The effect of heat on the induction and repair of DNA single (ssb) and double (dsb) strand breaks was studied in irradiated exponentially growing or plateau-phase CHO cells and their DNA dsb repair-deficient, radiation-sensitive counterpart, the xrs-5 cells. Induction and repair of DNA ssb was measured by the alkaline unwinding technique, whereas induction and repair of DNA dsb was measured by the non-unwinding filter elution technique. The results indicated that pre-exposure of cells to heat (45 x 5 degrees C) for 8-30 min did not affect the induction of DNA ssb or DNA dsb per Gy and dalton of DNA in CHO or xrs-5 cells, tested either in the exponential or in the plateau-phase of growth. On the other hand, pre-exposure to heat inhibited DNA repair processes and increased the fraction of unrepaired radiation-induced damage measured 2 h after irradiation. Repair of DNA dsb was more heat-sensitive than repair of DNA ssb in both cell lines. Repair of radiation-induced ssb or dsb was inhibited in xrs-5 cells to a larger extent than in CHO cells after a similar exposure to heat. These results complement those previously reported on heat-induced radiosensitization in these cell lines, and suggest that the reduction in heat-induced radiosensitization observed in xrs-5 cells is largely due to their deficiency in repairing DNA dsb, rather than to a reduction in the ability of heat to inhibit DNA repair processes in general. The data presented here provide further support to the hypothesis that DNA dsb repair proficiency is a prerequisite for heat-induced radiosensitization.

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.

Heat response and heat-induced radiosensitization were studied in plateau-phase cultures of CHO cells and their radiation-sensitive counterpart, the xrs-5 cells. The xrs-5 cells were more sensitive to heat alone than were CHO cells. A large enhancement in radiation-induced killing was observed in CHO cells pre-exposed to heat (43 degrees C), expressed as a reduction in the values of Do and Dq. Contrary to the results obtained with CHO cells, pre-exposure to heat of xrs-5 cells affected radiation sensitivity to a much lesser extent. D1, the radiation dose required to reduce cell survival to 1%, decreased in CHO cells from 8.7 Gy to 2.5 Gy with increasing heat damage (cell survival after exposure to heat alone), whereas it decreased from 1.4 Gy to 0.9 Gy in xrs-5 cells. These results suggest that heat-induced radiosensitization is compromised in plateau-phase xrs-5 cells. Since xrs-5 cells are deficient in DNA dsb repair, it is hypothesized that DNA dsb repair proficiency is a prerequisite for heat radiosensitization and that heat-induced inhibition of DNA dsb repair is likely to contribute to the radiosensitization observed in repair-proficient cell lines after exposure to high temperatures.

The effect of beta-arabinofuranosyladenine (araA) on the repair of radiation induced DNA damage, as measured by the DNA unwinding technique, was studied in exponentially growing and plateau-phase CHO-cells after exposure to x-rays. Induction of DNA damage by radiation was found to be similar in exponentially growing and plateau-phase cells. In the absence of araA, repair of radiation induced DNA damage proceeded with similar kinetics in exponentially growing and plateau-phase cells. AraA at concentrations between 0-1500 microM inhibited DNA repair both in exponentially growing and in plateau-phase cells. However, the degree of inhibition was significantly higher (by a factor of 3) in plateau-phase cells. A similar degree of repair inhibition by araA was observed in plateau phase cells treated in their conditioned medium, as well as in plateau phase cells that were transfered in fresh growth medium just before treatment initiation. These results indicate the importance of biochemical parameters associated with alterations in the growth state of the cells for the inhibitory effect of araA and may help in the elucidation of the molecular mechanism(s) underlying repair inhibition by inhibitors of DNA replication.

The purpose of this work was to investigate a possible correlation between DNA elution dose-response and cell radiosensitivity. For this purpose neutral (pH 9.6) DNA filter elution dose-response curves were measured with radiosensitive xrs-5 and the parental Chinese hamster ovary (CHO) cells in the logarithmic and plateau phase of growth. No difference was observed between the two cell types in the DNA elution dose-response curves either in logarithmic or plateau phase, despite the dramatic differences in cell radiosensitivity. This observation indicates that the shape of the DNA elution dose-response curve and the shape of the cell survival curve are not causally related. It is proposed that the shoulder observed in the DNA elution dose-response curve reflects either partial release of DNA from chromatin, or cell cycle-specific alterations in the physicochemical properties of the DNA.