The goal of these studies is to determine the molecular basis for the pronounced radioresistance of cultured TN-368 lepidopteran insect cells. The experimental approach is to examine several specific repair and recovery processess both on the molecular and cellular levels in the TN-368 cell line (and V79 mammalian cell line where appropriate) following gamma-ray and 254 nm ultraviolet light treatments. Molecular studies to be performed include an assessment of the effect of these treatments on DNA synthesis, the direct measurement of pyrimidine dimers, the measurement of pyrimidine-pyrimidone (6-4) photoproducts by radioimmunossay, postreplication repair studies using the alkaline elution technique, and the determination of oxidative protective enzyme levels by spectrophotometric methods. These molecular investigations will be performed in parallel with survival experiments concerned with cellular recovery processes, especially enhanced inducible recovery, assayed by colony formation. Cellular sensitivity to a variety of other agents such as 8-methoxypsoralen plus UVA light, heat, and UV and gamma- ray mimetic chemicals, will be determined. These agents will also be utilized to ascertain their ability to activate the inducible recovery process in the TN-368 cells. UV and gamma radiation as well as many of these other treatments will be used to determine whether the TN-368 cells demonstrate an adaptive response. TN- 368 cell mutants sensitive to ultraviolet light and gamma radiation will be isolated for the purpose of determining differences in the repair processes between wildtype and mutant cells. A major task related to the characterization of sensitive mutants will be the development of a mutation assay for determination of mutation frequencies in the TN-368 cells. TN- 368 cells will be fused with V79 or WR69-DM-1 cells to form hybrids in which it should be possible to focus on the genetic material responsible for radio resistance and make it easier to determine mechanisms of radioresistance. The results of the proposed experiments are expected to produce important information on molecular and cellular mechanisms for dealing with radiation damage which may be applied to mammalian cells. As repair mechanisms appear to play a significant role in radioresistance, these studies should be of value in better understanding mechanisms of mutagenesis and carcinogenesis. These studies should also be valuable in learning more about radioresistant tumors and mechanisms of multidrug resistance in tumor cell populations.
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