REJOINING OF X-RAY INDUCED CHROMOSOME BREAKS IN HUMAN CELLS AND ITS RELATIONSHIP TO CELLULAR REPAIR

A method was developed to improve the resolution for measuring breaks produced in interphase chromosomes by X-rays following the induction of premature chromosome condensation (PCC). It is based on the principle of 5-BrdU incorporation into the DNA of HeLa mitotic cells, which act as inducers of PCC when they are fused to diploid human fibroblasts. After a modified "Fluorescence Plus Giemsa" (FPG) protocol, the PCC stain intensely, while the mitotic inducer chromosomes stain faintly.; The dose response for density inhibited (G(,O)) human cells was linear from 10.9 to 600 rad, with a slope of .06 breaks per cell per rad. Upon incubation at 37(DEGREES)C, half of the breaks disappeared in 2 hours. Following a dose of 600 rad the initial rate of break rejoining mirrored the rate of increase in survival from post-irradiation incubation, due to the repair of potentially lethal damage (PLD). The X-ray induced PCC rejoining characteristics from two ataxia telangiectasia (A-T) cell lines were compared to profiles obtained with normal cells. Both normal and A-T cells apparently sustained the same initial level of radiation damage, and both cell types rejoined breaks at the same rate. However, while normal cells eventually rejoined all but about 5% of the breaks produced by 600 rad, the A-T lines were left with 5-6 times the level of residual damage. These experiments demonstrate that progression of cells into S phase is not a necessary condition for the measured frequency of chromosome fragments observed in X-irradiated A-T cells.; For normal cells the frequency of PCC breaks which did not rejoin after very long post-irradiation incubation (residual breaks) correlated with the frequency of chromosome aberrations seen in cells allowed to progress to mitosis. Furthermore, for cells released from contact inhibition (subcultured) 24 hours after irradiation, we observed a "one-to-one" correlation between the mean number of aberrations per cell seen at the first metaphase and cell survival, based on the premise that a single chromosome-type aberration represents a lethal event. Finally, we have attempted to explain the recovery from potentially lethal damage in terms of a model utilizing the kinetics of interphase (PCC) break rejoining.