BCR/ABL induces ROS-mediated DNA oxidative damage in hematopoietic cell lines. (A) Parental 32Dcl3 cells freshly transfected with empty plasmid (P) or p210BCR/ABL retroviral construct (B/A) were cultured continuously for 2 weeks (P-early [groups 1 and 2] and B/A-early [groups 3 and 4], respectively) or 10 weeks (P-late [groups 5 and 6] and B/A-late [groups 7 and 8], respectively) in the presence of IL-3. B/A-late cells were eventually pre-incubated for 48 hours with IM or PDTC (groups 9 and 10, or 11 and 12, respectively). ROS was measured by fluorescence in cells starved (-) or not (+) from IL-3 for 12 hours. Survival of cells was examined after 24 hours of incubation in the absence (starvation) or presence of IL-3 and represents the percentage of cells excluding trypan blue. P < .05 compared with other groups (^*), corresponding group incubated without IL-3 (^**), and groups 10 and 12 (^***). (B) 8-oxoG staining is detected by immunofluorescence in the nuclei, whose borders are marked in blue. (C) Oxidative DNA lesions were detected after addition of either EndoIII or Fpg to the comet reaction. Bars represent the enzyme-dependent increase of DNA damage over that detected in the undigested samples. ^*P < .05 compared with groups 1, 3, 4, 5, 9, and 11. (D) A DSB (arrow) detected in the BCR/ABL kinase sequence by LL-PCR followed by Southern analysis; parental 32Dcl3-late cells served as negative control. G/C-rich stretch at the predicted DSB site is listed. Numbering is consistent in the panels.