Figure 5

Simvastatin was more likely to induce chromosome instability and γ-H2AX expression in RB deficient or inactive tumor cells than in RB-proficient cells. (a) Simvastatin induced γ-H2AX expression in RB deficient or inactive tumor cells. U2OS, SaOS2, SiHa and C33A cells were treated with 5 μM Simvastatin for 72 h, and then analyzed by immunofluorescence staining with γ-H2AX antibody. (b and c) U2OS cells were transfected with siRB-1 and siRB-2 for 48 h to deplete RB. Then cells were treated with 5 μM Simvastatin for 72 h, incubated with PI and analyzed with FACS. The result was compared with that of control cells, statistical analysis of the difference in sub-diploid peak between drug-treated cells and control cells. (d and e) U2OS cells were depleted of RB by siRB and treated with Simvastatin (2.5, 5 or 10 μM) for 72 h. The resulting cells were analyzed by immunofluorescence staining or immunoblotting with γ-H2AX antibody. (f) U2OS, SaOS2, SiHa and C33A cells were treated with 2.5 or 5 μM Simvastatin for 24 or 72 h; then cells extracts were analyzed by immunoblotting with p-ATM, p-ATR, p-CHK1 or p-CHK2 antibodies. (g) Simvastatin induced more DNA damage in RB deficient or inactive tumor cells. U2OS and SaOS2 cells were treated with 5 μM Simvastatin for 72 h. Chromosomal instability was examined as described and chromosome gaps (white boxes) and breaks (black boxes) were scored