Figure 5.

Apoptosis and Noxa upregulation on CDDP/F-ara-A combination treatment are mediated by ROS. (a) p53-dysfunctional (n = 5; patient nos. 1–5) and p53-functional (n = 4; patient nos. 11, 12, 18 and 21) CLL samples were preincubated with 5 mM NAC for 30 min and subsequently treated with F-ara-A in increasing doses (5–50 μm), with or without 10 μm CDDP, for 48 h. Apoptosis was assessed by annexin-V/PI staining. Presented is mean + s.e.m. (b) CLL cells were treated for 48 h as indicated (M = control, C = 10 μm CDDP, F = 10 μm F-ara-A, CF = CDDP/F-ara-A both 10 μm) with or without preincubation with 5 mm NAC and analyzed for protein expression levels of Mcl-1, Puma, Noxa and actin. Experiments for protein collection were performed in the presence of Q-VD-OPh to prevent caspase-dependent breakdown of proteins. Percentage of apoptosis was assessed by annexin-V/PI staining (patient nos. 2 and 22; representative of six patients tested). (c) CLL cells, which have been cocultured with CD40L-expressing or control 3T3 fibroblast for 48 h, were treated for 48 h with 10 μm CDDP and 10 μm F-ara-A with or without preincubation with 5 mm NAC (n = 5; patient nos. 7, 15, 16, 21 and 22). Percentage of apoptosis was assessed by annexin-V/PI staining; bars represent mean + s.e.m. (**P<0.01; Mann–Whitney U-test). (d) Left panels: ROS content was determined by carboxy-H2-DCFDA staining and flow cytometry (as described in the Materials and methods section) after 6 h of treatment with 10 μm CDDP and 10 μm F-ara-A (C + F; open graph; upper panels) and compared with ROS content of untreated (gray graph) or CCCP (100 μm)-treated cells (open graph; lower panels) (patient no. 2). Loss of mitochondrial potential was assessed by MitoTracker staining. Right graph: summarized data of cellular ROS content after 6 h of treatment as indicated compared with ROS content of control sample (n = 4, patient nos. 1, 2, 3 and 5). Presented is mean + s.e.m. (*P<0.05, **P<0.01; Mann–Whitney U-test).