Vorinostat interacts synergistically with MK-0457 to induce apoptosis in CML cell lines. (A) K562 and LAMA84 cells were exposed to 1 μM MK-0457 (MK) for 24 hours (LAMA84) or 48 hours (K562), after which the percentage of cells exhibiting “low” Δψ_m or 7AAD positivity was determined by flow cytometry. (B) K562 and LAMA84 cells were treated as described in panel A, after which cells were lysed and subjected to Western blot using the indicated antibodies. (C) K562 and LAMA84 cells were incubated with increasing concentrations (5-300 nM) of MK in the absence or presence of vorinostat (Vor K562, 2 μm; LAM484, 1.5 μm) for 48 hours (K562) or 24 hours (LAMA84), after which the percentage of 7AAD⁺ cells was determined by flow cytometry. (D) K562 and LAMA84 cells were incubated with increasing concentrations (0.2-1 μM) of vorinostat in the absence or presence of 100 nM MK for 72 hours (K562) or 48 hours (LAMA84), after which the percentage of 7AAD⁺ cells was determined by flow cytometry. (E) K562 (top panel) and LAMA84 cells (bottom panel) were treated with a range of MK and Vor concentrations alone and in combination for 48 hours (K562) or 24 hours (LAMA84) at a fixed ratio as indicated. At the end of this period, the percentage of 7AAD⁺ cells was determined by flow cytometry; fractional effect values were determined by comparing results with those of untreated controls, and median dose effect analysis was used to characterize the nature of the interaction. Combination index (CI) values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (F) K562 and LAMA84 cells were treated with 5 to 100 nM MK with or without Vor (K562, 2 μM; LAMA84, 1.5 μM), after which Western blot analysis was performed to monitor cleavage of caspase-3 and PARP. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate (A,C,D). For each condition involving combined treatment, the net increase over treatment with Vor (C) or MK (D) alone was determined and evaluated for significance using the Student t test. Asterisks indicate significantly greater than values for treatment of cells with the single agent at the same concentrations (*P < .05; **P < .01). For panels B and F, each lane was loaded with 30 μg protein; blots were stripped and reprobed with β-actin or α-tubulin antibodies to ensure equal loading and transfer of protein. CF indicates cleavage fragment; Veh, vehicle. Two additional studies yielded equivalent results.