The objective of the proposed research is to determine what role(s), if any, the heat-induced increase in specific proteins associated with the nucleus is played in cell killing by hyperthermia. The specific approach will be to conduct multivariate analysis over an extensive series of modifications of thermal sensitivity to determine which parameters remain correlated with the extent of cell killing. Thermal sensitivity modifiers will be chemical, physiological, including thermotolerance and step-down heating, and genetic, namely, heat resistant variants. The logic used to test the hypothesis is essentially inductive, i.e., we will conclude that the parameter that remains tightly correlated with heat-induced cell killing over a wide range of thermal sensitivities is probably a measure of some step in the killing process. Specifically we will: 1. Determine if the correlation between heat-induced cell killing and excess nuclear protein content integrated over the time of its association with the nucleus conserved under conditions that modify thermal sensitivity.
This aim will be accomplished by exposing modified cells to graded doses of hyperthermia and measuring both clonogenic cell survival and the amount of heat-induced excess nuclear protein and its rate of removal. 2. Determine if the changes in the amounts of specific proteins associated with the nucleus after heat shock correlate with cell killing in normal and thermotolerant cells.
This aim will be accomplished by analyzing nuclear proteins by polyacrylamide gel electrophoresis (PAGE) immediately after various heat exposures. 3. Characterize the post-heat rate of removal of specific proteins associated with the nucleus in normal and thermotolerant cells. At various time intervals after heat shock nuclear proteins will be analyzed by PAGE. Determine if heat-induced excess nuclear proteins can be observed in cells heated in vivo and if such a change in nuclear associated protein correlates with cell killing by hyperthermia. 5. Determine roles of cell-cycle progression and spontaneous premature chromatin condensations and excess nuclear proteins in heat-induced cell killing.
This aim will be accomplished by monitoring cell-cycle progression, the frequency of SPCC, and the fraction clonogenic cells in G1, S and G2. 6. Determine if there is a correlation between excess nuclear proteins, SPCC, perturbation of cell-cycle progression and cell-killing by continuous exposure to low temperature (41-42.5 degrees C) hyperthermia.
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