Aurora kinase inhibition induces p53 and enhances p53-mediated mitochondrial apoptosis. (A) Cells were treated for 12 hours with 10 nM MK-0457 and 2.5 μM Nutlin-3 (1 μM in MOLM-13 cells), either as individual agents or in combination. DMSO-treated cells served as control. (B) Expression of p53-related proteins in OCI-AML-3 cells, which were treated for 24 hours with 2.5 μM Nutlin-3 and 10 nM MK-0457, either as individual agents or in combination. MK-0457 as well as Nutlin-3 induced increased protein expression of Mdm2, p21, and a BH3-only member of the Bcl-2 family protein Puma in OCI-AML-3 cells. The intensity of the p53 signals was quantified by densitometry using ImageJ 1.38x software, and the relative intensity compared with β-actin was calculated. (C) Western blot analysis of OCI-AML-3 cells electroporated with either control or Aurora B siRNA. Twenty-four hours after siRNA electroporation, Aurora B, p53, and β-actin levels were determined. (D) AML cells were cultured for 12 hours in the absence (□) or presence (■) of 10 nM MK-0457. Δψ_m was assessed by flow cytometry. Data are mean plus or minus SD of triplicate measurements. Comparable results were obtained in 2 other independent experiments. *P ≤ .01. (E) MK-0457 and Nutlin-3 synergistically induce Bax conformational change. MOLM-13 cells were treated with 10 nM MK-0457 and 2.5 μM Nutlin-3 for 6 hours, either as individual agents or in combination, and Bax conformational change was determined by staining with the active conformation-specific anti-Bax antibody YTH-6A7 (shaded histogram) or a corresponding isotype control (open histogram). To block caspase activation-mediated conformational change of Bax, cells were preincubated for 1 hour with 50 μM Z-VAD-FMK. Results are representative of 3 independent experiments. (F) After 1 hour of preincubation with 50 μM Z-VAD-FMK, MOLM-13 cells were treated with 10 nM MK-0457 and 2.5 μM Nutlin-3 for 6 hours, either as individual agents or in combination. Active Bax was immunoprecipitated (IP) from total cell lysates with the conformation-specific antibody YTH-6A7. The amount of immunoprecipitated Bax and the levels of Bax and β-actin in whole-cell lysates (WCL) were determined by Western blot analysis. (G) MK-0457 and Nutlin-3 synergistically induce Δψ_m loss. MOLM-13 cells were cultured for 6 hours in the presence of DMSO, 10 nM MK-0457, 2.5 μM Nutlin-3, or a combination of MK-0457 and Nutlin-3. Δψ_m was assessed by flow cytometry. Data are mean plus or minus SD of triplicate measurements. Comparable results were obtained in 2 other independent experiments. *P ≤ .01.