Mammalian cells in culture, and presumably tumor cells, adapt to an environment at low extracellular pH (pHe) by mechanisms that lead to reestablishment of a normal intracellular pH (pHi). Furthermore, preliminary data and results published elsewhere show that cells adapted to low pHe exhibit normal sensitivity to hyperthermia and a normal capacity to develop thermotolerance. Chemical agents that interfere with the membrane Na+/H+ antiport or the HCO3-/Cl- exchanger, or agents that inhibit the H+:lactate symport sensitize cells to hyperthermia if pHe is acutely reduced but not if pHe is within normal physiologic levels. The hypothesis to be tested is that inhibitors of H+, HCO3- or lactate transport, alone or combined, will preferentially sensitize cells adapted to low pHe. These agents may have utility in the clinic as tumor sensitizers to hyperthermia since tumor cells, and only few cells in normal tissues, are chronically exposed to low pHe. Furthermore, reducing pHi may inhibit the development of thermotolerance that would otherwise normally occur during exposure to temperatures less than 42.5 degrees C in tumor cells adapted to low pHe. This would be a second method of tumor cell sensitization since much of the tumor temperature distribution during clinical hyperthermia is lower than 42.5 degrees C. These hypotheses will be tested by adapting Chinese hamster ovarian carcinoma (OvCa) cells in vitro to pH 6.7 and determining hyperthermia sensitivity and thermotolerance development in the presence of amiloride, DIDS and quercetin. In addition, the hypothesis that the synthesis of major heat shock proteins hsp27, hsp70 and hsp110 are tightly coupled to the development of thermotolerance can be tested. Survival curves and thermotolerance development during exposure to 41 degrees or 42 degrees C to cells adapted to pHe of 6.7, acutely acidified to pH 6.7 or at physiologic pH 7.3 will be determined in the presence or absence of the H+ exchange inhibitors. The concentration dependence of the effects of the inhibitors on the kinetics of thermotolerance development will be determined and compared to the effects of step down heating (SDH) and inhibition of protein synthesis (PS) by cycloheximide to distinguish PS-dependent from PS-independent thermotolerance. The rates of synthesis of specific proteins will be determined by the incorporation of 3H- or 35S-labeled amino acids followed by separation by SDS-PAGE and autoradiography, or immunoblotting with antibodies to hsp27, 70 and 110. Changes in pHi will be monitored by intracellular fluorescence of BCECF. Cell cycle distribution at the time of treatment will be determined by flow cytometry. These results will help determine whether pH related hyperthermia sensitizers may have a potential for clinical trial, and will lead to a further understanding of the relationship between the synthesis and modification of heat shock proteins and the development of thermotolerance under clinically relevant conditions.
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