MEK1/2 inhibitors enhance DNA damage induced by AZD7762 in MM cell lines and primary CD138⁺ MM cells. (A) H929 and RPMI8226 cells were exposed to either AZD6244 (H929, 2.5μM; 8226, 5μM) or PD184352 (5μM for both lines) for 24 hours followed by the indicated concentrations of AZD7762 (H929, 24 hours; 8226, 32 hours). Cells were then lysed and subjected to Western blot analysis to assess γH2A.X (phosphorylated H2A.X at Ser139) expression. (B) Primary CD138⁺ MM cells isolated from the BM sample of a patient with MM were incubated with 250nM AZD7762 in the presence or absence of 5μM AZD6244 for 24 hours, and then immunofluorescently stained with AF 488–conjugated phospho-H2A.X (Ser139) Ab. Images were captured microscopically at 60×/1.40 under oil (top panels). In parallel, a comet assay was performed to assess DNA breaks (bottom panels), as described in “Methods.” (C) U266 and H929 cells were sequentially treated with 2.5μM AZD6244 for 24 hours, followed by AZD7762 (U266, 100nM; H929, 300nM) for 28 hours in the presence or absence of 20μM BOC-D-fmk. After treatment, the percentage of apoptotic (annexin V⁺) cells was determined by flow cytometry and was significantly lower (*P < .05 and **P < .02) than the values for cells treated with AZD6244/AZD7762 in the absence of BOC-D-fmk. (D) Alternatively, cells treated as described in panel C were lysed and subjected to Western blot analysis using the indicated primary Abs. For panels A and D, each lane was loaded with 20 μg of protein; blots were stripped and reprobed with anti-tubulin or anti-actin Ab to ensure equal loading and transfer. Two additional studies yielded equivalent results.