Recent evidence has identified a cathepsin B-like cysteine proteinase (CB) which may facilitate the processes of humor invasion and metastasis. CB from liver has been shown to degrade collagen and proteoglycans and to activate latent collagenase. Thus CB may act in tumor metastasis by enhancing the ability of tumor cells to invade surrounding tissue at primary and metastatic sites and to intravasate and extravasate. We will purify CB from subcutaneous tumors of the murine B16 amelanotic melanoma (B16a) and from B16a - conditioned culture media. CB released from tumors is apparently a proenzyme which has increased stability at neutral and alkaline pH. These enzymes will be characterized for their physical and kinetic properties. Proteolytic activation of proCB will be assessed using active CB, plasmin, plasminogen activator, kallikrein etc. Monoclonal antibodies to active CB, and to proCB will be developed and their cross-reactivities tested against one another as well as against CB purified from other murine and human tumors, other tissues, papain, and cathepsins H and L. Endogenous cysteine proteinases inhibitors will be purified and tested along with synthetic inhibitors for their ability to inactivate CB and proCB. Using purified CB and proCB we will determine their ability to degrade, in the presence and absence of monoclonal antibodies and cysteine proteinase inhibitors, components of the extracellular matrix [collagen (Types I-V), laminin, fibronectin]. The ability of CB and proCB to activate tumor-derived latent type IV collagenase will also be determined. In vitro assays to study invasion of tumor cells through an endothelial monolayer and through amnion-derived basement membrane will be established. Invasion by 125I-Udr labeled tumor cells will be followed in the presence and absence of monoclonal antibodies to CB and proCB and of cysteine proteinase inhibitors. These studies should enable us to assess the presumptive role of CB in tumor cell metastasis and to assess whether CB may be one proteolytic enzyme in a metastatic proteolytic cascade. For example, plasmin derived from the action of plasminogen activator (PA) on plasminogen may activate proCB and CB in turn may activate latent type IV collagenase. It might be possible to interrupt such a proteolytic cascade by directing therapeutic intervention toward preventing release of proCB or towards the pro frament of proCB (since proCB does not seem to be released from normal cells).

National Institute of Health (NIH)
National Cancer Institute (NCI)
Modified Research Career Development Award (K04)
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Cognition and Perception Study Section (CP)
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Wayne State University
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