The MK-0457/vorinostat regimen induces apoptosis in cells resistant to IM through disparate mechanisms. (A) IM-resistant K562-R cells were established as described in “Cells and reagents,” and Western blot analysis was performed to monitor expression of Bcr/Abl and phospho-CrkL, as well as levels of total and phosphorylated Lyn (inset). Parental K562 and K562-R cells were exposed to 5 μM MK for 48 hours, after which the percentage of 7AAD⁺ cells was determined by flow cytometry. (B) K562-R cells were incubated with 0.1 to 1 μM MK with or without 1.5 μM Vor for 48 hours, after which flow cytometry was performed to monitor the percentage of 7AAD⁺ cells. (C) K562R cells were treated as described in panel B, after which Western blot analysis was performed to monitor expression of total and phosphorylated Lyn, as well as processing of caspase-3 and cleavage of PARP. (D) Ba/F3 cells bearing wt or various point mutations, including T351I, M351T, and E255K, were exposed to 100 to 500 nM MK-0457 with or without 1.5 μM vorinostat for 48 hours, after which the percentage of 7AAD⁺ cells was determined by flow cytometry. (E) Alternatively, cells were lysed and subjected to Western blot analysis to evaluate cleavage of caspase-9 and PARP. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate (A,B,D). For each condition involving combined treatment, the net increase compared with treatment with Vor alone was determined and the significance of differences evaluated using the Student t test. Asterisks indicate significantly greater than values obtained after treatment of cells with MK alone at the same concentrations (*P < .05; **P < .01). For panels A inset, C, and E, each lane was loaded with 30 μg protein; blots were stripped and reprobed with antibodies to β-actin or α-tubulin to ensure equal loading and transfer. CF indicates cleavage fragment; Veh, vehicle. Two additional studies yielded equivalent results.