MODULATION OF RAS MEDIATED SIGNAL TRANSDUCTION

RAS 介导的信号转导的调节

• 批准号: 2414281
• 负责人: Bruce Montgomery
• 金额: $ 7.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-07 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414281
• 关键词: acyltransferase; antineoplastics; biological signal transduction; cell cycle proteins; diacylglycerols; enzyme activity; gene expression; guanine nucleotide binding protein; guanine nucleotides; lipid metabolism; mitogen activated protein kinase; neoplasm /cancer pharmacology; neoplastic transformation; oncogenes; oncoproteins; pentoxifylline; phosphatase inhibitor; phosphatidate phosphatase; phospholipid inhibitor; phosphonate; phosphorylation; point mutation; protein kinase C; tissue /cell culture; vasodilators

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.

这项建议的目标是分析 己酮可可碱及其相关化合物对H-ras诱发的致畸作用 体内、体外信号转导及其生物学表型。这个 提案的不同方面取决于初步证据，这些证据 表明激活的H-ras(12 Val)转换上调了 促有丝分裂和致癌磷脂二酰甘油(DAG)和磷脂 酸(PA)。暴露于己酮可可碱(PTX)等化合物可抑制 细胞增殖、集落形成能力与体内致瘤性 与转化的表型相关，并抑制 抑制赖氨酸PA酰基转移酶(LPAAT)产生DAG和PA 磷脂酸盐磷酸水解酶(PPH)。这表明存在特定的干扰 信号转导途径紊乱。PTX和PtX的水平 信号转导相关抑制剂对突变型H-ras的干扰 功能将从几个角度进行研究。第一，激活 暴露于抑制剂的突变体和亲本中ras p21的状态将 用结合p2l的鸟嘌呤核苷酸的32P标记进行检测 然后免疫沉淀p21和薄层层析 GDP/GTP。如果激活状态(即GTP/GDP界限RAS的比率)是 受影响，进一步分析药物和磷脂对p21的影响 调节剂，GTP酶激活和抑制蛋白，以及 将进行核苷酸交换。LPAAT/PPH抑制剂的作用能力 为了抑制H-ras的产生，将用放射性核素检查p21 免疫沉淀和凝胶联合标记ras-p21 正常蛋白和突变蛋白的电泳法。这些产品的潜力 影响ras p21亚细胞定位的化合物将通过 放射性核素标记、差示离心法和 免疫沉淀。如果p2l ras的本地化受到影响，分析 在LPAAT和PPH抑制剂存在下的RAS异戊二烯基化将是 被执行。抑制剂对H-ras介导信号的下游影响 转导将通过量化磷酸化来分析 由MAP、S6、CAM和蛋白质组成的人工底物 信号中正常和突变H-ras的必要性 转导过程将通过剔除正常、 并用特异性反义寡核苷酸激活p21-ras。这个 信号磷脂在高效液相色谱上的响应曲线 然后将检查不同的激动剂，并将其与所见信号进行比较 PTX和LPAAT/PPH抑制剂的特异性作用 我们将比较RAS在信号转导和肿瘤发生中的作用 对影响信号的其他癌基因家族的影响 转导。这些影响将通过对细胞的量化来分析 细胞增殖、集落形成能力和磷脂水平 用这些激活的癌基因转化成纤维细胞。这一比较 将允许定义异常信号转导的普遍存在 我们在H-ras(12val)转化的成纤维细胞中定义的系统和 潜在的正常ras在致瘤性中所起的作用 这些致癌基因。