The plasminogen activator urokinase (UK) is a member of a small group of proteinases which have been repeatedly implicated as mediators of tumor cell invasion and extracellular matrix destruction. Evidence from several laboratories suggests that UK may initiate a cascade of zymogen activations which culminate in the production of plasmin, collagenase, and heparinase activities. In this application I propose to investigate the effects on tumor cells of 4 human proteins that are putative regulators of UK. One of these proteins serves as a cell surface binding site for UK. We have recently shown that cultured cells load these sites with endogenous (cellular) UK (A. Bajpai and J. Baker, Biochem. Biophys. Res. Commun. 133,475, (1985)). The high affinity of these sites for UK and, especially, the extraordinarily slow rate that UK dissociates from them strongly suggest that these sites function to focus UK activity at the cell surface. The other 3 proteins are fast-acting inhibitors of UK that have recently been purified in small quantities from cell cultures: protease nexin I (PNI), beta plasminogen activator inhibitor (beta PAI), and placental plasminogen activator inhibitor (PPAI). These inhibitors vary in their rate of inactivation of UK, specificities for other proteinases and affinities for glycosaminoglycans I propose to examine the activity of UK bound to the cell surface UK-binding protein, purify the binding protein from human fibroblasts, partially sequence it, and generate a monoclonal antibody that blocks its binding of UK. (Both the sequence information and antibody will be useful for the eventual cloning of the gene for the cell surface UK binding protein). Simultaneously, we will use the services of Invitron, a company that specializes in large scale production of proteins secreted by mammalian cells, to generate greater than 100 mg quantities of PNI, beta PAI and PPAI. The ability of these inhibitors to inactivate UK bound to the cell surface will be investigated. We will examine the effects of the inhibitors and anti-binding site antibody on tumor cell growth, destruction of vascular extracellular matrices and invasion across placental amnion basement membranes. These latter studies are, in part, prompted by our recent finding that purified PNI at submicromolar concentrations prevents the destruction of vascular smooth muscle extracellular matrix by human fibrosarcoma cells and, transiently, inhibits the growth of these cells on the matrix (B.L. Bergman, et al Proc. Natl. Acad. Sci. U.S.A. (1986)).
