Hyperthermia has promise as a means of cancer therapy in combination with radiotherapy and chemotherapy for tumors which can be effectively heated. The molecular effects of hyperthermia are not well understood, in particular the target (if there is a single one) of hyperthermi and the molecular basis of thermotolerance are not known. These two problems are intimately related since thermotolerance is probably due to protection or stabilization of the target. Inhibition of repair of radiation damage to DNA b hyperthermia suggests that nuclear targets, specifically the nuclear matrix, are involved. This proposal is organized around a model for cell killing in which the initial, direct effect of hyperthermia is the denaturation of thermolabile proteins, which are the primary determinant of thermosensitivity, followed by co-aggregation of denatured and native proteins.
The specific aims of this proposal are to determine the identity of the thermolabile proteins of the nuclear matrix and nuclei that denature at the transition temperature (Tm =46 C for V79 cells and 47.4 C for human A549 cells) of the critical target by thermal gel analysis. If these proteins constitute the critical target, then chemical sensitizers to hyperthermia that increase the protein/DNA ratio of isolated nuclei from heated cells and protectors that lower this ratio should affect their thermostability (defined as the denaturation temperature Tm). In addition, we will determine if exogenous HSC 70, which translocates to the nucleus during hyperthermia, can stabilize the nuclear matrix proteins (i.e. increase Tm), reduce heat-induced aggregation, or facilitate the removal of excess nuclear proteins of isolated nuclei. The molecular mechanisms of sensitization by local anesthetics and low pH will be determined, specifically whether the conformational stability of thermolabile proteins or the extent of protein aggregation is altered by these sensitizers. These studies will determine the identity of the thermolabile proteins of the nuclear matrix, the roles of protein conformational stability and aggregation in thermal damage, and possible mechanisms for the involvement of HSC 70 in thermotolerance.
Showing the most recent 10 out of 29 publications
