DETERMINATION OF HYPERTHERMIC CELL KILLING MECHANISMS

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Preliminary data suggests that hyperthermia-induced increases in intracellular free calcium concentrations ((Ca+2)i) are cytotoxic and contribute to the resultant cell killing. Hyperthermia induced changes in (Ca+2)i and total intracellular calcium will be measured, respectively, with the fluorescent calcium dye indo 1 and atomic absorption. Calcium cytotoxicity will be induced into nonheated cells using the calcium ionophore ionomycin, such that the cell killing level is similar to that observed following hyperthermia. If changes in intracellular calcium are similar in the heated and nonheated cells at isosurvival, altered intracellular calcium will be implicated as the, or one of the, cellular responses to hyperthermia that renders cells reproductively nonviable. A disparity in survival levels will indicate to what degree (if any) calcium cytotoxicity contributes to hyperthermic killing. Agonists and antagonists of calcium and calcium-regulated functions will be used to further establish the role of calcium in hyperthermic cell death and elucidate how calcium mediates its cytotoxic effects. Whole and permeabilized heated cells will be used to determine if (Ca+2)i increases because of a calcium influx from the extracellular medium or a release from intracellular stores. The same system will be used to determine how treated cells attempt to re-regulate calcium to homeostatic levels. (Ca+2)i will be measured in individual cell that will also be scored for the ability to form a colony in order to establish the threshold increase in (Ca+2)i required to kill a cell. Changes in the morphology and cytoplasmic distribution of the endoplasmic reticulum, mitochondria, and cytoskeleton will be monitored and quantified in these same cells to establish which aspects of the cell are damaged by altered (Ca+2)i and represent lethal lesion sites. Answers to the questions posed by this study will help determine how hyperthermia interacts with cells. This in turn will lead to a more educated application of this modality in the clinic.