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MODULATION OF RAS MEDIATED SIGNAL TRANSDUCTION

RAS 介导的信号转导的调节

基本信息

批准号：
2700516
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
项目类别：
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

项目摘要

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.

本提案的目的是分析喷替福林和相关化合物消除H-ras诱导的异常信号转导及其生物学表型。的该提案的不同方面依赖于初步证据，表明激活的H-ras（12瓦尔）转化上调了促有丝分裂和致癌磷脂二酰基甘油（DAG）和磷脂酸（PA）。暴露于化合物如戊茶碱（PTX）抑制了细胞增殖、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）产生DAG和PA，磷脂酸磷酸水解酶（PPH）。这表明特定的干扰信号转导通路紊乱PTX和相关信号转导抑制剂干扰突变型H-ras 将从几个角度来审查功能。首先是激活暴露于抑制剂突变株和亲本株中的rasp 2l状态将使用结合到p2 l的鸟嘌呤核苷酸的32 p标记进行检查然后进行p2 l的免疫沉淀和国内生产总值/GTP。如果激活状态（即GTP/GDP结合ras的比率）为受影响，进一步分析药物和磷脂对p2 l的影响调节剂，GT3激活和抑制蛋白，以及将进行核苷酸交换。LPAAT/PPH抑制剂用放射性核素法检测H-ras p2 l的抑制作用 rasp 21标记结合免疫沉淀和凝胶正常蛋白和突变蛋白的电泳。这些的潜力影响rasp 21亚细胞定位的化合物将通过以下方法进行研究：放射性核素标记、差速离心和免疫沉淀。如果p2 l ras的定位受到影响，在LPAAT和PPH抑制剂的存在下，ras异戊二烯化将被抑制。贯彻抑制剂对H-ras介导信号的下游影响转导将通过定量磷酸化来分析， MAP激酶、S6激酶、CAM激酶和蛋白质的人工底物激酶C正常和突变型H-ras在信号中的必要性将通过消除正常，并使用特异性反义寡核苷酸激活p2 L-ras。的用以下物质刺激后，信号传导磷脂在HPLC上的响应曲线然后检查不同的激动剂并与所见的信号传导进行比较 PTX和LPAAT/PPH抑制剂的特异性作用， ras对信号转导的调节与肿瘤发生的关系对其他影响信号传导的癌基因家族有影响转导这些影响将通过定量细胞增殖，集落形成能力和磷脂水平，成纤维细胞转化这些激活的癌基因。该比较将允许定义异常信号转导的普遍性我们已经在H-ras（12瓦尔）转化的成纤维细胞中定义了该系统，可能正常ras在肿瘤诱导中的作用这些致癌基因。