DNA DAMAGE & REPAIR IN IRRADIATED NORMAL & TUMOR CELLS

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The overall objective of this proposal is to determine the effect(s) of radiosensitizing hyperthermic treatments on cell nuclear structure and function. In particular we will determine the effect(s) of: 1) acute heating (AH; 43 to 45 degrees); 2) step- down-heating (SDH; AH followed by chronic exposure to 41 degrees); 3) chronic heating at 41 degrees (tolerance development) before X-irradiation; and 4) chronic heating at 41 degrees subsequent to X-irradiation on the induction and/or repair of radiation-induced DNA double strand breaks (dsb), apurinic (AP) sites, DNA protein crosslinks (DPC) and chromosome aberration induction in CHO cells grown in monolayer. The hypothesis we will test is that a hyperthermia-induced increase in the induction frequency, and/or inhibition of repair, of one or more of these radiation-induced DNA lesions correlates with radiosensitization at the cellular level. To date, this hypothesis has not proven generally true in the case of DNA single strand breaks (ssb). The induction frequency and repairability (rate and extent) of radiation-induced lesions (ssb and dsb) in specific DNA sequences will be measured in control and heated cells. Bulk DNA will be isolated by alkaline/neutral filter elution or alkaline sucrose gradient sedimentation from irradiated cells, and cells during the repair of DNA lesions. Bulk DNA collected onto nitrocellulose filters will be probed by filter hybridization against nucleic acid probes for transcriptionally active and/or inert DNA sequences. The possible role heat-induced alterations of nuclear structure and/or composition in the inhibition of DNA damage removal will also be assessed. The kinetics of increase, and removal, of polyeptides in nuclesomal and nunnucleosomal chromatin DNA and in cell nuclear matrices will be determined by exogenous nuclease digestion of DNA, centrifugal isolation of nuclear components and polypeptide quantitation and characterizations by polyacrylamide gel electrophoresis. The extent to which hyperthermic exposure alters the number and/or conformation of nuclear matrix DNA binding sites will be determined by assessing the DNA "domain" size and frequency in control and heated cells.