ONCOGENE REGULATION OF DIACYLGLYCEROL METABOLISM

甘油二酯和蛋白激酶C似乎参与了这一调节 生长因子和肿瘤促进剂对细胞增殖的影响。这个 这项提议的长期目标是确定蛋白质是否 某些癌基因的产物也可能激活这一途径。我们有 开发了一种新的检测方法来测量绝对浓度 组织培养细胞中的SN-1，2-二酰甘油。这项化验将被用于 测量由sis，fms，src， ROS、N-ras、K-ras和H-ras癌基因。脂肪酸组成和 二酰甘油的代谢将在对照和 转化的细胞。初步数据显示，SIS增加了30%到70% 转化的NRK细胞和K-ras转化的NRK细胞增加140%到170% 细胞。我们将尝试确定这些增长是否会在 甘油二酯激活蛋白激酶C从而导致易位 蛋白激酶C从胞浆到细胞膜或增加 胞浆中发现的80 kDa蛋白激酶C底物的磷酸化 3T3和NRK细胞。甘油二酯的增加可由以下因素引起 磷脂酶C介导的肌醇磷脂的水解性 降解酶活性降低，包括二酰基甘油酶 或者是脂肪酶。甘油二酯也是磷脂的一种常见中间体 和甘油三酯的合成和降解，所以这些变化 加工过程可能会导致二酰基甘油增加。这些路径将是 比较了对照细胞和转化细胞。将制备膜 从对照中，K-ras和ts-K-ras转化的细胞和膜结合 测定磷脂酶C活性以确定ras基因产物 导致磷脂酶C的直接激活。这项建议是健康的 相关，因为要检测的几种癌基因(尤其是ras) 与人类的恶性行为有牵连。二酰基甘油似乎 作为许多细胞表面受体的第二信使，所以 从这些研究中获得的信息将广泛应用于 激素作用机制、细胞分化和增殖、创伤 治疗和血管疾病。

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.