Figure 5

DCQ induces DNA damage and increases ATM expression in p53^+/+ HCT116 cells. SSB and DSB induced by DCQ in p53^+/+ cell line. (A) Examples of comets induced by DCQ in cells subjected to the alkaline comet assay. Cells treated with DCQ for 6 hrs in normoxia or hypoxia were collected directly after treatment, subjected to the alkaline comet assay and images were taken using a fluorescent microscope at 40× (oil immersion) magnification. The comets observed by each treatment are directly proportional to the amount of SSBs induced. (B) The mean of the parameters (% DNA in comet' s tail and tail moment) are shown in the graphs above. More than 50 cells per treatment were photographed and quantified using TriTek CometScore software. (*) indicates p < 0.05 (one way ANOVA) with respect to control. (C) DCQ-induced phosphorylation of ATM in p53^+/+ cells at 6 hrs as an indication of DSB. After treatment, cells were fixed and subjected to immunocytochemical detection of ATM phosphorylated on Ser-1981, and stained with PI to detect at the same time p-ATM in each phase of the cell cycle. The mean of the FL-1 intensity ± SD (reflecting the level of p-ATM expression) at the G₁, S and G₂M phases of the cell cycle are shown in the table. The experiment was repeated twice each in duplicates.