The purpose of this proposal is to determine the effects of protease inhibitors in the abrogation of invasion by human bladder tumor cells. To study the biochemical mechanisms of bladder tumor invasion, we have previously analyzed invasive transitional cell carcinoma cell line EJ and non-invasive transitional cell carcinoma cell line RT4 which had been implanted into the bladders of nude mice. Our results demonstrated that cathepsin B, a cysteine proteinase which has the ability to degrade basement membrane laminin, is found in the plasma membrane of invasive tumor cells whereas non-invasive tumor cells have cathepsin B confined to their lysosomes. This suggests that cathepsin B is redistributed and may be used by invasive cells to degrade basement membrane components. Our finding further suggests that protease inhibitors may play a significant role in limiting the invasive potential of human bladder tumor cells. In this proposal, we will determine the effects of cysteine proteinase inhibitors in inhibiting the activity of plasma membrane bound cathepsin B at a biochemical level using enzyme overlay membranes and isoelectric focusing of subcellular fractions. Furthermore, we will determine the effects of proteinase inhibitors in inhibiting the degradation of laminin, a basement membrane component, by membrane bound cathepsin B. After our initial biochemical analyses, we will determine the effects of proteinase inhibitors in inhibiting the invasion of human bladder tumor cells through an in vitro artificial basement membrane. And finally, we will develop an in vivo model to determine the effects of protease inhibitors in bladder tumor invasion. We will attempt in these studies to determine not only the possible effectiveness of such intervention at a biochemical, cellular, and in vivo level, but also address the fundamental biochemical mechanisms of tumor cell invasion.
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