Recovery from a DNA-damage-induced G2 arrest requires Cdk-dependent activation of FoxM1

Abstract

Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.

Figure 1

FoxM1 is transcriptionally active during a DNA-damage-dependent arrest and checkpoint recovery. (A) Scheme of the recovery-induced experimental setting. (B) U2OS cells were transfected with siRNA control (GAPDH) and two independent siRNAs targeting FoxM1. After thymidine treatment for 24 h, cells were released for 6 h and doxorubicin (dox) was applied for 1 h. Caffeine (caff) was added 18 h after doxorubicin washout. The protein level of FoxM1 and FoxM1 targets was determined by western blotting at the indicated time points. (C) Reverse transcription PCR of FoxM1 target genes 18 h after doxorubicin treatment. Cells were transfected with either GAPDH or siFOXM1 #1 and treated as described in (B). The graphs in panels (B) and (C) represent the relative protein or mRNA level, respectively, of the FoxM1 targets in siFoxM1 #1-treated cells as compared with that in control cells 18 h after doxorubicin treatment. (D) U2OS cells were transfected with an empty vector (mock) or a plasmid encoding FoxM1 wild type, and co-transfected with the pBP-1 luciferase reporter plasmid. Relative luciferase expression was measured at the indicated time points during the DNA-damage response. Expression of endogenous FoxM1 was analysed by western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, small inhibitory RNA.

Figure 2

FoxM1 is essential for checkpoint recovery. (A) U2OS cells transfected with an siRNA against GAPDH and with two siRNAs against FoxM1 were synchronized and treated as in Fig 1 and the amount of mitotic cells was determined by pH3 positivity by FACS. The graph shows the percentage of mitotic cells at the indicated time points. The level of FoxM1 depletion was assessed by western blot analysis. (B) Cells transfected and treated as in (A) were followed by time-lapse microscopy after caffeine addition and scored for entry into mitosis. (C) Cells were transfected and synchronized as described in (B) and subjected to 5 or 10 Gy γ-irradiation. Immediately after irradiation, paclitaxel was added for 24 h before harvesting and the mitotic index was determined by pH3 staining. (D) U2TR cells stably expressing inducible RNAi-resistant FoxM1 wild type were co-transfected with GFP-spectrin, pBabe-Puro and empty pSuper (pS) or pS-FoxM1. The cells were synchronized and treated as described in (A) and doxycycline was added immediately (early) or 18 h (late) after doxorubicin washout. Cells were harvested 8 h after caffeine addition and the percentage of mitotic cells was determined by scoring GFP-positive cells for pH3 reactivity by FACS. The data on the graph are the average of three independent experiments and the error bars represent the standard deviation. The protein levels of FoxM1 and its targets were assessed by western blot analysis of cells selected with puromycin at the indicated time points. (E) Cells were transfected and synchronized as described in (D) and fixed at the indicated time points after caffeine addition. The mitotic index of GFP-positive cells was determined by FACS analysis. Cyclin B protein levels were determined by immunofluorescence and automated image analysis was performed as described under Methods. At least 200 GFP-positive cells were counted per well. Measurements were performed in triplicate and error bars represent the standard deviation. FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; RNAi, RNA interference; siRNA, small inhibitory RNA.

Figure 3

Regulation of FoxM1 activity during a DNA-damage-induced G2 arrest. (A) U2OS cells were transfected with FoxM1 wild type, FoxM1 3A (FoxM1 T600A/T611A/S638A), together with short-hairpin RNA-targeting vectors against cyclin A (pS-cycA) or cyclin B (pS-cycB). The cells were synchronized in G2 and transactivation of BP-1 reporter by FoxM1 was determined 18 h after doxorubicin washout. The efficiency of cyclin depletion was analysed by western blotting of puromycin-selected cells. (B) Cells transfected with FoxM1 and the pBP-1 luciferase reporter were treated with doxorubicin and incubated with the indicated Cdk inhibitors for 18 h before harvesting. The graph shows the relative luciferase activity. The data shown in the graphs (A,B) are the average of three independent experiments and the error bars represent the standard deviation. Cdk, cyclin-dependent kinase; pS, pSuper.

Figure 4

Cdk inhibition induces loss of recovery competence after DNA damage. (A) U2OS cells were synchronized by thymidine treatment (24 h). Roscovitine was added 6 h after release from the thymidine block, for the indicated periods of time. The mitotic index was determined 12 h after roscovitine washout in the presence of nocodazole. (B) U2OS cells were synchronized as described in (A) and treated with doxorubicin for 1 h. After doxorubicin washout, roscovitine and RO3306 were added for 18 h. Subsequently, the Cdk inhibitors were washed out extensively and caffeine was added for 8 h. Mitotic entry after caffeine addition was analysed by time-lapse microscopy. Lysates from cells harvested 18 h after doxorubicin and Cdk inhibitor treatment were subjected to immunoblotting with the indicated antibodies. (C) U2TR cells stably expressing the inducible RNAi-resistant FoxM1ΔN/ΔKEN mutant were treated as described in (B). Doxycycline was added during release from the thymidine block. Cells were collected 18 h after doxorubicin treatment for western blot analysis and the amount of mitotic cells was determined 8 h after caffeine addition. The graph shows the average of three independent experiments and the error bars represent the standard deviation. Cdk, cyclin-dependent kinase; RNAi, RNA interference.