The focus of this project is to examine the mechanism(s) by which prostaglandins and linoleic acid metabolites potentiate the EGF mitogenic signal in fibroblast cell lines. In our previous work with Syrian hamster embryo (SHE) cells, we found that linoleate products stimulate EGF- dependent DNA synthesis in normal cells, but do not enhance the mitogenic response in variant SHE cells that had lost tumor suppressor gene function. This altered response to EGF in the variant cell line cannot be accounted for by differences in EGF receptor number or in production of linoleic acid metabolites between the tumor suppressor (+) and (-) phenotypes. One plausible interpretation of these results is that the tumor suppressor (-) cell line lacks the responsive element in the EGF signal transduction pathway which interacts with linoleate compounds. Our studies with SHE cells showed that loss of tumor suppressor phenotype correlates with an inability of the variant cell to increase EGF-mediated DNA synthesis in response to linoleic acid metabolites. These results suggest that linoleate products may act at control points where tumor suppressor genes interact with EGF-mitogenic signals, in particular, the protein tyrosine phosphorylation pathway. We plan to examine the effects of linoleic acid metabolites and prostaglandins on cellular protein phosphotyrosine by studying the interaction of these lipid compounds with the regulatory tyrosine phosphatase and kinase enzymes. Our preliminary data suggest that the oxygenated linoleic acid products may act by inhibiting the tyrosine phosphatase activity involved in regulation of the EGF receptor tyrosine kinase. Treatment of normal SHE cells with linoleate metabolites resulted in an increase in the magnitude and duration of EGF receptor tyrosine phosphorylation as assessed by Western analysis using anti-phosphotyrosine immunoblotting techniques. Similar effects were observed with the tyrosine phosphorylation of GAP (120 kDa) and MAP kinase (42 and 44 kDa). Using 32P-labeled synthetic substrate, direct enzyme assays in cell lysates revealed that the linoleic acid metabolites inhibited 60-70% of vanadate sensitive tyrosine phosphatase activity. In inhibiting tyrosine phosphatase activity, the effective concentrations and rank order of potency among the class of linoleic products correlates well with the previously observed actions on potentiating EGF-dependent DNA synthesis. We will also evaluate and characterize by 2-dimensional phosphoamino acid analysis and phosphopeptide mapping the effects of linoleic metabolites and prostaglandins on the tyrosine phosphorylation of cellular proteins that are activated by the EGF receptor tyrosine kinase.
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