Crossref PubMed Scopus (51) Google Scholar, 35Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (607) Google Scholar, 36Knudsen B.S. Hapel P.C. Nachman R.L. J. Clin. Invest. 1987; 80: 1082-1089Crossref PubMed Scopus (112) Google Scholar, 37Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 38Deng G. Curriden A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (432) Google Scholar) to bind to VN. Both uPAR and PAI-1 compete mutually and with integrins for VN binding (32Deng G. Royle G. Wang S. Crain K. Loskutoff D.J. J. Biol. Chem. 1996; 271: 12716-12723Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar,37Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 38Deng G. Curriden A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (432) Google Scholar, 39Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (143) Google Scholar). PAI-1 bound to VN exerts its full inhibitory activity toward uPA; however, the resulting uPA·PAI-1 complex displays a markedly diminished affinity for VN (40Deng G. Royle D. Seiffert D. Loskutoff D.J. Thromb. Haemostasis. 1995; 74: 66-70Crossref PubMed Scopus (76) Google Scholar); uPA binding to uPAR enhances the interaction of uPAR with VN. The strength of the association of PAI-1 and uPAR with VN and their level of expression in concert with uPA and integrins influence the balance between tumor cell adhesion and detachment (30Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 31Kanse S. Kost C. Wilhelm O. Andreasen P.A. Preissner K.T. Exp. Cell Res. 1996; 224: 344-353Crossref PubMed Scopus (232) Google Scholar, 32Deng G. Royle G. Wang S. Crain K. Loskutoff D.J. J. Biol. Chem. 1996; 271: 12716-12723Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Loskutoff D.J. Curriden S.A. Hu G. Deng G. APMIS. 1999; 107: 54-61Crossref PubMed Scopus (144) Google Scholar, 34Lauffenburger D.A. Nature. 1996; 383: 390-391Crossref PubMed Scopus (51) Google Scholar, 35Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (607) Google Scholar, 36Knudsen B.S. Hapel P.C. Nachman R.L. J. Clin. Invest. 1987; 80: 1082-1089Crossref PubMed Scopus (112) Google Scholar, 37Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar, 38Deng G. Curriden A. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Crossref PubMed Scopus (432) Google Scholar, 39Van Meijer M. Pannekoek H. Fibrinolysis. 1995; 9: 263-276Crossref Scopus (143) Google Scholar, 40Deng G. Royle D. Seiffert D. Loskutoff D.J. Thromb. Haemostasis. 1995; 74: 66-70Crossref PubMed Scopus (76) Google Scholar). Because integrins are known to affect gene expression via intracellular signaling (3Giancotti F.G. Rouslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar), we asked whether cross-talk occurs between both systems by investigating the impact of ECM/integrin interaction on expression levels of uPA, uPAR, and PAI-1. The focus is on the effects of αvβ3-mediated adhesion of human ovarian cancer cells to the ECM protein VN as the shared ligand between αvβ3, uPAR, and PAI-1. Cell culture media, supplements, Lipofectin®, VN, and laminin (LN) were obtained from Life Technologies, Inc./Becton Dickinson. [γ-32P]dATP (3000 Ci/mmol) and ECL Western blot kit were from Amersham Pharmacia Biotech. Fibronectin (FN) was purchased from Collaborative Research (Bedford, MA). Polyclonal rabbit antibodies directed to RelA (sc-109-X) were bought from Santa Cruz Inc. (Santa Cruz, CA). Monoclonal antibodies (mAb) directed to αvβ3 (clone LM609) or αvβ5 (mAb 2019Z) were purchased from Chemicon (Hofheim, Germany). The polyclonal antibody directed to αv was obtained from Boehringer Ingelheim Bioproducts (Heidelberg, Germany); the mAb directed to β3 (clone PM6/13) was from Southern Biotechnology Associates (Birmingham, AL). Fluorescein isothiocyanate- and Alexa-488-labeled goat anti-mouse and goat anti-rabbit IgG were from Molecular Probes (Eugene, OR). The cyclic peptides cRGDfV and cRADfV were synthesized at the Institute of Organic Chemistry and Biochemistry (Technische Universität München, Garching, Germany) according to previous publications (41Aumailley M. Gurrath M. Müller G. Calvete J. Timpl R. Kessler H. FEBS Lett. 1991; 291: 50-54Crossref PubMed Scopus (529) Google Scholar, 42Gurrath M. Müller G. Kessler H. Aumailley M. Timpl R. Eur. J. Biochem. 