The importance of collagenases in tumor invasion and metastasis is well establised but mechanisms able to control the activity of these enzymes in tumors have not yet been explored. Studies of mechanisms of inhibition of these enzymes may be of importance to prevent tumor invasion. We have recently shown that bovine endothelial cells inhibit the collagenolytic activities normally displayed by human tumor cell lines. Subsequently we found 2 types of collagenase inhibitors in serum free medium conditioned by endothelial cells. Whereas one of these inhibitors with a molecular weight of 28,500 is similar to the ubiquitous tissue inhibitor of metalloproteinase (TIMP), the second has a molecular weight of approximately 70,000 and may be unique to endothelial cells. The role of these inhibitors in tumor invasion and metastasis will be investigated. The high molecular weight inhibitor will be isolated and purified from endothelial cell conditioned medium using various chromatographic procedures and its inhibitory activity against various collagenases including type IV specific collogenase will be tested. This will allow further comparision with other known inhibitors of collagenases. The low molecular weight inhibitor (TIMP) will be obtained as a recombinant protein. The ability of the purified high molecular weight and the recombinant TIMP to suppress the degradative and invasive activities of human tumor cells in vitro will then be explored. Rabbit polyclonal antibodies against these inhibitors will be raised and used to study their production by bovine endothelial cells of various origins (venous, arterial, capillary). The effect of phorbol diesters and cytochalasin D on the production of these inhibitors by endothelial cells will be investigated and the ability of tumor cells to secrete soluble factors influencing their biosynthesis by endothelial cells will be analyzed. These studies, will bring important and new information on the inhibition and modulation of tumor cell invasion and/or metastasis by endothelial cells.
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