Our objective is to delineate the mechanisms by which hyperthermia damages cell structures and/or functions and to determie how this damage kills normal and malignant cells. A subset of this objective is to understand the mechanism by which cells develop resistance to subsequent hyperthermic exposures after an initial high temperature treatment. This latter phenomenon has been termed thermotolerance. The hypothesis to be tested is that the synthesis of specific proteins (e.g. the heat shock proteins) or cellular factors (specifically the ubuquitously occurring polyamines), and/or degradation of specific normal or abnormal proteins or polyamines is causatively related to sensitivity and resistance tot he cytotoxic and cytostatic effects of hyperthermia. Proposed experiments, using recombinant DNA techniques, will probe the possible role(s) of the known heat shock proteins in phenomena such as thermotolerance, the repair of hyperthermic damage and temperature sensitive growth. We will construct mammalian cells which either overproduce or do not express (using the novel technique of """"""""antisense"""""""" RNA production) these proteins at either normal or hyperthermic temperatures. Specific inhibitors and novel cell growth conditions will be used to study the synthesis of the polyamines in heat shocked cells, and investigate the possible function of the eukaryotic protein synthesis initiation factor eIF-4D, which is post-translationally modified by spermidine, in the heat shock-induced inhibition of protein synthesis and its relation to cell death. Experiments will further explore recent findings that polyamines, especially spermidine, are integral factors in regulating the degradation rate of certain proteins and will study the role of polyamines in protein degradation in cells treated with elevated temperatures. Finally, the effects of polyamine degradation by amine oxidases on cell viability responses after heat shock will be evaluated, since oxidation of these amines can lead to the production of toxic radical species. Realization of the objective of this proposal will impact current cancer therapy, as hyperthermia is becoming an accepted form of treatment for human malignancies, by discovering methods of regulating normal and tumor tissue responses to hyperthermia.
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