Hyperthermia is increasingly used in conjunction with radiotherapy for the treatment of human malignancy. The rationale for combining these two modalities in clinical protocols for the treatment of cancer rests on the observation that heat radiosensitizes and kills preferentially cells that are resistant to radiation alone. Elucidation of the molecular mechanism(s) underlying heat radiosensitization might provide information useful in the development of such combined treatment protocols. The long term objective of the present proposal is, therefore, to identify the molecular processes that underlie the phenomenon of heat induced radiosensitization and to elucidate the mechanism(s) involved. The central hypothesis on which this proposal rests is that heat sensitizes cells to radiation by increasing the probability of conversion of DNA double strand breaks (dsb) to lethal chromosome aberrations. The enhancement in the conversion probability of DNA dsb to chromosome aberrations is assumed to be caused either by alterations in chromatin structure and function, known to occur after acute exposures to heat, and/or by a reduction in the efficacy of repair. Alterations in chromatin structure and function, invoked to explain radiosensitization induced by pre-irradiation exposure to heat at 43-45.5 degrees C, may lead either to structural instability of irradiated DNA molecules, or to a decrease in the accessibility of radiation induced DNA dsb to repair enzymes that results in an increase in residual chromosome damage. Reduction in the efficacy of the repair system is mainly invoked to explain radiosensitization by postirradiation exposure to heat at 41-43 degrees C. The importance of DNA dsb in heat radiosensitization will be evaluated with radiation sensitive mutants showing reduced ability to repair this lesion, whereas alterations in the conversion of DNA dsb to chromosome damage will be assessed by measuring, on the one hand, induction and repair of DNA dsb using the non-unwinding filter elution technique or pulse field gel electrophoresis and, on the other hand, induction and repair of chromosome damage in interphase cells using the technique of premature chromosome condensation (PCC). Specifically, experiments will be carried out to investigate heat radiosensitization in radiation sensitive mutants of CHO (xrs-5) and V-79 (irs-1, irs-2, irs-3) cells that show different ability to repair DNA dsb, and the effect observed at the cell level will be correlated with the degree of repair deficiency as well as with the effect of heat on DNA and chromosome repair. Furthermore, experiments will be carried out to investigate heat radiosensitization in repair proficient cells and to correlate the effect of heat on the induction and particularly on the repair of radiation damage in the DNA (dsb) and the chromosomes. The results obtained are expected to evaluate the importance of DNA dsb in the phenomenon of heat radiosensitization and will be combined to construct models of the molecular processes underlying this phenomenon.
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