The overall goal of our ongoing research is to delineate the mechanisms of cell killing and radiosensitization by hyperthermia, in both acute 43 degree C - 45 degreeC, and moderate, 41 degree C - 42 degree C, temperature ranges. We propose to test one hypothesis on heat-induced cell killing and one on radiosensitization. The first hypothesis addresses the mechanism of S-phase cell killing because S-phase cells are the most sensitive to acute hyperthermia and appear to be the only cells that are killed at moderate temperatures. Hypothesis 1: heat-induced aggregation of proteins to DNA replication factories and/or the altered binding of proteins normally associated with these structures is a key step in S-phase cell killing. For the purposes of this proposal, we will use """"""""protein aggregation"""""""" to refer to a process where proteins not normally targeted to a subnuclear structure become bound to that structure due to heat shock. Similarly, """"""""altered binding of proteins"""""""" refers to proteins that are functionally part of a subnuclear structure and are solubilized under normal isolation conditions, but coisolate with that structure and/or the nuclear matrix (NM) after heat shock. Hypothesis 2: proteins thermally aggregated or bound to the NM contribute to radiosensitization at 41 degree C by inhibiting or altering DNA repair in a manner analogous to that at 43 degree C - 45 degree C.
The specific aims are: 1. Determine if protein targeting sequences specific for DNA- replication-factories affect heat-induced aggregation and/or protein binding to DNA replication factories in vivo. 2. Determine if the association of heat-labile proteins with DNA replication factories sensitizes cells to (or mimics) heat- induced cell killing. 3. Determine if DNA replication factories are a target for heat-induced cell killing at 45 degree C. 4. Determine if DNA replication factories are a target for heat- induced cell killing at 41.1 degree C. 5. Determine if heat- induced alterations in protein binding and/or aggregation to the NM and/or DNA repair complexes inhibit or alter DNA repair and thereby contribute to radiosensitization by 41.1 degree C hyperthermia in a manner analogous to that at 43 degree C - 45 degree C.
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