EXPRESSION OF ENZYMES AND INHIBITORS OF METASTASIS

DEGRADATION OF COLLAGEN IV IN BASEMENT MEMBRANES HAS BEEN IMPLICATED IN THE PATHOGENESIS OF METASTASIS, AND IN THE PROCESS OF ANGIOGENESIS. THE 72 KD AND THE 92 KD TYPE IV COLLAGENASES ARE METALLOPROTEASES CAPABLE OF DEGRADING COLLAGEN IV WHICH HAVE BEEN SUGGESTED TO PLAY A CRITICAL ROLE IN THE ABILITY OF CELLS TO PENETRATE BASEMENT MEMBRANES. A NATURALLY OCCURRING INHIBITOR OF METALLOPROTEASES, TIMP-2 (TISSUE INHIBITOR OF METALLOPROTEASE-2), FORMS A NONCOVALENT COMPLEX WITH THE 72 KD ENZYME AND INHIBITS ITS ACTIVITY AND THE ACTIVITY OF THE 92 KD ENZYME. TO INVESTIGATE THE BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF THESE PROTEINS AND TO TEST THEIR EFFICACY FOR THE DIAGNOSTIC AND TREATMENT OF METASTASIS AND ANGIOGENIC DISEASES WE INTEND TO EXPRESS ANALYTICAL AMOUNTS OF TIMP-2 AND TYPE IV COLLAGENASES IN MAMMALIAN CELLS USING A RECOMBINANT VACCINIA VIRUS EXPRESSION SYSTEM (VAC/T7). SINCE ACTIVE TYPE IV COLLAGENASES CANNOT BE ISOLATED FROM NATIVE ENZYME-INHIBITOR COMPLEXES, PRODUCTION OF THESE PROTEINS IN THE VAC/T7 SYSTEM AS SINGLE POLYPEPTIDES WILL PROVIDE THE REAGENTS TO CHARACTERIZE THEIR BIOCHEMICAL PROPERTIES, BINDING SITES, AND INHIBITORY ACTIVITY. ALSO, THE RECOMBINANT PROTEINS WILL BE VALUABLE FOR THE CHARACTERIZATION OF MONOCLONAL ANTIBODIES. THE ABILITY TO DETECT AND TO INHIBIT TYPE IV COLLAGENASE ACTIVITY WILL BE IMPORTANT FOR THE TREATMENT AND DIAGNOSIS OF MALIGNANT DISEASE.