Hyperthermia is an accepted approach to cancer therapy where it can cause tumor regression and sensitization to radiational killing. The usefulness of hyperthermia is limited, however, by the development of thermotolerance. Thermotolerance occurs in cells which have been previously heated to 42 degrees C (or higher), causing them to become refractile to further heat treatments. Experimentally, it correlates with the induction and continued presence in the cell of heat-shock proteins, in particular the 70,000 dalton protein (HSP70) This study will examine the molecular mechanisms regulating HSP70 expression immediately after heat-shock, the period when the greatest proportion of HSP70 protein is synthesized. Two key processes controlling the post-transcriptional levels of HSP70 will be studied - preferential translation of HSP70 mRNA and the HSP70 mRNA turnover rate. The approach is to introduce deletions and mutations into two clones HSP70 genes, mouse and human, then determine the transcripts half-life by Northern blot analysis or the translational efficiency by protein gel analysis. RNA regulatory sequences delineated by this analysis will be cross-linked with UV irradiation to any contacting proteins and the proteins identified by gel electrophoresis. Because these proteins may contribute to HSP70 synthesis, antibodies will be produced and used to screen a cDNA expression library for regulatory protein clones. A second approach will attempt to obtain clones by directly binding the RNA elements to the cDNA encoded peptides. Beyond its direct involvement in thermotolerance, the mechanisms regulating HSP70 RNA may be responsible for the observed heat-shock stabilization of some proto-oncogene transcripts. In the future, it will be interesting to examine whether the resulting increase in oncogene expression plays a role in post-hyperthermic cell growth or survival.
|Goswami, P C; Roti Roti, J L; Hunt, C R (1996) The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation. Mol Cell Biol 16:1500-8|
|Gasser, D L; Sternberg, N L; Pierce, J C et al. (1994) P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci. Immunogenetics 39:48-55|
|Hunt, C R; Gasser, D L; Chaplin, D D et al. (1993) Chromosomal localization of five murine HSP70 gene family members: Hsp70-1, Hsp70-2, Hsp70-3, Hsc70t, and Grp78. Genomics 16:193-8|