Proteolytic enzymes have been implicated in invasive steps of the metastatic cascade. Our long term objective is to determine the role(s) of cysteine proteinases (in particular cathepsin B) and endogenous low Mr cysteine proteinase inhibitors (CPI) in tumor cell metastasis. Increases activities of several cysteine proteinases, e.g., cathepsin B and cancer procoagulant, have been reported in both human and animal tumors. Such enhanced activities could be due to several factors including changes in tumor CPI. The CPI that are the most effective inhibitors of cathepsin B are CPI of the stefin family (stefin A being more potent than stefin B). The present proposal is designed to test the hypothesis that changes in CPI in tumors may be responsible for enhanced cysteine proteinase activities in tumors. Specifically, we will 1) purify tumor CPI and determine their inhibitory properties towards cysteine proteinases from normal and tumor tissue, 2) determine whether the subcellular loclaization of CPI in tumors is changed from the cytoplasm (as in normal tissue) to the plasma membrane and 3) determine whether such a change affects the inhibitory properties of tumor CPIs. Recently, the ras gene product p21 was shown to possess CPI activity against partially purified rat kidney cathepsins B and L. Therefore we will determine the immunological relatedness of p21s and tumor CPI, including membrane-associated tumor CPI, and the mechanism of interaction of p21s with cysteine proteinases (normal and tumor). Using cell lines transfected with ras (of known metastatic capability) we will compare the lines for levels of p21 and CPI and for CPI activities. The same cell lines will be injected concurrently in nude mice in our laboratories to allow us to directly compare their tumorigenicity and metastatic (spontaneous) capabilities with CPI levels and activities. In collaboration with Dr. Guy Salvesen we will obtain cDNA probes to enable us to address the question of changes in the expression of CPI in these same ras transfected cell lines. The proposed characterization of tumor CPIs should further our understanding of the role(s) of cysteine proteinases in a proteolytic casade resulting in metastasis. Our eventual goal is to interrupt this cascade by designing more effective inhibitors or by altering the expression and subcellular localization of endogenous CPI.
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