The plasminogen activator urokinase is overexpressed in many malignancies including that of the colon. Urokinase may be involved in tumor metastases as evidenced by the ability of anti-urokinase antibodies to block this phenomenon in chicken embryos. A specific binding site for the plasminogen activator has been identified and may be crucial for the actions of urokinase. Although the cellular controls of urokinase and its receptor are poorly understood, they represent ideal therapeutic targets and may ultimately yield anti-metastatic agents. The regulation of expression of urokinase and its receptor in colon cancer will be investigated. Poorly differentiated colon cancer cells, but not their well differentiated counterparts, express elevated levels of urokinase in a constitutive manner. These cells are characterized as secreting and responding to TGF- a. The objective of specific aim #1 is to determine whether constitutive production of this plasminogen activator in some colon cancers is a consequence of stimulation by autocrine TGF-a. Poorly differentiated cells display 10X more urokinase receptors than well differentiated cell types. Preliminary studies show that urokinase is a negative regulator of its receptor in well differentiated cells. The ability of urokinase to reduce receptor number in poorly differentiated cells will be determined in specific aim #2. Studies will be undertaken to determine what part of the urokinase molecule is responsible for receptor modulation. These binding sites may fix the plasminogen activator on the cell surface, thereby facilitating local proteolysis. Indeed, all of the cultured colon cancer cell lines degrade laminin, in the presence of plasminogen. The anti- laminin degrading activity of a peptide which binds to the receptor, but has no active site, will be tested in specific aim #3. Analysis of receptor expression is currently hampered by the lack of molecular probes such as antibodies and cDNA's. This is due, in part, to the lack of purified receptor.
In specific aim #4, the urokinase receptor will be purified to homogeneity from a colon cancer cell line. Monoclonal antibodies will be raised against the binding site.
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