Proteolysis induced by plasminogen activation plays a central role in tumor cell invasion and metastasis. The tight control of extracellular plasminogen activator (PA) activities excerted by normal cells through the release of specific inhibitors (PAI) is deficient in tumor cells resulting in free PA activities with subsequent plasmin generation. Clonal variation of expression of the different genes coding for PA and PAI was recently detected in human sarcoma cells. This suggests that tumors consist of invasive clones expressing high levels of PA and low levels of PAI and non-invasive clones with high PAI gene expression. Thus, decrease of PA production or increase of PAI synthesis should abolish the invasive behavior of tumor cells. This hypothesis is evaluated by investigation of the effects of purified PAI proteins and expression of full length PAI cDNAs in invasive tumor cells. Due to rapid inactivation of natural PAI-1 by oxidation and low yield of purified PAI-2 from cell culture medium, purified recombinant proteins will be used. Full length cDNA of PAI-1 and PAI-2 have been obtained. Site specific mutation of PAI-1 with substitution of MET 347 by Val and Leu, respectively, has been generated. This mutation prevents oxidative inactivation of PAI-1 resulting in a stable inhibitor. Recombinant PAI- 1 (wild and mutated types) and PAI-2 will be isolated and purified from E. coli cell lysates transformed with full length cDNAs. Physicochemical properties, kinetics and spectrum of protease inhibition by recombinant PAI will be established and compared with those obtained with natural PAI-1 and PAI-2. Inhibition by recombinant PAI-1 and PAI-2 of tissue degradation and invasion by human tumor cells is investigated by using complex, radiolabelled extracellular matrices produced in vitro. Full length PAI cDNAs will be transfected into invasive tumor cells to obtain increased PAI expression resulting in abolishment of their invasive and tissue degradative behavior. Transfected and untransfected tumor cells will also be tested for their invasive and metastatic behavior in a novel animal model system. These studies will elucidate the effects and roles of PA and PAI in tumor cell invasion which ultimately may lead to a novel adjuvant treatment of cancer.
