Protease inhibitor studies utilizing the Rous sarcoma virus transformed fibroblast (RSVCEF) culture system have indicated that the serine protease plasminogen activator acts in concert catalytically with one or more undefined metalloproteases in degrading extracellular matrix and modulating normal and malignant cell behavior. A 70 kDa metalloprotease has recently been purified from cultures of RSVCEF transformed fibroblasts and is a prime candidate for having a direct role in RSVCEF-mediated matrix degradation. A number of other metalloproteases have been detected and shown to be elevated in RSV transformed fibroblasts and their role in transformed cell behavior are at present unknown. All of these metalloproteases appear to be secreted as inactive zymogens but the mechanisms involved in their activation in vivo remain unknown.
The aims of the present application is to purify and characterize a family of metalloproteases and their inhibitors in RSVCEF transformed fibroblasts. Specific anticatalytic monoclonal antibodies will be made against the metalloproteases and used as direct probes to examine the role of these enzymes in the transformed, invasive phenotype. A full length cDNA corresponding to the 70 kDa metalloprotease will be cloned from an RSVCEF cDNA expression library. The cDNA will be used to deduce the primary structure of the metalloprotease and comparatively analyzed for elucidation of the catalytic and zymogen activation sites, substrate and inhibitor binding sites and regulatory domains of the molecule. A search for the natural activator(s) of the 70 kDa metalloprotease will be initiated focusing on endogenous activators that might be present in conditioned medium, isolated cell membranes and preparations of extracellular matrix. The studies on metalloproteases, their inhibitors and activators will be extended to a human tumor cell system. We recently have purified and characterized a human 92 kDa metalloprotease and have isolated a unique, anticatalytic monoclonal antibody to this enzyme. This antibody will be added into specific assays that manifest the degradative, migratory, invasive and metastatic aspects of selective human tumor cells. Direct experimental approaches on the involvement of metalloproteases in two separate cell systems using immunological and molecular probes will help to evaluate the functional role of these enzymes in the malignant phenotype.
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