Figure 6
DNA damage response incurred by MK-1775 and MK-8776 is dependent on CDK activity. A, Resistant (H460) or sensitive (LoVo) cells were treated with concentrations of MK-1775 and MK-8776 described for Figure 4, or 1 uM nocodazole for control. After 24 hours, cells were harvested and lysates analyzed by Western blot for caspase-dependent cleaved PARP (PARP*). B, A2058, HT-29, and LoVo cells were treated for 30 minutes with either DMSO or the indicated concentration of CDK inhibitor (SCH-727965). Following this pretreatment, further DMSO or concentrations of MK-1775 and MK-8776 used in Figures 3 and 4 (125 nM MK-1775 plus 150 nM MK-8776 in A2058; 125 nM MK-1775 plus 300 nM MK-8776 in HT-29, and 40 nM MK-1775 plus 75 nM MK-8776 in LoVo) were added to the cells for an additional 2 hours before cells were harvested and lysates analyzed by Western blot for phosphorylated CHK1S345, indicative of activated DNA damage response. C, LoVo cells were treated for 2 hours with 75 nM MK-1775 alone or in combination with 150 nM MK-8776, as indicated. Cells were harvested and lysates analyzed by Western blot for the proteins and phosphoproteins indicated.