The repression of FoxM1 is p21 dependent. (a) H1299-derivative cell lines containing tetracycline (tet)-regulated wild-type p53 (H24 cell line), transcriptionally impaired p53^Q22/S23 or p21 were grown in the presence or absence of tetracycline for 48 h before extraction for protein and RNA analysis. Left panel: immunoblot shows levels of FoxM1, p53, p21 proteins with actin as loading control. Right panel: relative FoxM1 mRNA expression was assessed using quantitative reverse transcription–polymerase chain reaction (QRT–PCR) and was normalized to 1 for each +tet condition (light gray, +tet (−p53 or p21); black, −tet, (+p53 or p21)). (b) MCF7 cells were treated with control, p53 or p21 small interfering RNA (siRNA) for 72 h. Daunorubicin (dauno, 0.22 μM) was added for the final 24 h before extracting cells for protein and RNA analysis. MCF7 shp53-expressing cells were also treated with daunorubicin for 24 h, but were not transfected with siRNA (Unt, untransfected). Right panel: immunoblot shows FoxM1, p53 and p21 protein levels with actin shown as a loading control. Left panel: relative FoxM1 mRNA expression was assessed using QRT–PCR and was normalized to 1 for each untreated condition (−dauno), (light gray, −dauno; black, +dauno). (c) HepG2 cells were treated with control, p53, or p21 siRNA for 72 h and 0.22 μM daunorubicin was added for the final 24 h before extraction of cells for protein and RNA analysis. Left panel: immunoblot shows levels of FoxM1, p53 and p21 with actin as a loading control. Middle panel: relative FoxM1 mRNA expression, assessed using QRT–PCR, was normalized to 1 for the untreated control siRNA condition and compared with FoxM1 mRNA levels in the untreated sip53 condition. Right panel: FoxM1 relative mRNA expression was assessed using QRT–PCR and was normalized to 1 for each untreated condition (−dauno) (light gray, −dauno; black, +dauno).

Nutlin is a potent FoxM1 repressor in two cancer cell lines that do not effectively repress FoxM1 in response to daunorubicin (dauno). (a and b) U2OS (osteosarcoma) or HCT116 (colon carcinoma) cells (wild-type, p53−/−, p21−/−) cells were treated with either dimethylsulphoxide (DMSO), 10 μM nutlin-3 or 0.22 μM daunorubicin for 24 h before extraction for RNA and protein analysis. Left panels: immunoblots show levels of FoxM1, p53 and p21 with actin as loading controls. Right panels (top, U2OS; bottom, HCT116) show relative FoxM1 mRNA levels determined using quantitative reverse transcription–polymerase chain reaction (QRT–PCR). The mRNA levels were normalized to 1 for the DMSO-treated condition (for HCT116, light gray, DMSO; dark gray, nutlin; Black, dauno) Statistical significance (P<0.05) is denoted with an asterisk. (c) MCF7-24 cells were treated with control or p21 small interfering RNA (siRNA) for 72 h. At 48 h after siRNA transfection, tetracycline (tet) was withdrawn from the cell culture medium to allow for 24 h of p53 induction. Left panel: relative FoxM1 mRNA levels were assessed using QRT–PCR and were normalized to 1 for each + tet (−p53) condition, (light gray, −p53; black, +p53). Right panel: immunoblot shows p53 levels and p21 levels.

FoxM1 mRNA is downregulated in a p53-dependent manner. (a) Left panel: p53 was induced in H24 and in MCF7-24 cells for 48 h by withdrawal of tetracycline (tet) from the growth medium, and p53 in cell extracts was detected using immunoblotting. Right panel: relative FoxM1mRNA levels were determined using quantitative reverse transcription–polymerase chain reaction (QRT–PCR) and were normalized to 1 for each+tet (−p53) condition (light gray, −p53; black, +p53). (b) MCF7 wild-type cells or MCF7 cells that express a short hairpin RNA (shRNA) to p53 (shp53) were either untreated or treated with daunorubicin (0.22 μM) for 24 h. Left panel: immunoblot shows FoxM1, p53 and p21 levels with and without daunorubicin (dauno) treatment. As a loading control, extracts were probed for actin. Right panel: changes in FoxM1 mRNA levels were assessed using QRT–PCR in three independent experiments (light gray, −dauno; black, +dauno). Relative mRNA expression was normalized to 1 for each untreated (−dauno) condition. (c) The average percentage of cells in G2/M phases of the cell was obtained using fluorescence-activated cell sorting (FACs) analysis (also graphically represented in Figure 5a).

Retinoblastoma (Rb) family members have a role in FoxM1 mRNA repression (a) MCF7 cells were treated with a combination of small interfering RNA (siRNA) to Rb, p130 and p107 or control siRNA for 72 h and 0.22 μM daunorubicin (dauno) was added for the final 24 h before extraction for protein and mRNA analysis. Left panel: relative FoxM1 mRNA expression was assessed using quantitative reverse transcription–polymerase chain reaction (QRT–PCR) and was normalized to 1 for each untreated condition (−dauno), (light gray, −dauno; black, +dauno). Right panel: immunoblot shows levels of p130, p107, Rb, p53 and p21 with actin as a loading control in untreated and daunorubicin-treated cells. (b) MCF7 cells were treated with either control or E2F1 siRNA at a final siRNA concentration of 100 nM for 48 h before extraction for RNA and protein analysis. Right panel: immunoblot shows FoxM1 and E2F1 proteins with actin as a loading control. Left panel: QRT–PCR was used to determine both FoxM1 and E2F1 relative mRNA expression (light gray, FoxM1 mRNA levels; black, E2F1 mRNA levels). Relative mRNA expression for each primer set was normalized to 1 for the control siRNA condition. Statistical significance (P<0.05) is denoted with an asterisk.

The p53-mediated repression of FoxM1 has a role in stable G2 arrest. (a) MCF7 wild-type and shp53-expressing cells were treated with control, FoxM1 no. 1, or FoxM1 no. 2 small interfering RNA (siRNA) for 72 h. Alternatively, cells were untransfected (Unt) but treated with daunorubicin (dauno, 0.22 μM) for 24 h. Left panel: cells were subjected to fluorescence-activated cell sorting (FACs) analysis and the percentage of cells with a 4 N content in each of the above-mentioned conditions was determined and plotted on the y axis of the graph as percent G2/M. Right panel: representative immunoblot shows FoxM1, p53 and p21 protein levels after siRNA or daunorubicin treatment. Cells were untransfected (lane 1), untransfected but treated with daunorubicin (lane 2), treated with control siRNA (lane 3) or treated with siRNA no. 1 (lane 4) or siRNA no. 2 (lane 5) versus FoxM1. (b) MCF7 wild-type and shp53-expressing cells were treated for 2 h with 0.22 μM daunorubicin, which was washed out, and cells were allowed to recover for 72 h. FACs analysis was performed and the percentage of cells with a 4 N content is plotted on the y axis of the graph as percent G2/M. (c) The same time course/treatments as in (b) were applied with the addition of taxol for the final 20 h of the time course to trap cells in mitosis. The mitotic index was determined by the percentage of cells that were positive for phosphorylated ser10H3 as assessed using FACs analysis. (d) Immunoblot analysis of cell extracts of replicate cultures corresponding to the conditions in (c), −/+ acute daunorubicin treatment + taxol. In all graphs in this figure, light gray bars represent wild-type MCF7 cells and black bars represent MCF7 shp53 cells.