1992; 210: 911-921Crossref PubMed Scopus (310) Google Scholar, 43Pfaff M. Tangemann K. Müller B. Gurrath M. Müller G. Kessler H. Timpl R. Engel J. J. Biol. Chem. 1994; 269: 20233-20238Abstract Full Text PDF PubMed Google Scholar). Poly-l-lysin (PL), collagen type I (Col-I), collagen type IV (Col-IV), andp-nitrophenyl-N-acetyl-β-d-glucosaminide were purchased from Sigma. The dual luciferase reporter gene assay kit, the firefly luciferase reporter gene plasmids pGL2-Basic, and theRenilla luciferase reporter gene vector pRL-SV40 were obtained from Promega (Madison, WI). The expression plasmid pcDNA3.1/myc-His and mAb directed to c-Myc (R950–25) were purchased from Invitrogen (San Diego, CA). Oligodeoxynucleotides (ODN) were synthesized at Metabion (Martinsried, Germany). The origin and cultivation of the human ovarian cancer cell line OV-MZ-6 (44Möbus V. Gerharz C.D. Press U. Moll R. Beck T. Mellin W. Pollow K. Knapstein P.G. Kreienberg R. Int. J. Cancer. 1992; 52: 76-84Crossref PubMed Scopus (88) Google Scholar) as well as the preparation of OV-MZ-6 cell lysates for ELISA have been published (22Reuning U. Wilhelm O. Nishiguchi T. Blasi F. Graeff H. Schmitt M. Nucleic Acids Res. 1995; 23: 3889-3893Crossref Scopus (48) Google Scholar, 23Reuning U. Guerrini L. Nishiguchi T. Page S. Seibold H. Magdolen V. Graeff H. Schmitt M. Eur. J. Biochem. 1999; 259: 143-148Crossref PubMed Scopus (20) Google Scholar). uPA, uPAR, and PAI-1 antigen concentrations in cell lysates and cell culture supernatants were determined as described (22Reuning U. Wilhelm O. Nishiguchi T. Blasi F. Graeff H. Schmitt M. Nucleic Acids Res. 1995; 23: 3889-3893Crossref Scopus (48) Google Scholar) using ELISA kits IMUBIND uPA 894, IMUBIND uPAR 893, and IMUBIND PAI-1 821 from American Diagnostica (Greenwich, CT). The adhesive capacity of OV-MZ-6 cells was assessed by a modification of the assay described by Landegren (45Landegren U. J. Immunol. Methods. 1984; 67: 379-388Crossref PubMed Scopus (603) Google Scholar); 96-well cell culture plates were coated overnight at 4 °C or alternatively for 2 h at room temperature with FN, VN, Col-I, Col-IV, LN (10 µg/ml each), and PL (0.01% w/v), respectively. After three washes in phosphate-buffered saline (PBS), cell culture plates were blocked for 2 h at room temperature in PBS containing 2% (w/v) bovine serum albumin (BSA) and washed twice prior to cell plating. Then OV-MZ-6 cells in 50 µl of cell culture medium containing 0.5% (v/v) fetal calf serum were added. At different time points, cells were washed again in PBS, and then 50 µl of substrate (15 mm p-nitrophenyl-N-acetyl-β-d-glucosaminide, 0.5% (v/v) Triton X-100, 100 mm sodium citrate, pH 5.0) were added to determineN-acetyl-β-d-hexoaminidase activity. At distinct time intervals, the reaction was stopped by the addition of 100 µl of 0.2 m NaOH, 5 mm EDTA, and the changes in optical density were recorded at 405 nm as a measure of number of adherent cells. Linearity of the absorption range to the number of adherent cells has been proven by establishing standard curves with increasing cell numbers. The involvement of αvβ3 in OV-MZ-6 cell adhesion was investigated by applying the αvβ3-directed synthetic cyclic peptide cRGDfV as a competitor (41Aumailley M. Gurrath M. Müller G. Calvete J. Timpl R. Kessler H. FEBS Lett. 1991; 291: 50-54Crossref PubMed Scopus (529) Google Scholar, 42Gurrath M. Müller G. Kessler H. Aumailley M. Timpl R. Eur. J. Biochem. 1992; 210: 911-921Crossref PubMed Scopus (310) Google Scholar, 43Pfaff M. Tangemann K. Müller B. Gurrath M. Müller G. Kessler H. Timpl R. Engel J. J. Biol. Chem. 1994; 269: 20233-20238Abstract Full Text PDF PubMed Google Scholar). Dose-response curves using the peptide in a concentration range between 10 and 500 µg/ml were established and revealed that cRGDfV was most efficient at a concentration of 250 µg/ml without compromising cell viability, which is in accordance with previously published results by others (46Nowlin D.M. Gorcsan F. Moscinski M. Chiang S.