The long term objective of this grant proposal is to provide a basic understanding of hyperthermia in order to increase its therapeutic effectiveness. The mechanisms which regulate thermal sensitivity and hyperthermic enhancement of radiation sensitivity are not well understood. Both, however, can be regulated by the intracellular glutathione concentration and redox ratio.
Specific aim 1 tests the hypothesis that glutathione acts in an antioxidant capacity during hyperthermic treatment. The intracellular hydrogen peroxide concentration will be measured in control and glutathione depleted cells incubated at temperatures ranging from 37o to 46oC. The role of iron redox cycling in thermal sensitivity will also be investigated. Compounds which mimic superoxide dismutase and human embryonic kidney cells transfected with superoxide dismutase will be used to investigate the relationship between glutathione concentrations and superoxide dismutase activity during hyperthermia.
Specific aim 2 tests the hypothesis that enhanced synthesis of heat shock proteins and the development of thermotolerance protects cells from oxidative damage. This hypothesis will be tested using the glucocorticoid receptor as a model. The results obtained will be compared to those obtained when the receptor is inactivated by heat shock.
Aim 3 seeks to determine if thiol oxidation triggers the synthesis of heat shock proteins and/or the development of thermotolerance. Experiments have been designed to determine if there is a correlation between the rate of GSSG formation measured during oxidant challenge and enhanced synthesis of the 110, 90, 73/72, and 27 kDa heat shock proteins. Northern analysis of mRNA, nuclear run-offs, and in vitro translation will be used to determine if enhanced synthesis is due to increased transcription of heat shock genes. If transcription is increased, then a gel mobility-shift assay will be used to determine if thiol oxidation can induce binding activity specific to a region of the element of the heat shock 70 promoter.
Aim 4 is designed to investigate the regulation of the intracellular glutathione concentration. Cells will be exposed to xenobiotics such as BSO or adriamycin and the rate of GSH synthesis, the activity and mRNA levels of gamma-glutamyl transpeptidase and gamma-glutamyl cysteine synthetase, the activity of GSH reductase, rate of GSH turnover and efflux will be quantified.
Aim 5 will test the hypothesis that glutathione metabolism is involved in the enzymatic repair of aerobic radiation damage. The role of iron and Se-dependent and independent peroxidase activity in the expression of radiation damage, as measured by cell survival and the formation and repair of DNA double strand breaks, will be determined. The purpose of this aim is to obtain information which can be applied toward understanding how glutathione depletion can increase thermal radiosensitivity.
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