Transcriptional activation of proapoptotic Bcl-2 family proteins and transcription-independent apoptosis mediate Nutlin-induced apoptosis. (A) Expression of apoptosis- and cell-cycle–associated proteins in OCI-AML-3 cells, which were treated with 5 μM Nutlin-3a for the indicated times. Nutlin-3a induced increased protein expression of p53, MDM2, and p21 in OCI-AML-3 cells in a time-dependent fashion. Nutlin-3a induced Noxa, a BH3-only member of the Bcl-2 family, followed by caspase activation. β-actin was used to confirm equal loading of proteins. Arrowheads indicate cleared caspases. (B) Expression of proapoptotic Bcl-2 family proteins in primary AML cells, which were treated with 5 μM Nutlin-3a for the indicated times. Nutlin-3a induced at least 1 proapoptotic Bcl-2 family member protein in cells from 3 primary AML samples (nos. 6, 14, and 18 in Table 1) examined. Nutlin-3a induced Noxa, Puma, and Bax up-regulation in case 14 (top panel), Puma in case 18 (bottom panel), and Noxa in case 6 (not shown). β-actin was used to confirm equal loading of proteins. (C) OCI-AML-3 cells or MOLM-13 cells at a starting concentration of 2 × 10⁵ cells/mL were cultured for 24 hours in the presence of DMSO (□), 3.5 μM cycloheximide (▧), 10 μM Nutlin-3a (▦), or a combination of cycloheximide and Nutlin-3a (▪). Δψ_m was assessed by flow cytometry. Results are expressed as mean ± SD of triplicate measurements. Comparable results were obtained in 2 other independent experiments. *P < .05.