ONCOGENE REGULATION OF DIACYLGLYCEROL METABOLISM

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Diacylglycerol and protein kinase C appear to be involved in the regulation of cell proliferation effected by growth factors and tumor promoters. The long range objective of this proposal is to determine whether the protein products of certain oncogenes may also activate this pathway. We have developed a novel assay to measure absolute concentrations of sn-1,2-diacylglycerol in tissue culture cells. This assay will be used to measure diacylglycerol levels in cells transformed by the sis, fms, src, ros, N-ras, K-ras and H-ras oncogenes. The fatty acid composition and metabolism of the diacylglycerols will be determined in the control and transformed cells. Initial data demonstrate a 30 to 70% increase in sis transformed NRK cells and a 140 to 170% increase in K-ras transformed NRK cells. We will attempt to determine whether these increases in diacylglycerol activate protein kinase C and thereby lead to translocation of protein kinase C from the cytosol to the membrane or to increased phosphorylation of an 80kDa protein kinase C substrate found in the cytosol of 3T3 and NRK cells. An increase in diacylglycerol can be caused by phospholipase C-mediated hydrolysis of inositol phospholipids or by decreased activity of degradative enzymes including diacylglycerol kinase or lipase. Diacylglycerol is also an ordinary intermediate in phospholipid and triglyceride synthesis and degradation, so alterations in these processes could lead to increased diacylglycerol. These pathways will be compared in the control and transformed cells. Membranes will be prepared from control, K-ras and ts-K-ras transformed cells and membrane-bound phospholipase C activity measured to determine if the ras gene product leads to direct activation of phospholipase C. The proposal is health related because several of the oncogenes to be tested (particularly ras) have been implicated in human malignancy. Diacylglycerol appears to function as a second messenger for many cell surface receptors, so information gained from these studies will have wide application to mechanisms of hormone action, cell differentiation and proliferation, wound healing and vascular disease.