DSBs induced by CNDAC are products of replication, rather than a consequence of induction of apoptosis. (A-C) ML-1 cells were pulse-labeled with various concentrations of CNDAC in the presence of IdU for 10 minutes and chased in to fresh media; CldU was added 12 hours after washing. Samples were taken at different times and subjected to flow cytometry and PFGE. (A) Samples were fixed and stained with FITC-IdU antibody and allophycocyanin-CldU antibody. Flow cytometric analysis determined the time at which double-labeled cells appeared, indicating cells in the second S-phase. (B) Absolute cell number was quantitated by electronic particle counter at different times. (C) The generation of DNA DSBs was assessed by PFGE. The intensity of each band was quantitated, and the percentage of DSB fragment versus total input in each lane was expressed in the graph. Note that 0.8μM CNDAC did not cause DSBs beyond the background level. (D-F) ML1 cells were pulse-labeled with 2μM CNDAC in the presence of IdU for 10 minutes and chased into fresh media, CldU was supplemented 12 hours after wash with or without aphidicolin. Samples were taken at different time points and subjected to flow cytometry and PFGE. (D) Absolute cell number was counted by Coulter counter at different time points, and cell growth was inhibited in the presence of aphidicolin. (E) The generation of DNA DSBs was assessed by PFGE. The intensity of each band was quantitated, and the percentage of DSB fragment versus total input in each lane was expressed in the graph. Note that aphidicolin prevented the induction of DSBs at 18 to 24 hours after treatment. Data are 1 representative of 2 independent experiments. (F) Time of onset of apoptosis in ML-1 cells after 2μM of CNDAC treatment for 10 minutes. Apoptosis was detectable after 31 hours after treatment, as assessed by annexin V staining.