The Chk1/MEK1/2 inhibitor strategy kills MM cells in G0/G1 phase. (A) RPMI8226 cells cultured in 10% FBS were sequentially exposed to 1.5μM AZD6244 for 24 hours, followed by 400nM AD7762 for another 24 hours or 5nM Taxol as a control. The distribution of apoptotic cells in various cell-cycle phases was then determined by flow cytometry combining staining for cleaved (activated) caspase-3 (y-axis) and DNA content (PI; x-axis). The representative results are shown to indicate caspase-3 activation in specific populations of cell-cycle phases, including G0/G1, S, and G2M. Values indicate the -fold increases (treated vs untreated control) of cells displaying caspase-3 activation within each phase of the cell cycle after gating out the subdiploid population. Inset shows the corresponding results of cell-cycle analysis. Taxol-treated cells were incubated with IgG instead of anti-cleaved caspase-3 Ab to demonstrate the specificity of the immunostaining. (B) H929 cells were cultured in low- or high-serum–containing medium (0.1% vs 10% FBS) for 42 hours, and cell-cycle profiles were analyzed by flow cytometry. (C) After being cultured in 0.1% or 10% FBS medium for 42 hours, H929 cells were exposed to various agents, including the Chk1 inhibitors AZD7762 (300nM) and UCN-01 (150nM), the microtubulin-stabilizing agent Taxol (5nM), and the topoisomerase inhibitor VP-16 (4μM) for 24 hours. Cell death was then assessed by 7-AAD staining and flow cytometry and was significantly greater than (**P < .01) or less than (#P < .02) values for cells cultured in 10% FBS. (D) U226 cells were stably transfected with constructs encoding shRNA against human Chk1 (shChk1) or scramble control shRNA (shCtrl) and clones selected with G418. Western blot analysis demonstrates down-regulation of Chk1 expression in 2 shChk1 clones (C4 and E7) compared with shCtrl cells (inset). Cells were then cultured in medium containing 0.05% or 10% FBS for 48 hours, after which time the percentage of apoptotic (annexin V+) cells was determined by flow cytometry and was significantly greater (**P < .02 and ***P < .01) than values for cells cultured in 10% FBS. (E) H929 cells were cultured in 0.1% FBS for 26 hours, and then treated with 1.5μM AZD6244 for 18 hours, followed by the indicated concentrations of AZD7762 for another 24 hours. The extent of apoptosis was analyzed by annexin V staining and flow cytometry and was significantly greater (**P < .005 and ***P < .001) than values for cells cultured in 10% FBS. (F) Alternatively, cells were subjected to Western blot analysis using the indicated primary Abs.
