The hypothesis of this project is that reduced intracellular pH (pHi) will increase, and the development of thermotolerance (TT) will decrease, heat-induced critical damage to the cytoskeleton and nuclear matrix. The experimental plan involves correlating the changes in structural lesions to the cytoskeleton and nuclear matrix with the changes in survival as a function of holding time in conditioned medium (CM) at 37 degrees C following heat shock (i.e. rate of repair of heat-induced potentially lethal damage [H-PLDR]). As a result, conclusions can be drawn regarding the relationship of residual structural damage to the cytoskeleton and in the nucleus to heat-induced cytotoxicity. Intracellular pH will be reduced by acutely reducing pHe to 6.7 in the presence or absence of amiloride and/or quercetin. Thermotolerance will be induced by prior heating. Amiloride and/or quercetin will also be used to reduce the pHi of cells chronically adapted to growth at reduced pHe (i.e., a condition representative of cells in a tumor like environment). G1 CHO cells at pHe 7.3 or adapted to growth at pH 6.7 will be exposed to heat after reduced pHi or after development of TT, and the presence of structural lesions win be correlated with surviving fraction during H-PLDR. Finally, these experiments will be performed using G1 OvCa cells from Project #2 that have been chronically adapted to pHe 6.7. pHi will be reduced by exposure of cells to pHe 6.7 (McCoys 5A buffered with MES/HEPES) combined with inhibitors that prevent regulation of pHi: amiloride and quercetin (inhibitors of Na+ dependent and independent pHi regulating processes). pHi will be measured using the BCECF fluorescence technique. The morphological integrity of the microtubular cytoskeleton (area of microtubular network fluorescently stained with specific monoclonal antibodies) and the nuclear matrix (architecture and electron density) will be studied using light microscopy (phase and fluorescence) and transmission electron microscopy (emphasizing resinless section electron microscopy). The heat-induced increase and subsequent removal of nuclear proteins will be monitored by flow cytometry, image analysis and by conventional biochemical and polyacrylamide gel electrophoretic techniques. The """"""""halo assay"""""""" of Roti-Roti will be used to test the functional integrity of the nuclear matrix/chromatin complex. Video microscopy will be performed using an optical memory disc recorder combined with computerized digital image enhancement and analysis for capture and subsequent quantification of all fluorescence images. These studies to establish the effect of altering conditions of heat sensitization and protection on structural damage leading to the loss of reproductive integrity may also provide a basis for using the response of the cytoskeleton and NM as prognostic indicators of the response of a tissue to hyperthermia.
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