Synergistic induction of p53 signaling by F-ara-A and Nutlin-3a in CLL cells with wild-type p53. (A) Synergistic induction of p53 by F-ara-A and Nutlin-3a in CLL cells with wild-type p53. Samples from 4 wild-type p53 patients (nos. 4, 10, 11, and 30; no. 10 showed reduced Atm expression) and from a mutant p53 patient (no. 28) were treated for 24 hours with 5 μM F-ara-A (F) and 5 μM Nutlin-3a (3a) either as individual agents or in combination. DMSO-treated cells served as control (C). Shaded histograms represent isotype controls. Results show that F-ara-A and Nutlin-3a synergistically induce p53 in cases with wild-type p53, irrespective of Atm status. p53 expression levels were expressed as mean fluorescence intensity (MFI) ratio calculated by the following formula: MFI ratio = (MFI for anti-p53 antibody)/(MFI for isotypic control). WT indicates wild-type; MUT, mutant; N, normal; R, reduced; and ND, not done. (B) A synergistic activation of Bax by F-ara-A and Nutlin-3a in CLL cells with wild-type p53. Cells from patient no. 27 (wild-type p53) and from patient no. 28 (mutant p53) were treated for 24 hours with 5 μM F-ara-A (F) and 5 μM Nutlin-3a (3a) either as individual agents or in combination. Bax conformational change was determined by staining with the active conformation-specific anti-Bax antibody YTH-6A7 or a corresponding isotype control (shaded histogram). Z-VAD-FMK (200 μM) was used to inhibit caspase activation–mediated conformational change of Bax, and cells treated with Z-VAD-FMK alone served as control (C). Results show a synergistic activation of Bax by F-ara-A and Nutlin-3a in wild-type p53 but not in mutant p53 cells.