The goal of this research is to evaluate the sequence of events from DNA damage to cytogenetic damage to cell death in mammalian cells grown in vitro in the presence of halogenated pyrimidines (HP) (mainly BrdUrd and IdUrd) at concentrations that are clinically achievable. The hypothesis is that HP sensitize cells to radiation damage by enhancing fixation of potentially lethal damage (PLD). This cellular fixation process will be related to fixation and misrepair of chromosome damage and to repair inhibition events at the DNA level. Studies will be carried out with transformed (CHO,TCL-15) and nontransformed (10T1/2) cell lines as in vitro models for tumor and normal tissue, respectively. At the cellular level, incorporation of HP into DNA will be correlated with relaxation of growth control mechanisms, and modification of the D0 and Dq of the radiation survival curve in growing vs. nongrowing cell as well as in cells irradiated in various phases of the cell cycle. The possibility that repair of PLD compromises radiosensitization induced by HP will be examined. Very little is known about IdUrd-induced radiosensitization at the endpoints examined here. A comparison will be made between BrdUrd and IdUrd mediated radiosensitization and the enhancement of killing caused by post-irradiation treatment with araA and caffeine. At the chromosome level, the induction of chromosome damage by radiation will be assayed by the Premature Chromosome Condensation (PCC) technique in plateau- phase or synchronized G1-phase cells grown in the presence of HP. A relationship will be established between the amount of thymidine replaced by HP and rate of repair of PCC fragments as well as amount of misrepair induced (rings and dicentrics). A similar relationship using chromosomal aberrations as classically measured at metaphase will be established in irradiated plateau-phase cells and cells synchronized and irradiated at various phases of the cell cycle. Repair at the chromosome level (PCC) will be compared to the repair of PLD observed under similar conditions and a correlation will be established between cell killing and chromosome damage. At the DNA level, DNA damage and repair (total breaks and double-strand breaks specifically) will be measured in exponentially growing cells, plateau-phase cells, and cells synchronized in G1-, S-, and G2+M-phase as a function of HP incorporation. A relationship will be established between amount of thymidine replaced by HP and induction and repair of DNA damage and residual damage.
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