Translation termination a chemotherapeutic target?

描述：（申请人提供） 翻译终止是基因表达中普遍存在的步骤， 作为癌症化疗的潜在靶点几乎未被探索。的 本研究的总体目标是检验阻断 翻译终止将选择性地抑制肿瘤细胞生长。的 这一假设得到了经验观察的支持，即广谱 抗肿瘤药物吉罗达唑抑制翻译终止。此外，由于 异常高比例的mRNA编码的蛋白质参与细胞 生长对照在其上游含有开放阅读框（uORF）， 转录前导序列，翻译过程中阻断的药理学诱导 预计终止将对许多翻译造成障碍， 致癌蛋白 该项目的第一个具体目标是确定和/或设计 翻译终止抑制剂，并测试其对基因表达的影响。 在无细胞提取物中表达。专门设计的人类突变体 真核细胞释放因子1和3将被表达并测试其 诱导核糖体在终止位点停滞的能力， 肽基tRNA水解。这些药剂和药物的相对影响 吉罗达唑对含有和缺乏uORF的mRNA的细胞自由翻译的影响将 评估以确定具有uORF的mRNA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， 完整细胞中的表达和细胞生长。吉罗达唑和 真核细胞释放因子突变体，通过瞬时转染或 在诱导型启动子的控制下稳定导入细胞， 含有和缺乏uORF的mRNA的翻译以及对细胞生长的影响将 进行评估。终止抑制剂逆转恶性肿瘤的能力 表达时由HER 2/neu癌蛋白转化的细胞的性质 将确定来自含有或缺乏uORF的mRNA。

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.