Of the many heat shock proteins (hsp's) preferentially synthesized after a heat shock, the concentration of hsp 70 appears to correlate best with heat resistance, either permanent or transient. The long-term goal of this project is to establish the molecular basis related to the role that hsp 70 plays in modulating cellular responses to heat and drugs, and to enable clinicians to use this knowledge in a predictive manner. During the current period, emphasis is placed on three specific aims. (1) To develop practical assays using hsp 70 to predict the thermal response, development and decay of thermotolerance in various tissues. To accomplish this, we plan to investigate various techniques to quantify this protein, and to establish correlations between the intracellular concentration of hsp 70 and the survival kinetics of thermotolerance in model systems. (2) To use both microinjection and genetic manipulation to vary intracellular concentration of hsp 70, then to probe more directly the relationship between hsp 70, transient thermotolerance, and thermal resistance. In mammalian cells, microinjection of hsp 70 and antibodies against hsp 70 will be followed by survival and cellular localization studies of the microinjected cells after heat exposure. In yeast, clones will be constructed in which the concentration of hsp 70 can be modified by expressing the cloned gene from an inducible promoter gal 1 on a 2u multicopy plasmid. Transcription of gal 1 promoter can be modulated by varying ratios of glucose to galactose in the growth medium. Again, the thermal survival response will be related to concentration of hsp 70. (3) To establish the interrelationship between heat response, thermotolerance, drug resistance, and heat-drug interactions. This work will utilize human tumor cells resistant to either heat or drugs. SYSTEMS: Mammalian cell lines (CHO, human tumor lines), murine tumors (RIF-1, SCC VII/SF) and normal tissues (bone marrow, intestine). TECHNIQUES: Cell survival, gel electrophoresis, antibody production, enzyme-linked immunosorbent assay radioimmunoassay, immunoblotting, recombinant DNA and flow cytometry.
Showing the most recent 10 out of 64 publications
