The present proposal builds on several significant findings resulting from the research supported by this award. During the course of these studies we have isolated a 37KD chymotryptic fragment, containing the so-called """"""""synergistic"""""""" cell adhesion-promoting activity of fibronectin (FN). We now plan to map and characterize this site using monoclonal antibodies (MAbs), recently generated in our laboratory, and soluble proteolytic fragments or recombinant peptides in adhesion/motility promoting or blocking experiments. The putative novel cell adhesion site within the 40KD recombinant peptide, described in our progress report, will be similarly characterized; important questions are whether it acts synergistically or independently of the RGD (Gly-Arg-Asp) site, and the nature of its cell-type specificity. Once the two new cell adhesion promoting sites have been precisely mapped, fusion peptides containing multiple adhesion sites will be generated by recombinant-DNA technology and pre-screened in adhesion/motility assays for possible testing in metastasis assays. Based on our finding that murine melanoma and embryo neurites, both neural crest derived cells, bind to FN via the sites within the IIICS, an important question to be addressed is whether these sites can also promote the adhesion of cells, normal or cancer, derived from non-neuronal tissue. For example, if we are successful in identification of a novel or cell-type specific adhesion site that does not interact with receptors on bone marrow progenitor cells, then peptides containing this site will be used in bone marrow purging experimental protocols. Finally, the above peptides with adhesion/motility-promoting activity comparable to that of intact FN, e.g., the 37KD and 40KD peptides, will be tested for their antimetastatic activity; peptides effective in inhibiting metastasis will be used to investigate the potential for visualizing the disruption of FN-mediated interactions following intravenous injection of B16-F1O melanoma cells using light and electron microscopic techniques. These studies will be important in elucidating the mechanism(s) of tumor cell adhesion to fibronectin and the role of these interactions in metastasis and invasion.
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