Figure 1
P73α is required for CDDP-induced apoptosis in OVCA cells. (a) TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in OVCA cell lines. The effect of ΔNp73α over-expression was cell-type specific. (a) OV2008 and C13*, (b) A2780s and A2780cp and (c) Hey cells were transfected with either TAp73α cDNA, ΔNp73α cDNA (2 μg; 24 h) or an empty vector (CTL) followed by CDDP (10 μM; 24 h). TAp73α and ΔNp73α over-expression was assessed by western blot (top), and apoptosis by Hoechst staining (bottom). Effective over-expression of TAp73α and ΔNp73α was confirmed by western blot using anti-HA antibody. The expression of HA-TAp73α sensitized all the tested cell lines to CDDP-induced apoptosis when compared with the control groups (OV2008, A2780s, A2780cp and Hey; P<0.01 and C13*; P<0.001). The effect of HA-ΔNp73α over-expression was variable. As observed with TAp73α, the over-expression of ΔNp73α promoted basal and CDDP-induced apoptosis in OV2008 and C13* compared with controls (OV2008 (P<0.05); C13* (P<0.01)). In contrast, ΔNp73α failed to sensitize A2780s and Hey cells to CDDP-induced apoptosis but it did increase the basal level of apoptosis in A2780cp cells. The enhancement of CDDP-induced apoptosis by either isoform was associated with increased cleavage of PARP, a substrate of caspase-3. (d) P73α downregulation by p73α siRNA attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008. OV2008 cells were transfected with p73α siRNA (0–100 nM; 48 h) and treated with CDDP (10 μM; 24 h). TAp73α, ΔNp73α, PUMA and NOXA contents (Top) and apoptosis (Bottom) were determined by western blot and Hoechst stain, respectively. P73α siRNA markedly downregulated TAp73α and ΔNp73α contents (lane 2 vs 1 and lane 4 vs 3) and had no effect on p53 content. TAp73α and ΔNp73α downregulation significantly attenuated CDDP-induced apoptosis compared with siRNA controlled-group treated with CDDP (P<0.001). Figures indicate the mean±s.e.m. of three independent experiments assessed by two way-ANOVA (*P<0.05, **P<0.01, ***P<0.001).
