Figure 2.

Conditional expression of c-Myc downregulates Ku DNA binding activity and suppresses DSB repair leading to increased genetic instability. (A) HO15.19 cells bearing inducible Tet-Off/Tet-On c-Myc gene were established as described in Materials and Methods section. c-Myc was turned on in three independent stable clones by the addition of DOX (1 µg/ml) for 24 hours. Expression levels of c-Myc, Ku70, and Ku86 were analyzed by Western blot. (B) DNA binding activity of Ku70 or Ku86 in c-Myc-Off or c-Myc-On cells was measured using a Ku70/86 DNA Repair Kit. Error bars represent ± SD. (C and D) The c-Myc-Off and c-Myc-On HO15.19 cells were exposed to 5 Gy of IR. Cells were harvested at various time points. DSBs were determined by PFGE or analysis of γ-H2AX by immunostaining. The number of γ-H2AX foci per cell was determined on a cell-to-cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [58]. The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [34]. Error bars represent ± SD. (E and F) c-Myc was turned on by addition of DOX for 4 weeks. Then, percentage of abnormal metaphases in the c-Myc-Off or c-Myc-On cells was quantified by T-FISH analysis. At least 30 metaphases per culture were analyzed. Error bars represent ± SD. Frequency of cytogenetic abnormality per metaphase in c-Myc-Off or c-Myc-On cells. Each experiment was repeated three times and error bars represent ± SD.