EVIDENCE OF THE INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE IN THE MIGRATION, ACTIN STRESS FIBER FORMATION, AND ALPHA(V)BETA(3)-INTEGRIN-MEDIATED ADHERENCE OF HUMAN-MELANOMA CELLS

Tumor invasion and formation of metastases are major obstacles for a s uccessful therapy of melanomas. Metastasis is thought to require multi ple steps such as alpha(v) beta(3)-integrin-mediated adhesion, proteol ytic digestion of extracellular matrix by metalloproteinase-2, and reo rganization of the actin cytoskeleton. To analyze the functional role of phosphatidylinositol 3-kinase in these processes, melanoma cells we re treated with the fungal metabolite wortmannin. Wortmannin inhibited phosphatidylinositol 3-kinase activity in melanoma cells and migratio n in an equally concentration-dependent fashion. Flow cytometric analy sis of itrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-stained actin networ k indicated reduction of actin filaments by wortmannin. Fluorescence l aser confocal microscopy experiments revealed breakdown of actin stres s fibers. In addition, wortmannin inhibited alpha(v) beta(3)-integrin- mediated adhesion of melanoma cells to vitronectin. Since how cytometr ic measurements did not show altered expression of the alpha(v) beta(3 )-integrin at the cell surface, avidity changes of the alpha(v) beta(3 )-integin by wortmannin are suggested. In contrast to the actin analys is and adhesion assays, wortmannin had no influence on mRNA expression or on protein secretion of metalloproteinase-2. These data provide ev idence that phosphatidylinositol 3-kinase is an essential signal trans duction protein required for migration of melanoma cells, regulating f ormation of the actin stress fiber as well as alpha(v) beta(3)-integri n-mediated adhesion.