With the increasing use of hyperthermia in the clinic, heat-induced thermal resistance (thermotolerance) presents an important consideration in the design of efficacious fractionated hyperthermia protocols. The objective of the proposed research is to investigate the role of hsp70 in the phenomenon of thermotolerance by a systematic characterization of heat resistant variants of Chinese hamster fibroblasts that overproduce the hsp70. More specifically, we wish to obtain molecular probes for the expression and localization of hsp in wild-type and heat resistant cells. We plan to accomplish this by isolating the DNA sequences coding for hsp70 and by generating monoclonal antibodies against the hsp70. We will then use these probes for a detailed characterization of the expression and localization of the hsp70 mRNA and protein under normal culture conditions, throughout the cell cycle and during the development of thermotolerance. The cloned Chinese hamster hsp70 gene will also be fused to selectable antibiotic resistance markers and used for transfection of suitable recipients. The transfected cells will then be tested for altered heat resistance. We will take advantage of the availability of heat resistant variants, which are permanently thermotolerant to further understand the basic mechanisms involved in heat-induced cell killing. We propose to accomplish this by determining whether or not any modifications in the heat-induced perturbations of several organelles associated with the cellular heat response, including the plasma membrane, nucleus and cytoskeleton, are altered in heat-resistant cells relative to wild-type cells. This series of experiments will also determine what, if any, role the hsp70 has in the physiology and function of normal cells. The overall results of the proposed research will test the hypothesis that the hsp70 plays a pivotal role in protecting cells from thermal damage. A positive demonstration of the role of hsp70 in thermal protection would suggest that the manipulation of the expression of this protein could eventually be utilized to differentially alter the thermal response of normal and tumor tissues. The further understanding of the molecular basis of thermotolerance could also contribute substantially to the manipulation of therapeutic efficacy of fractionated hyperthermia in the clinic.
