Hyperthermia, either alone or in combination with radiation or chemotherapeutic drugs, is currently being developed as a new modality for killing tumor cells. In order to increase hyperthermic killing of tumor cells, and increase the synergism between heat and radiation/chemotherapeutic drugs, a better understanding is required of the precise molecular interactions which become altered by heat. Definition of those interactions is also needed for an understanding of thermotolerance, the phenomenon of acquired resistance to heat. This proposal addresses these issues by focusing on the heat-induced alterations to the centrosome, a recently identified target for thermal killing. Specifically, the centrosomal protein pericentrin will be studied to determine the following:
Specific Aim #1 : To identify the molecular basis of thermal damage to centrosomal proteins.
Specific Aim #2 : To distinguish between direct or indirect modes of action of thermal sensitizers and protectors.
Specific Aim #3 : To determine the molecular basis for enhanced centrosome recovery in thermotolerant cells. Lastly, the relationship between centrosome damage and heat killing will be examined:
Specific Aim #4 : To establish whether heat-induced alterations to centrosome organization, and the resultant mitotic dysfunction, cause loss of proliferative potential through chromosome loss or gain Aims 1 and 2 will be accomplished through an ultrastructural and molecular analysis of the immediate, heat-induced alterations to the centrosome. Methods include electron microscopy, western blotting, immunoprecipitation, and 2-D gel electrophoresis.
Aim 3 is an evaluation of a role for heat shock protein 70 (HSP7O) in the enhanced centrosomal recovery of thermotolerant cells. Interaction between HSP7O and pericentrin will be studied in cells by immunoprecipitation, and in vitro by adding purified HSP7O to heated centrosomes. The methodology for performing Aim 4 involves synthesis of chromosome-specific fluorescent probes, followed by fluorescence in situ hybridization (FISH). This allows detection of chromosome loss or gain, and will test the hypothesis that thermal damage to the centrosome causes cell killing by induction of aneuploidy.
