The long term objective is to provide a basic understanding of hyperthermia in order to increase its therapeutic effectiveness. This investigation focuses on how changes in the glutathione concentration, the oxidation of GSH to GSSG and the oxidation of protein thiols effect thermal sensitivity as well as thermal enhancement of radiation damage.
The specific aims are: (1) To determine if depletion of intracellular glutathione modifies protein thiol/disulfide (SH/S-S) ratios during hyperthermia thereby enchancing thermal sensitivity. Changes in the number of sodium borohydride reducible protein thiols and protein mixed disulfides will be correlated to alterations in cell survival. (2) To determine the role of thiol oxidation in the regulation of intracellular calcium homeostasis during hyperthermia. Calcium levels, measured spectrophotometrically using Arsenazo III, will be determined as a function of GSH level, GSH oxidation, and protein thiol oxidation. (3) To determine if thermal sensitivity and thermal enhancement of radiation damage are affected by glutathione depletion during reoxygenation after acute chronic hypoxia. Heating plus or minus BSO will occur as a function of time after reoxgenation and cell survival determined. In some experiments, the cells will be irradiated while hypoxic and then heated after reoxygenation. (4) To determine why depletion of GSH increases thermal sensitivity in CHO cells but not in mouse LM cells. The thermal sensitivity of the 2 cell lines measured in the presence of a GSH depleting agent will be contrasted with regard to duration of exposure and the mitochondrial GSH concentration. (5) To determine if GSH depletion increases the thermal sensitivity of cells derived from a patient with 5- oxoprolinuria. The thermal sensitivity of the GSH mutant will be measured in the presence and absence of BSO and the results compared to those obtained from wild type cells. Changes in cell survival will be correlated with changes in protein thiols, mitochondrial GSH levels and calcium levels. (6) To determine if thiol oxidation triggers the synthesis of heat shock proteins and/or the development of thermotolerance. Heat shock proteins (HSP) will be measured by gel electrophoresis. (7) To determine whether depletion of GSH increases thermal enhancement of radiation damage by inhibiting repair. Single strand DNA breaks will be measured using alkali unwinding and the results correlated to changes in cell survival.
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