-L. Lobl T.J. Cardarelli P.M. J. Biol. Chem. 1993; 268: 20352-20359Abstract Full Text PDF PubMed Google Scholar,47Cardarelli P.M. Cobb R.R. Nowlin D.M. Scholz W. Gorcsan F. Mosciniki M. Yasuhara M. Chiang S.-L. Lobl T.J. J. Biol. Chem. 1994; 269: 18668-18673Abstract Full Text PDF PubMed Google Scholar). The cDNA of the full-length human uPA promoter was a kind gift of F. Blasi (Department of Genetics and Microbiology, University of Milan, Italy) (11Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar). Rel protein-binding sites contained within the human uPA promoter (11Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar, 17Novak U. Cocks B.G. Hamilton J.A. Nucleic Acids Res. 1991; 19: 3389-3393Crossref PubMed Scopus (82) Google Scholar, 18Hansen S.K. Nerlov C. Zabel U. Verde M. Johnson M. Baeuerle P.A. Blasi F. EMBO J. 1992; 11: 205-231Crossref PubMed Scopus (135) Google Scholar) were mutated according to Kunkel et al. (49Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar) and subcloned into the luciferase reporter gene plasmid pGl2-Basic as published (23Reuning U. Guerrini L. Nishiguchi T. Page S. Seibold H. Magdolen V. Graeff H. Schmitt M. Eur. J. Biochem. 1999; 259: 143-148Crossref PubMed Scopus (20) Google Scholar). The human PAI-1 promoter (−798 to +142) (25Riccio A. Lund L.R. Sartorio R. Lania A. Andreasen P.A. Danφ K. Blasi F. Nucleic Acids Res. 1988; 16: 2805-2824Crossref PubMed Scopus (60) Google Scholar) was amplified by polymerase chain reaction (PCR) using genomic DNA of OV-MZ-6 cells as a template and applying the primers 5′-AAA AAC CCG GGA AGC TTA CCA TGG TAA CCC CTG G-3′ and 5′-AAA AAG GTA CCC TGA AGT TCT CAG AGG TGC CTT GC-3′. The amplicon was subcloned into the SmaI andKpnI sites of the plasmid pGl2-Basic, and the amplified insert was sequenced. Full-length cDNAs for integrin subunits αv and β3, kindly provided by J. Loftus (Mayo Clinic, Scotsdale, AZ), were subcloned into the expression vector pcDNA3.1/myc-His. The plasmid containing three κB consensus sequence-binding elements for Rel proteins following the sequence of the thymidine kinase promoter in front of the reporter gene luciferase was a generous gift of K. Brand (Department of Clinical Chemistry, Munich, Germany) (50Haas M. Page S. Page M. Neumann F.-J. Marx N. Adam M. Löms Ziegler-Heitbrock H.W. Neumeier D. Brand K. J. Leukocyte Biol. 1998; 63: 395-404Crossref PubMed Scopus (62) Google Scholar). Transient transfections of OV-MZ-6 cells with the reporter gene constructs and the different expression vectors were conducted using Lipofectin® (23Reuning U. Guerrini L. Nishiguchi T. Page S. Seibold H. Magdolen V. Graeff H. Schmitt M. Eur. J. Biochem. 1999; 259: 143-148Crossref PubMed Scopus (20) Google Scholar). Stable OV-MZ-6 cell transfectants overexpressing the integrin αvβ3 were generated and isolated upon neomycin selection (G418, 1 mg/ml) (51Fischer K. Lutz V. Wilhelm O. Schmitt M. Graeff H. Heiss P. Nishiguchi T. Harbeck N. Kessler H. Luther T. Magdolen V. Reuning U. FEBS Lett. 1998; 438: 101-105Crossref PubMed Scopus (61) Google Scholar). Stable OV-MZ-6 cell transfectants carrying the empty plasmid served as controls. From each transfection, several OV-MZ-6 cell clones were isolated after immunocytochemical verification of αvβ3 overexpression by confocal laser scanning microscopy (CLSM) (Leica, Heidelberg, Germany) using the mAb LM609 directed to αvβ3. Promoter activity measurements were performed with the dual firefly Renillaluciferase reporter gene assay kit according to the manufacturer's recommendation. Transient transfections were performed in the presence of 1 µg of the uPA and PAI-1 promoter-luciferase reporter gene constructs, respectively. To normalize for varying transfection efficiency, OV-MZ-6 cells were cotransfected with 30 ng of theRenilla luciferase reporter gene vector pRL-SV40. Relative uPA promoter activity was calculated upon normalizing the results from firefly luciferase activity measurements to the values obtained forRenilla luciferase activity (23Reuning U. Guerrini L. Nishiguchi T. Page S. Seibold H. Magdolen V. Graeff H. Schmitt M. Eur. J. Biochem. 