Figure 7. Synchronous S phase progression as a potential cause of SN-38 sensitization.

A, After transfection with luciferase siRNA (open circles) or Chk1 siRNA (filled circles) were treated for 24 h with diluent (0.1% DMSO) or the indicated concentration of SN-38 for 24 h, Ovcar-5 cells washed and incubated in drug-free medium for 8 d to allow colonies to form. Error bars, ± s.d. from triplicate plates. B, cell cycle distribution of Ovcar-5 cells transfected as described above and exposed for 24 h to diluent or 20 nM SN-38. Histograms are representative of at least 4 independent experiments, with cell cycle distributions summarized as mean ± s.d of those experiments in the inset of each histogram. C, BrdU incorporation assessed during a 30 min incubation that started 12 h after addition of diluent or 50 nM gemcitabine. Alternatively, cells were treated with 50 nM gemcitabine for 24 h, washed, and incubated in drug-free medium for 12 or 24 h. D, quantification of BrdU incorporation after the treatments shown in panel C. Error bars, ± S.E.M. from 4-6 independent experiments. E, DNA histograms showing cell cycle progression after a 24 h treatment with 50 nM gemcitabine followed by removal of the drug. Samples were taken every 3 h after gemcitabine removal. For clarity, only histograms obtained 3, 9, 15, 18, 21 and 24 h after gemcitabine removal are shown. F, histograms showing time-course of cell cycle progression in cells sequentially exposed to gemcitabine followed by SN-38. After a 24-h treatment with 50 nM gemcitabine, cells were washed and exposed to 20 nM SN-38 for the indicated length of time before PI staining and analysis as indicated in panel E. Panels E and F come from the same experiment and are representative of 3 independent experiments.