The working hypothesis of the proposed study is that heat-denatured proteins represent the rudimentary hyperthermic lesion that causes cell killing and induces thermotolerance. Denatured proteins initiate the formation of protein aggregates which subsequently disrupt normal cellular structure and function. Ultimately denaturation aggregation related damage prevents cell from undergoing a successful mitosis and they are rendered clonogenically dead. Thermotolerance is induced by the presence of denatured and/or aggregated proteins and protects cells from hyperthermic killing by inhibiting protein denaturation-aggregation and facilitating the disaggregation process.

The experiments are designed to demonstrate unequivocally that protein denaturation-aggregation is a lethal hyperthermic lesion and to determine how it causes cell death. The cytotoxicity of denatured proteins in the absence of other hyperthermic damage will be determined by electroporating heat-denatured proteins into nonheated cells and quantitating the resultant cytotoxicity. Complementary experiments will involve electroporating mammalian cells with thermolabile, nonmammilian proteins. The ability of the thermolabile proteins to either sensitize cells to heat or lower the threshold temperature for cell killing will support protein denaturation as a lethal hyperthermic lesion. Experiments will then be performed to confirm protein aggregation as a direct consequence of protein denaturation and to determine if the magnitude of protein aggregation, integrated over both the heating time and post heating recovery serves as a measurement of the cytotoxic dose. Efforts will then be directed towards identifying specific cellular targets that express lethal hyperthermic damage. Experimental approaches will include cell enucleation and refusion techniques to determine the cytotoxity associated heating the cytoplasm nor nucleus. We will determine if aggregated proteins interfere with normal nuclear function in a manner that results in micronuclei or aberrant chromosomes. Heat damage to the centrosome will also be investigated for its effect on cell viability. Finally, we will determine the mechanisms by which thermotolerance protects cells against protein denaturation/aggregation. This will include experiments to determine the relative efficacy of the major heat shock proteins in inhibiting aggregate formation and facilitating the disaggregation process.

The results of this study will provide fundamental knowledge concerning the interactions of hyperthermia with biological cells which can be used in the development of hyperthermia as a clinical modality for treating human cancers. Protein denaturation/aggregation has also been implicated as a lethal lesion in other stresses of clinical import, e.g., ischemia. Thermotolerance protects against these other stresses which themselves induce thermotolerance. This cross-resistance provided by thermotolerance suggest that is part of a more generalized mechanism that has evolved to protect cells from stress. Thus, the information obtained from this study may have broader medical implications than the narrow application of hyperthermic oncology.

R01CA060331

1994-02-01

2000-01-31