Figure 2.

The p21^Waf1/Cip1 upregulation and activation corresponding to apoptotic process induced by zinc: (a) zinc-augmented p21^WAF1/Cip1 expression. (a) Zinc ion chelating agents blocked zinc-induced apoptotic effect. LNCaP and PC3 cells were exposed to 150 μM ZnSO₄ in the presence or absence of zinc inhibitor of 150 μM or 1 mM CaEDTA for 24 h. Sub-G1 populations were detected using flow cytometry. Columns, mean (n=3); bars, S.D.; ***P<0.001 (one-way ANOVA). (b) Zinc-augmented p21^WAF1/Cip1 expression. LNCaP and PC3 cells were exposed to 150 μM ZnSO₄ in the presence or absence of zinc inhibitor of 150 μM or 1 mM CaEDTA for 24 h. The p21^WAF1/Cip1 levels were examined by immunoblot analysis with either p21^WAF1/Cip1 or β-actin antibody. (c) Zinc increased p21^WAF1/Cip1 transactivation in a Smad-dependent manner. LNCaP cells were cotransfected with 25 ng of Renilla luciferase reporter and 100 ng of luciferase reporter, p21P-luc or p21PΔp53-luc (c), pp53-luc (d) or 4*SBE-luc (e) as indicated, and then were exposed to various concentrations of ZnSO4 for 24 h. Relative luciferase activities were measured and calculated by the ratio of the firefly luciferase activity to Renilla luciferase. Columns, mean (n=5); bars, S.D.; **P<0.01 (one-way ANOVA)