Translation termination a chemotherapeutic target?

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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.