The molecular mechanisms by which the model fibroblast cell, Balb/c 3T3, adheres and forms specialized contacts on fibronectin (FN)- containing extracellular matrices are being investigated, as well as alteration of these adhesion processes upon oncogenic virus (Kirsten murine sarcoma virus) transformation or oncogene (Ras/Sis/erbB) transfection. Primary and lung metastatic tumors have been isolated from nude mice using four different injection routes; the Ras oncogene display special properties in these cells. Untransformed 3T3 cells require two binding activities of FN in an intact polypeptide chain--i.e., to both heparin sulfate proteoglycan (HSPG) and to the glycoprotein complex integrin on the cell surface--in order to generate focal contacts with FN on the substratum. Adhesion processes will be investigated using a panel of complementary proteolytic fragments generated from individual plasma or transformed cellular FN subclasses containing permutations of alternately-spliced sequences. The roles of HSPG- and/or integrin-binding reactions of FN are also being tested with various tumor populations to relate changes in FN adhesion mechanisms (attachment, spreading, cytoskeletal reorganization, etc.) to the tumor-progressing phenotype and/or the state of the oncogene. We will also test whether dermatan sulfate PG inhibits attachment or facilitates detachment of tumor cells on FN more or less effectively than 3T3 cells (also tested in vivo). Using two other tumor systems as precedents, tumor-specific binding domains on FN will be assayed that may be HSPG- and integrin-independent (evaluated with sensitivity to soluble RGDS (Arg-Gly-Asp-Ser) peptide in the medium). A rapidly-progressing lung tumor cell whose adhesion of FN is resistant to high concentrations of RGDS peptide will be evaluated for its adhesion mechanisms. In parallel, RGDS sensitive primary tumor cells will be used to select cell variant that are RGDS resistant; the FN adhesion mechanisms and tumor-progressing properties of these cells will then be compared. For biochemical correlation with adhesion function studies, PGs and chains in tumor adhesion sites will be tested by affinity chromatography using various HS-binding ligands to relate reduced functional dependence upon HSPG to specific changes in the structure/catabolism of these molecules. Of related importance is the identification of non-proteoglycan protein(s) in adhesion sites that can selectively modulate binding of HSPG to FN; such a factor(s) may serve as HSPG-binding or """"""""link"""""""" proteins in a variety of functional contexts and these proteins(s) will be purified and mechanism(s) of action determined for the cells described here. In conclusion, these studies are based on the premise that understanding of FN adhesion mechanisms of these cell populations in vitro is required to provide approaches and reagents for studying and eventually regulating tumor cell/FN matrix interactions in vivo during the metastatic progression sequence.
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