Radiation therapy is an important treatment modality for cancer. Its effectiveness has been restricted, however, by the inherent resistance of certain tumor cells to ionizing radiation. Although diverse factors dictate the response to radiation, this study will focus on the importance of the product of the human proto-oncogene c-raf-1 (Raf-1) in the cellular response to gamma radiation. The observation of the negative regulation of radiation resistance in human tumor cells transfected with antisense c-raf-1 cDNA provides a basis for the hypothesis that radiation resistance is linked to the function of Raf-1 serine/threonine protein kinase. Because radiation induces transcription of certain genes, and because an important transcription factor, AP-1, requires Raf protein kinase, the second hypothesis is that the gamma-radiation modulates Raf-1 protein kinase. The kinase, in turn, may play a role in the post-irradiation changes in cell cycle, DNA synthesis and/or transcription. Renal cell carcinoma (RCC), a progressive and relatively radioresistant disease, will be used as a human tumor cell model to examine the role of Raf-1 in the regulation of radiation resistance (AIM 1), to determine the effects of gamma-radiation on Raf-1 biochemistry and function (AIM 2), and to examine the radiobiological oncogene-mediated transformation of normal renal cells (AIM 3). Raf-1 function will be regulated by the expression of novel Raf-specific dominant negative mutants containing deletions or subtle amino acid substitutions, and sense/antisense c-raf-1 cDNA. The expression, posttranslational modification, serine/threonine kinase activity, subcellular localization and the cell cycle regulation of Raf-1 will be examined. Radiobiological responses analyzed will include the radiation survival dose response, and the induction and repair of sublethal damage, potentially lethal damage, and DNA strand breaks. These studies will (i) generate a better understanding of the role of Raf-1 protein kinase in the regulation of radiation resistance and (ii) advance our knowledge of the molecular targets and mechanically, of the action of ionizing radiation. The current investigations should lead us to begin to elucidate substrates of Raf-1 protein kinase in the intracellular signal transduction pathway(s) to radioresistance. The information gained may facilitate the development of new strategies for effective radiotherapy.
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