Aurora kinase inhibition contributes functionally to the lethality of MK-0457/vorinostat in CML cells. (A) K562 (top) and LAMA84 (bottom) cells were treated with 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 24 hours (K562) or 16 hours (LAMA84), after which cytospin slides were prepared and stained with FITC-conjugated antibodies to phosphorylated (Ser 10) histone H3 (top panels) as described in “Immunocytochemistry.” Bottom panels exhibit DAPI staining. Images were captured with an Olympus BX40 microscope at 20×/0.50 (Olympus America, Center Valley, PA) and a CE digital camera (Alpha Innotech, San Leandro, CA) with RS Image software version 1.7.3. (Roper Scientific Photometrics, Tucson, AZ). (B) LAMA84 and K562 cells were incubated with 100 nM MK with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 16 hours (LAMA84) or 24 and 48 hours (K562), after which cells were stained with PI and DNA content was analyzed by flow cytometry. N indicates number of ploidy. (C) K562 cells were exposed to 10 μM of a selective aurora kinase A and B inhibitor (Aur K inh) in the absence or the presence of Vor (2 μM) for 48 hours, after which cells were lysed and subjected to Western blot to monitor expression of phospho-Bcr/Abl and phospho-CrkL, as well as levels of total and phosphorylated histone H3. Each lane was loaded with 30 μg protein. (D) K562 and LAMA84 cells were treated with 10 μM Aur K inh with or without Vor (K562: 2 μM; LAMA84: 1.5 μM) for 48 hours, after which the percentage of 7AAD⁺ cells were determined by flow cytometry. Results represent the means plus or minus SD for 3 separate experiments performed in triplicate. Asterisks indicate significantly greater than the value for treatment of cells with Aur K inh alone at same concentrations (**P < .01). The results of a representative experiment are shown (A-C); 2 additional studies yielded equivalent results.