HCT116 cells that had been grown in the presence of 6-TG (1μM) DNA for 24h were treated with (a) H2O2 (1h) or (b) KBrO3 (3h) or (c) γ-irradiated at the doses indicated. Survival of HCT116 in absence of 6-TG (black line) or containing 6-TG in their DNA (grey line) was determined by clonogenic assay (a - c). The data represent the mean ± SD of 4 independent experiments. In parallel, the same HCT116 cells were loaded with the ROS reactive dye CM-H2DCFDA prior to exposure to H2O2, KBrO3 or IR. Changes in fluorescence intensity were analysed by FACS (insert a - c). The solid histogram represents untreated cells.
d) DNA replication. Control (black line) or 6-TG treated HCT116 cells (grey line) were treated with H2O2 for 1h and [3H]-TdR incorporation into TCA-insoluble material was measured. Incorporation is expressed as a percentage of that in untreated cells. Data are the mean ± SD from 2 separate experiments.