Our specific aims are to analyze whether transfection of vascular endothelial cells with bFGF cDNA expression vector could lead to autonomous proliferation of vascular endothelial cells and their transformation into neoplastic cells. We will first analyze whether bFGF would act intracellularly (as most oncogene products do) or need to be released by the cells in order to interact with exposed cell surface receptors. We will also analyze its cellular localization, in order to determine whether it could ultimately become associated with the nuclear, cytosol, or plasma membrane fraction. Since cell transformation can be the direct result of oncogene expression, we will analyze in transfected vascular endothelial cells whether constitutive expression of c fos, c myc and c ras oncogenes can be observed. Whether the bFGF gene product could be released from the cells in association with ECM components such as heparan sulfate GAG, for which the mitogen should have a high affinity, will be assessed. The effect of TGFB and TNF on the growth of parental and transfected cells in soft agar, as well as PA expression, will be analyzed in order to determine whether they could have effects similar to those reported when cells are grown on solid substrates. Monoclonal antibodies against the FGF receptor will be obtained and used for the development of sensitive RIA for the FGF receptor, and studies of its expression. They will also be used to isolate FGF receptors by immunoaffinity chromatography. The FGF receptor will then be characterized structurally, and the information gained will be used to develop sepecific oligonucleotide probes, which will be used for the cloning of 2 FGF receptor cDNA.
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