An ionizing radiation-sensitive mutant of CHO cells: irs-20. I. Isolation and initial characterization

We have isolated a stable mutant of CHO cells, designated irs-20, that is hypersensitive to ionizing radiation. The selection system was designed to select for mutants unable to proliferate at the low dose rate of 0.06 Gy per hour, a dose rate that has little influence on the effect of radiation on the cell cycle of wild-type cells during 1- to 2-week exposures. The irs-20 mutant cells irradiated continuously at 0.06 Gy/h showed a cell cycle redistribution, with an increasing G2 + M-phase fraction, but underwent approximately four doublings before cell population growth was completely inhibited. Dose rates three to four times higher were required to produce similar perturbation in the cell cycle of wild-type cells. Asynchronous log-phase irs-20 cells were approximately twofold more sensitive than the parental CHO cells as measured by comparing the doses required to reduce survival to 10%. The survival response of synchronized irs-20 cells after a single radiation dose of 3.8 Gy at different times during the cell cycle was qualitatively similar to the pattern for wild-type CHO cells for an approximately isosurvival dose of 7.4 Gy. The irs-20 cells were hypersensitive to the "radiomimetic" drug bleomycin but showed the wild-type sensitivity to ethyl methane sulfonate, ultraviolet light (254 nm) and mitomycin C. The irs-20 mutant cell has maintained its phenotype for over 1 year in continuous culture, indicating that the defect is genetically stable. The karyotype of the mutant cells is not different from that of its parent. Further evidence of stability is that clonal lines derived from cells surviving high radiation doses also had the irs-20 phenotype, and treatments with 5-azacytidine sufficient to cause high reversion (approximately 2 x 10(-1)) to proline independence resulted in no measurable reversion to wild-type radiosensitivity.