Angiostatin is a potent inhibitor of metastatic growth of some solid tumors. It is derived by proteolysis of plasminogen, and consists of the kringle 1-4 domain of plasminogen. Although the enzyme that converts plasminogen to angiostatin is expressed by some primary tumors, the identity of this enzyme, and which tumors express the enzyme remain unknown. We have demonstrated that the PC-3 and DU-145 human prostate cancer lines express the enzyme (or enzymes) necessary and sufficient for angiostatin generation, and this is tentatively designated as plasminogen- angiostatin converting enzyme (PACE). Additional data indicate that PACE activity is a serine protease. In preliminary experiments several purification tools have been identified and characterized which will allow for purification of PACE during this project. These include Reactive Red 120-Agarose (Dye-Ligand chromatography), Hi-Q-Sepharose (anion exchange chromatography) and soybean trypsin inhibitor-sepharose (affinity chromatography). The angiostatin generated in vitro by PC-3 cells is bioactive based on inhibition of endothelial cell proliferation and migration. Angiostatin antigen has been demonstrated in the plasma of athymic mice bearing PC-3 tumors. This proposal is to utilize the purification tools identified to complete the purification of the PACE enzyme or enzymes. If the enzyme is novel, the cDNA will be cloned. Monoclonal antibodies to PACE will be generated which will facilitate development of immunoassays and immunohistochemical staining for PACE. The kinetics of the interaction between PACE and plasminogen in vitro, as well as potential antiangiogenic and anti-metastatic effects of PACE administration in vivo will be analyzed.
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