1999; 259: 143-148Crossref PubMed Scopus (20) Google Scholar). PAI-1 promoter activity was measured by the same procedure. Rel DNA binding activity was determined by utilizing a luciferase reporter gene plasmid containing three tandem κB repeats upstream of the thymidine kinase promoter (50Haas M. Page S. Page M. Neumann F.-J. Marx N. Adam M. Löms Ziegler-Heitbrock H.W. Neumeier D. Brand K. J. Leukocyte Biol. 1998; 63: 395-404Crossref PubMed Scopus (62) Google Scholar). For transient overexpression of αv and/or β3, OV-MZ-6 cells were cotransfected with the expression plasmids pcDNA3.1/myc-His encoding either αv or β3 using concentrations ranging from 0.5 to 3 µg/well. These dose-response curves revealed that cotransfections using the expression plasmids at a concentration of 1 µg/well was most efficient, so that for all further experiments these plasmid concentrations were applied in all reporter gene assays. Nuclear and cytosolic extracts of OV-MZ-6 cells were prepared, and gel retardation analysis was performed as described (52Guerrini L. Blasi F. Denis-Donini S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9077-9081Crossref PubMed Scopus (259) Google Scholar). The double-stranded ODN 5′-CAGAGGGGACTTTCCGAGA-3′ encompassing the Rel consensus motif was radiolabeled by using the Klenow fragment. Reverse transcriptase-PCR analyses for the determination of uPA mRNA concentrations in OV-MZ-6 cells as a function of αvβ3 overexpression was performed as described (22Reuning U. Wilhelm O. Nishiguchi T. Blasi F. Graeff H. Schmitt M. Nucleic Acids Res. 1995; 23: 3889-3893Crossref Scopus (48) Google Scholar). PAI-1 transcript concentrations were also evaluated by reverse transcriptase-PCR applying the ODN 5′-GGC GCA GGC CTC CAA GGA CCG-3′ and 5′-GGT CCA CGG TCC TTT CCC AAG C-3′. As control for equal RNA loading and for normalization of reverse transcription efficiency, glyceraldehyde-3-phosphate dehydrogenase (amplicon size, 194 base pairs) was amplified in the same PCR reaction. Expression of αv and β3 by OV-MZ-6 cells was evaluated by flow cytofluorometry using the FACSCalibur instrument (Becton Dickinson). Viable cells were blocked in PBS containing 2% (w/v) BSA for 2 h at room temperature and then incubated for 1 h in PBS containing 1% (w/v) BSA with polyclonal antibody directed to αv (20 µg/ml) or mAb directed to β3 (1 µg/ml). The cells were again washed in PBS and incubated with fluorescein isothiocyanate-labeled anti-mouse IgG or anti-rabbit IgG. Fluorescent signals in the presence of the secondary Ab alone as well as in the presence of control IgG of rabbit or mouse origin instead of the primary Ab served as controls (53Chucholowski N. Schmitt M. Goretzki L. Schüren E. Moniwa N. Weidle U. Kramer M. Wagner B. Jänicke F. Graeff H. Biochem. Soc. Trans. 1992; 20: 208-216Crossref PubMed Scopus (7) Google Scholar). OV-MZ-6 cells were grown on microchamber slides, fixed with 2% (w/v) paraformaldehyde for 30 min at room temperature, and blocked for 2 h at room temperature in PBS containing 2% (w/v) BSA. The mAb LM609 directed to αvβ3 (1 µg/ml) was incubated with the cells in blocking buffer for 1 h at room temperature. Detection of the specific antigens was conducted by applying secondary Alexa-488-labeled anti-mouse IgG (45 min at room temperature). After three washes in PBS, the slides were mounted and the fluorescence intensity was evaluated by CLSM. Staining in the absence of antibody (as a measure of OV-MZ-6 cell autofluorescence), in the presence of Alexa-488-labeled IgG alone, or in the presence of irrelevant control IgG served as background controls. Cells lysis was performed in a buffer containing 50 mm HEPES, pH 7.5, 150 mm" @default.
