DNA double-strand breakage by bleomycin in Ehrlich ascites tumor cells as measured by nondenaturing filter elution

DNA double-strand breakage by bleomycin (Bleo) in Ehrlich ascites tumor cells was examined by nondenaturing filter elution using 0.2% SDS at pH 9.6 as the eluant. The majority of damaged DNA from cells treated with 5 to 100 microM Bleo and for durations up to 6 h eluted in the first two or three 1.5-h fractions. This was significantly different from the multiphasic elution pattern of DNA from cells irradiated with 30 or 50 Gy 137Cs gamma rays, which showed a more gradual elution. Relative elution with respect to 50 Gy 137Cs gamma rays produced by 1-h treatments (about 0.35) showed no differences over the range of concentrations from 5 to 50 microM. Elution at pH 7.2 did not detectably reduce the fraction of DNA eluting from Bleo-treated cells, while reducing the elution of DNA from 50 Gy-irradiated cells by 35%. An experiment examining specificity for double-strand breakage by Bleo in different stages of the cell cycle demonstrated no differences in relative elution between G1, S and G2/M phases. Overall, the results of 1-h treatments are consistent with net production of high levels of double-strand breaks restricted to a portion of cellular DNA. However, extended treatments (up to 4 h) produced a nearly proportional increase in relative elution.