The goal of this study is to understand how cells resist killing by heat and how they develop increased levels of thermotolerance after surviving hypothermia. In a collaborative project between the Weber and Landry laboratories, we have found that human gene encoding the 27 kDa heat shock protein is expressed constitutively in rodent cells and produces a heat resistant phenotype. Using specific antibody to the human hsp27 protein, we find a good correlation between the amount of human hsp27 in stably transformed hamster and mouse cell lines and the degree of thermal resistance. These cell lines will be used in the present study to learn whether overexpression of hsp27 in the absence of the other heat shock proteins can inhibit formation of accelerate the repair of specific lesions in cell function and structure that are known to be caused by heat. These include inhibition of transcription, RNA processing, translation, increased protein catabolism, and alterations in the structure of the cytoskeleton. We will also investigate the importance of phosphorylation of he hsp27 protein in determining thermal resistance in response to thermoprotecting and thermosensitizing agents. The sites of phosphorylation in the protein will be determined and the significance of each site for thermoprotection following heat shock will be evaluated by site-directed mutagenesis studies. Finally, the high constitutive activity of the human hsp27 gene in rodent cells will be used to develop transformed cell lines that overexpress other genes and cDNAs encoding different heat shock proteins. The phenotype of each type cell line will be studied with respect to thermoresistance and protection from known heat induced lesions. Cell lines that overexpress multiple heat shock proteins will also be developed to test for functional interactions between the different proteins. Information obtained from this study will provide a better understanding of how cells respond the hyperthermia which could lead to more rational approaches to thermotherapy in cancer treatment.
