DETECTION OF DNA DOUBLE-STRAND BREAKS THROUGH THE CELL-CYCLE AFTER EXPOSURE TO X-RAYS, BLEOMYCIN, ETOPOSIDE AND (125)IDURD

Ionizing radiation-induced DNA double-strand breaks (DSBs) are generally more difficult to detect in S-phase cells than in cells from other phases of the cell cycle. To explore the basis for this observation, other double-strand breaking agents were examined: etoposide, bleomycin and I-125dUrd. DSBs induced by these agents in single cells in S, G1 or G2/M-phases of cell cycle were measured using the neutral comet assay. Regardless of the nature or location of the DSBs, Chinese hamster V79 cells with S-phase DNA content showed about 2-3 times less damage by all agents than cells with G1 or G2/M-phase DNA content. Residual protein content measured after lysis of S-phase cells embedded in agarose did not differ significantly from the protein content of asynchronous cells, and removal of proteins prior to irradiation did not enhance S phase migration. The number of DSBs, the physical nature of the DSB, or the presence of residual proteins, did not appear to influence migration. Therefore, we conclude that differences in DNA structure are responsible for reduced sensitivity for detecting DSBs in S-phase cells.