Induction of Bim by vorinostat plays a functional role in interactions between vorinostat and MK-0457. (A) K562 (top) and LAMA84 (bottom) cells were treated with 5 to 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours (K562) or 24 hours (LAMA84). (B) IM-resistant K562-R cells were treated with 0.1 to 1 μM MK with or without 1.5 μM Vor for 48 hours. (C) Ba/F3 cells with wt or T315I Bcr/Abl were treated with 0.1 to 0.5 μM MK with or without 1.5 μM Vor for 48 hours. (D) MNCs from bone marrow samples obtained from CML patients 1 and 3 were exposed to 100 to 500 nM MK with or without 1.5 μM Vor for 48 hours. (A-D) After drug treatment, cells were lysed and subjected to Western blot analysis to monitor expression of Bim. (E) K562 cells were stably transfected with constructs encoding siRNA against Bim (siBim) or nonspecific sequence (siCtrl). Cells were exposed to 5 μM of Vor (top panel) or MK (bottom panel) for 48 hours, after which cells were stained with annexin V–FITC and subjected to flow cytometry. (F) K562 cells transfected with siBim or siCtrl were treated with 100 nM MK with or without 2 μM Vor for 48 hours, after which cells were lysed and Western blot analysis was performed to monitor expression of Bim, Bcr/Abl, and phosphohistone H3. (G) Alternatively, cells were stained with DiOC6 (top panels) or annexin V–FITC (bottom panels), respectively, and subjected to flow cytometry. Each lane was loaded with 30 μg (A-C,F) or 100 μg (D) of protein; blots were stripped and reprobed with β-actin or α-tubulin antibodies to ensure equal loading and transfer of protein. Two additional studies yielded equivalent results. The results of a representative experiment are shown (E,G); 2 additional studies yielded equivalent results.
