Arrest of the cell cycle by doxorubicin and its impact on VCR-induced apoptosis. (A-B) Cell-cycle analysis was performed using propidium iodide staining of DNA in CEM cells (A). Corresponding cell-cycle histograms are presented in supplemental Figure 3A. To discriminate between G₂ and M arrest, double staining for phospho-histone H3 (Ser10) and propidium iodide was performed after 24 hours (B). co indicates unstimulated control cells. (C) CEM cells were preincubated with caffeine (300 μg/mL) for 8 hours, followed by doxorubicin together with VCR for 48 hours. *P < .05 (ANOVA). NS indicates not significant. (D) CEM cells were stably transfected with a shRNA targeting cyclin A (shcyclin A) or a control mock sequence and were analyzed for cell-cycle distribution of spontaneously growing cells (left panel) or for apoptosis induction by VCR (3 ng/mL) after 48 hours (right panel). *P < .05 (ANOVA). NS indicates not significant. (E) Scheme summarizing the data presented in Figures 4 to 6: Doxorubicin-mediated activation of p53 and G₂ arrest inhibits VCR-induced cell death, abrogating VCR-induced phosphorylation of Bcl-2 family members, the distal apoptosis signaling pathway, and cell death. The concentrations of doxorubicin and VCR, measurement of apoptosis, presentation of data, and statistical analysis were performed as described in Figure 1A.