Matrix metalloproteinases (MMPs) have long been implicated in the degradation of the extracellular matrix (ECM) during tumor invasion and metastasis. One of the early steps to the invasion process is the localized dissolution of the ECM, to create a pathway for cell migration. A site-specific cleavage of fibrillar collagen by collagenase is necessary to begin the degradation process and to prepare the collagen for further proteolysis. Our focus in this project is on the in vivo events that activate collagenase from its normally latent form to its enzymatically active form. Although our laboratory and others have studied the in vitro activation of collagenase and other MMPs, little is known about this process in tumor tissue. The hypothesis to be tested is that the collagenase secreted by oral tumor cells is more easily activated than in normal oral tissue. This alteration in the activation process could result from secretion by tumor cells of an activator of collagenase (for example, a proteolytic activator, such as another MMP like stromelysin-1 or gelatinase) or by disruption of the balance between collagenase and its natural inhibitors such as the tissue inhibitors of metalloproteinases; TIMPs). Over the last eight years, we have accumulated both cDNA probes and unique antibody reagents for studying this process, and we propose to apply these materials to this study of collagenase activation in vivo in oral tumors. We propose three specific aims to address the hypothesis proposed for this study: 1. Do tumors contain activated collagenase and other MMPs? We will combine previous in situ hybridization and immunochemistry approaches to detect MMP expression in oral tumor material with a novel new spot degradation assay to measure dissolution of collagen films underneath spots of primary cells grown from oral tumors. Preliminary results obtained with SCC-25 cells (derived from an oral squamous cell carcinoma) have shown detectable levels of activated collagenase and we propose to determine whether most oral tumors contain activated collagenase. 2. How is collagenase activated in oral tumor cells? We will purify intermediates in the activation process. If the intermediates are proteolytically processed, we will determine the sites of cleavage and identify the activating agent (collagenase itself, other MMPs, other proteases). 3. Do other MMPs play a role in collagenase activation in oral tumors? We will use inhibitory antibodies that we have on hand to block specific MMPs or MMP inhibitors that might be found in the cell culture medium of our spot collagen degradation assay. Synthetic MMP inhibitors will also be used as a confirmatory approach.
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