MODULATION OF RAS MEDIATED SIGNAL TRANSDUCTION

RAS 介导的信号转导的调节

• 批准号: 2101546
• 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/2101546
• 关键词: acyltransferase; antineoplastics; biological signal transduction; cell cycle proteins; diacylglycerols; enzyme activity; gene expression; guanine nucleotide binding protein; guanine nucleotides; high performance liquid chromatography; immunoprecipitation; lipid metabolism; neoplasm /cancer pharmacology; neoplastic transformation; oncogenes; oncoproteins; 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.

该提案的目的是分析其机制 己酮可可碱和相关化合物消除 H-ras 诱导的异常 信号转导及其体外和体内生物学表型。这 该提案的不同方面依赖于初步证据 表明激活的 H-ras(12 val) 转化上调 促有丝分裂和致癌磷脂二酰甘油 (DAG) 和磷脂 酸（PA）。接触己酮可可碱 (PTX) 等化合物会抑制 细胞增殖、集落形成能力和体内致瘤性 与转化的表型相关以及抑制 通过抑制溶血 PA 酰基转移酶 (LPAAT) 生成 DAG 和 PA 磷脂酸磷酸水解酶（PPH）。这表明特定的干扰 信号转导途径紊乱。 PTX 和的水平 相关信号转导抑制剂干扰突变型 H-ras 功能将从多个角度进行考察。首先是激活 暴露于抑制剂的突变体和亲本系中 ras p2l 的状态将 使用与 p2l 结合的鸟嘌呤核苷酸的 32p 标记进行检查 然后进行 p2l 的免疫沉淀和薄层色谱 GDP/GTP。如果激活状态（即 GTP/GDP 结合 ras 的比率）为 影响，进一步分析药物和磷脂对p2l的影响 调节剂、GTPase 激活和抑制蛋白，以及速率 将进行核苷酸交换。 LPAAT/PPH抑制剂的能力 抑制 H-ras p2l 的产生将通过放射性核素进行检查 结合免疫沉淀和凝胶标记 ras p21 正常和突变蛋白的电泳。这些的潜力 将研究影响 ras p21 亚细胞定位的化合物 放射性核素标记、差速离心和 免疫沉淀。如果p2l ras的定位受到影响，则分析 在 LPAAT 和 PPH 抑制剂存在下，ras 异戊二烯化将 执行。抑制剂对 H-ras 介导信号的下游影响 将通过定量磷酸化来分析转导 MAP 激酶、S6 激酶、CAM 激酶和蛋白质的人工底物 激酶 C. 信号中正常和突变 H-ras 的必要性 将通过消除正常的产生来分析转导过程， 并使用特定的反义寡核苷酸激活p2l ras。这 刺激后信号磷脂在 HPLC 上的响应曲线 然后将检查不同的激动剂并与所观察到的信号进行比较 PTX 和 LPAAT/PPH 抑制剂的特异性对 ras 的影响 ras对信号转导的调节和致瘤性将进行比较 对影响信号的其他癌基因家族有影响 转导。这些影响将通过细胞定量来分析 增殖、集落形成能力和磷脂水平 用这些激活的癌基因转化的成纤维细胞。这个比较 将允许定义异常信号转导的普遍性 我们在 H-ras(12 val) 转化的成纤维细胞中定义的系统和 正常 ras 在致瘤性中所起的潜在作用 这些致癌基因。