Figure 5.

CHK1 inhibition sensitizes only cells with resistance towards MMC. (A) Percentage and representative examples of cells with pan-nuclear yH2AX signal (red) after treatment with MMC alone or combined with CHK1 (MK8776) inhibition. Exponentially growing cells were treated with 0.5 µg MMC for 1 h after being incubated with 2 µM MK8776 for 2 h. The 24 h after treatment immunofluorescence staining for γH2AX was performed and nuclei were counterstained with DAPI. Cells with pan-nuclear γH2AX signal indicating replication stress were microscopically evaluated. Means + SEM of three independent experiments are shown. Asterisks represent significant differences (* p < 0.05, ** p < 0.001, *** p < 0.0001, Student’s t-test). (B) Cellular survival after treatment with MMC alone or in combination with the CHK1 inhibitor MK8776. Cells were plated, treated with MK8776 (2 µM) for 2 h and/or MMC for 1 h, fixed and stained after 14 days and the number of colonies was counted. Adding MK8776 sensitized the two resistant cell lines to MMC (p = 0.003 and 0.007 at 1.5µg/mL MMC). For the two MMC sensitive cells there was no sensitizing effect by the CHK1 inhibitor. Shown are means of three independent experiments ± SEM. Asterisks represent significant differences (* p < 0.05; ** p < 0.01, Student’s t-test). Induction of the replicative catastrophe should result in cell death, which was investigated by the colony formation assay. Figure 5B shows that adding the CHK1 inhibitor MK8776 sensitized the two resistant cell lines to MMC. The IC50 values for the two cell lines were reduced by an enhancement factor of 4.3 and 2.9. For the two MMC sensitive cells there was no sensitizing effect by the CHK1 inhibitor. Thus, only cell lines resistant to MMC could be sensitized by inhibition of CHK1 (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n.s. not significant; Student’s t-test).