PHOSPHATIDYLCHOLINE METABOLISM IN RAS-TRANSFECTED CELLS

这项提议试图调查改变的机制。 人H-ras基因转导细胞的磷脂酰胆碱代谢 致癌基因。磷脂酰胆碱的降解与多种途径有关 Ras癌基因的蛋白产物是 被认为在信号转导途径中起作用。我们最近 C3H10T1/2小鼠成纤维细胞磷脂酰胆碱代谢的研究 与H-ras细胞相比，H-ras细胞有四个显著差异 正常和ras转基因细胞之间的差异：磷胆碱水平升高 胆碱激酶活性升高，甘油磷胆碱水平升高， 胞苷转移酶活性降低。我们的工作假设是 RAS转换直接或间接地导致激活 磷脂酶A作为增加磷脂酰胆碱的机制 合成反应磷脂酰胆碱的降解增加， 胆碱激酶活性增加。胞苷转移酶活性为 然后通过微调途径降低。 在这个提案中，我们将测试这一假设的各个方面。我们会 将ras基因置于可诱导启动子的控制下，以便 确定这些变化发生的时间顺序 Ras的表达。磷脂酶A、C和D的活性将是 在对照C3H10T1/2细胞和ras感染细胞中检测到。这个 胆碱激酶和胆碱酯酶激活的机制 将研究ras转化细胞胞苷转移酶的变化。 此外，我们还将讨论磷脂酶A的可能作用 Ras在转化机制中的激活。我们将确定是否 抑制因子或ras中磷脂酶A活性的抑制 转化还可以抑制ras-1的磷脂酶A活性。 转化的细胞。这些实验的结果将有助于阐明 磷脂酰胆碱生物合成的协调调节机制 并有助于理解磷脂酶活性在 RAS变换。

This proposal seeks to investigate the mechanisms of altered phosphatidylcholine metabolism in cells transfected by the human H-ras oncogene. Phosphatidylcholine hydrolysis has been implicated in pathways of signal transduction, and the protein product of the ras oncogene is thought to play a role in a signal transduction pathway. Our recent studies on phosphatidylcholine metabolism in C3H10T1/2 murine fibroblasts transfected with the H-ras cells have revealed four marked differences between normal and ras-transfected cells: elevated phosphocholine levels elevated choline kinase activity, elevated glycerophosphocholine levels, and decreased cytidylyltransferase activity. Our working hypothesis is that ras transformation results, directly or indirectly, in activation of a phospholipase A. As a mechanism for increasing phosphatidylcholine synthesis in response to the increased degradation of phosphatidylcholine, choline kinase activity is increased. Cytidylyltransferase activity is then decreased by way of fine-tuning the pathway. In this proposal we will test various aspects of this hypothesis. We will put the ras gene under the control of an inducible promoter to allow determination of the temporal order in which these changes take place upon expression of ras. The activities of phospholipases A, C, and D will be determined in control C3H10T1/2 cells and in ras-infected cells. The mechanisms by which the activities of choline kinase and cytidylyltransferase change in ras-transformed cells will be investigated. In addition, we will address the possible role of phospholipase A activation in the mechanism of transformation by ras. We will determine if inhibition of phospholipase A activity in a suppressor or ras transformation can also suppress phospholipase A activities in ras- transformed cells. Results from these experiments will help elucidate mechanisms of coordinate regulation of phosphatidylcholine biosynthesis, and lead toward an understanding of the role of phospholipase activity in ras transformation.