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W2129127779 title "Integrin αvβ3/Vitronectin Interaction Affects Expression of the Urokinase System in Human Ovarian Cancer Cells" @default.
W2129127779 cites W143133239 @default.
W2129127779 cites W1544975530 @default.
W2129127779 cites W1548286725 @default.
W2129127779 cites W1555563546 @default.
W2129127779 cites W1560207700 @default.
W2129127779 cites W1571549380 @default.
W2129127779 cites W1607041879 @default.
W2129127779 cites W1670322156 @default.
W2129127779 cites W1926488772 @default.
W2129127779 cites W1973270637 @default.
W2129127779 cites W1982849453 @default.
W2129127779 cites W1983212899 @default.
W2129127779 cites W1989434876 @default.
W2129127779 cites W1995412965 @default.
W2129127779 cites W1996722746 @default.
W2129127779 cites W2002082738 @default.
W2129127779 cites W2003593894 @default.
W2129127779 cites W2006678973 @default.
W2129127779 cites W2007026034 @default.
W2129127779 cites W2010275471 @default.
W2129127779 cites W2012710893 @default.
W2129127779 cites W2013813330 @default.
W2129127779 cites W2019728202 @default.
W2129127779 cites W2020567039 @default.
W2129127779 cites W2023242965 @default.
W2129127779 cites W2023285074 @default.
W2129127779 cites W2027077889 @default.
W2129127779 cites W2035830356 @default.
W2129127779 cites W2041111722 @default.
W2129127779 cites W2042155824 @default.
W2129127779 cites W2042274047 @default.
W2129127779 cites W2043513099 @default.
W2129127779 cites W2043648660 @default.
W2129127779 cites W2047013487 @default.
W2129127779 cites W2048653605 @default.
W2129127779 cites W2049631846 @default.
W2129127779 cites W2055884349 @default.
W2129127779 cites W2063147847 @default.
W2129127779 cites W2064484362 @default.
W2129127779 cites W2064517965 @default.
W2129127779 cites W2068561314 @default.
W2129127779 cites W2073993982 @default.
W2129127779 cites W2074012988 @default.
W2129127779 cites W2075172358 @default.
W2129127779 cites W2081475030 @default.
W2129127779 cites W2092492313 @default.
W2129127779 cites W2099213932 @default.
W2129127779 cites W2102577298 @default.
W2129127779 cites W2106761248 @default.
W2129127779 cites W2113185415 @default.
W2129127779 cites W2123764828 @default.
W2129127779 cites W2130580559 @default.
W2129127779 cites W2139625377 @default.
W2129127779 cites W2164921896 @default.
W2129127779 cites W2167280914 @default.
W2129127779 cites W2256510372 @default.
W2129127779 cites W2411677906 @default.
W2129127779 cites W2414052497 @default.
W2129127779 cites W2419154990 @default.
W2129127779 cites W264579827 @default.
W2129127779 cites W2888511473 @default.
W2129127779 cites W4213141530 @default.
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