Figure 1. Functional p53 is required for FoxM1 suppression by Nutlin-3.

(A) In cell lines with functional p53, p53 expression is induced, and FoxM1 expression is suppressed by Nutlin-3. Cell lines with known TP53 mutations are indicated by “mt” and wild type cell lines are indicated by “wt”. Note, although OVCAR10 contains a mutant allele (V172F), p53 expression is induced, and FoxM1 expression is suppressed by Nutlin-3, suggesting the wild type copy is sufficient to suppress FoxM1 expression. Significant down-regulation of FoxM1 was observed in NCI-H23 (**, P ≤ 0.01) and A2780 (*, P ≤ 0.05). FoxM1 expression was normalized with β-actin and was expressed relative to DMSO-treated controls. (B-D) Time-course experiments indicate that p53 is induced within 3 hours of Nutlin-3 treatment in A2780 and NCI-H23 cells with wild type p53, and FoxM1 downregulation is observed at 24 hours in these cells. In HEC-1A cells with mutant p53, neither p53 nor FoxM1 was affected by Nutlin-3 at various time points. These results indicate functional p53 is required for FoxM1 downregulation by Nutlin-3. Cells were treated with vehicle (0.05% DMSO), 10 μM Nutlin-3 for 3, 6 or 24 h, or with CHX, CHX+Nutlin-3, ActD or ActD+Nutlin-3 for 24 h. Proteins were isolated and subjected to Western analysis. β-actin was used for normalization of loading. Downregulation of FoxM1 at 24 hours following Nutlin-3 treatment is highlighted in the dotted box.