SSBs induced after CNDAC incorporation during the first S-phase were converted into DSBs in the second S-phase. ML-1 cells were synchronized in S phase using 1μM aphidicolin for 24 hours, followed by a 4-hour pulse with CNDAC and chase in to fresh media. Samples were taken at indicated times and subjected to flow cytometric analysis, comet assay, or electronic counting. (A-B) Samples were fixed by 4% paraformaldehyde solution and stained with FITC-conjugated γ-H2AX antibody and PI. Flow cytometry showed both cell-cycle progression (A) and γ-H2AX–positive profile (B) that was CNDAC dose-dependent (●, 0μM; ▾, 0.8μM; ♦, 2μM). (C) Generation of DNA DSBs was assessed by single-cell gel electrophoresis (comet assay) under neutral conditions. The level of DSBs was quantitated by determination of Olive tail moment before and after washing out of CNDAC (●, 0μM; ▾, 0.8μM; ♦, 2μM). (D) Absolute cell number was scored by an electronic particle counter at indicated time points, and cell growth curve was plotted for each drug concentration (●, 0μM; ▾, 0.8μM; ♦, 2μM). Dashed lines indicate time of drug washing out. Data are 1 representative of 2 independent experiments.