The goal of this proposal is to analyze the mechanism by which pentoxifylline and related compounds abrogate H-ras induced aberrant signal transduction and its biological phenotype in vitro and in vivo. The different aspects of the proposal rely on preliminary evidence which demonstrates that activated H-ras(12 val) transformation upregulates the mitogenic and oncogenic phospholipids diacylglycerol(DAG) and phosphatidic acid(PA). Exposure to compounds such as pentoxifylline(PTX) inhibits the cell proliferation, colony forming capabilities and in vivo tumorigenicity associated with the transformed phenotype as well as suppressing the generation of DAG and PA by inhibiting lyso PA acyl transferase(LPAAT) and phosphatidate phosphohydrolase(PPH). This suggests specific interference with a disordered signal transduction pathway. The level at which PTX and related inhibitors of signal transduction interfere with mutant H-ras function will be examined from several perspectives. First the activation state of ras p2l in mutant and parental lines exposed to inhibitors will be examined using 32p labeling of guanine nucleotides bound to p2l followed by immunoprecipitation of p2l and thin layer chromatography of GDP/GTP. If activation state (i.e. ratio of GTP/GDP bound ras) is affected, further analysis of drug and phospholipid effects on the p2l regulators, GTPase activating and inhibiting protein, as well as rate of nucleotide exchange will be performed. The ability of LPAAT/PPH inhibitors to inhibit production of H-ras p2l will be examined by radionuclide labeling of ras p21 in conjunction with immunoprecipitation and gel electrophoresis of normal and mutant protein. The potential for these compounds to affect subcellular localization of ras p21 will be studied by radionuclide labeling, differential centrifugation and immunoprecipitation. If localization of p2l ras is affected , analysis of ras isoprenylation in the presence of LPAAT and PPH inhibitors will be carried out. Downstream effects of inhibitors on H-ras mediated signal transduction will be analyzed by quantitating phosphorylation of artificial substrates by MAP kinase, S6 kinase, CAM kinase and Protein Kinase C. The necessity for normal and mutant H-ras in the signal transduction process will be analyzed by eliminating production of normal, and activated p2l ras using specific antisense oligonucleotides. The response profile of signaling phospholipids on HPLC after stimulation with different agonists will then be examined and compared with signaling seen with intact ras.The specificity of PTX and LPAAT/PPH inhibitor effects on ras modulation of signal transduction and tumorogenictiy will be compared with effects on other families of oncogenes which affect signal transduction. These effects will be analyzed by quantitation of cell proliferation, colony forming capability and phospholipid levels in fibroblasts transformed with these activated oncogenes. This comparison will allow definition of the ubiquity of the aberrant signal transduction system that we have defined in H-ras(12 val) transformed fibroblasts and potentially the role which normal ras plays in tumorigenicity induced by these oncogenes.
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