Translation termination a chemotherapeutic target?

描述：(申请人提供) 终止翻译是基因表达中普遍存在的一个步骤，它具有 作为癌症化疗的潜在靶点，几乎没有人研究过。这个 这项研究的总体目标是检验阻断的假设 翻译终止将选择性地抑制肿瘤细胞的生长。这个 这一假设得到了广谱的经验观察的支持 抗肿瘤药物格罗达唑抑制翻译终止。同样，由于一个 编码与细胞有关的蛋白质的mRNAs比例异常高 生长控制包含上游开放阅读框架(UORF) 转录引导者，翻译过程中药物诱导的封锁 预计终止协议将对许多 致癌蛋白。 该项目的第一个具体目标是识别和/或设计 翻译终止抑制剂及其对基因的影响 在无细胞提取物中表达。专门设计的人类突变体 真核释放因子1和3将被表达并检测其 在终止部位诱导核糖体停滞和抑制的能力 肽基tRNA水解液。这些药物和药物的相对影响 格罗达唑对含和不含uORF的mRNAs自由翻译的影响 评估以确定带有uORF的mRNAs是否对 终止抑制。 第二个目的是确定抑制终止对基因的影响 在完整细胞中的表达和细胞生长。格罗达唑和阿司匹林的药效 真核释放因子突变体，通过瞬时转染法或通过 在可诱导启动子的控制下稳定地导入细胞，on 含有和缺乏uORF的mRNAs的翻译以及对细胞生长的影响 被评估。终止抑制物逆转恶性肿瘤的能力 HER2/neu癌蛋白转化细胞的表达特性 从含有或不含有uORF的信使核糖核酸中确定。

DESCRIPTION: (provided by applicant) Termination of translation is a ubiquitous step in gene expression that has been virtually unexplored as a potential target for cancer chemotherapy. The overall goal of this research is to test the hypothesis that blocking translation termination will selectively inhibit tumor cell growth. The hypothesis is supported by the empirical observation that the broad spectrum anti-tumor drug girodazole inhibits translation termination. As well, since an unusually high percentage of mRNAs that encode proteins involved in cellular growth control contain upstream open reading frames (uORFs) in their transcript leaders, pharmacological induction of a blockade during translation termination would be expected to create a barrier to translation of many oncogenic proteins. The first specific aim of this project is to identify and/or engineer inhibitors of translation termination and to test their effects on gene expression in cell free extracts. Specifically designed mutants of human eukaryotic release factors 1 and 3 will be expressed and tested for their ability to induce ribosomal stalling at termination sites and to inhibit peptidyl tRNA hydrolysis. The relative impact of these agents and of the drug girodazole on cell free translation of mRNAs containing and lacking uORFs will be evaluated to determine whether mRNAs with uORFs are hypersensitive to termination inhibition. The second aim is to determine the impact of inhibiting termination on gene expression and cell growth in intact cells. The effects of girodazole and of eukaryotic release factor mutants, expressed by transient transfection or by stable introduction into cells under control of an inducible promoter, on translation of mRNAs containing and lacking uORFs and on cellular growth will be assessed. The ability of termination inhibitors to reverse the malignant properties of cells transformed by the HER2/neu oncoprotein when expressed from an mRNA containing or lacking a uORF will be determined.