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. 2003 Jan 24;278(4):2101-5.
doi: 10.1074/jbc.M211027200. Epub 2002 Nov 8.

Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage

Hong S Yoon  1 , Xinming Chen, Vincent W Yang
Affiliations

Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage

Hong S Yoon et al. J Biol Chem. .

Abstract

The tumor suppressor p53 is required for the maintenance of genomic integrity following DNA damage. One mechanism by which p53 functions is to induce a block in the transition between the G(1) and S phase of the cell cycle. Previous studies indicate that the Krüppel-like factor 4 (KLF4) gene is activated following DNA damage and that such activation depends on p53. In addition, enforced expression of KLF4 causes G(1)/S arrest. The present study examines the requirement of KLF4 in mediating the p53-dependent cell cycle arrest process in response to DNA damage. We show that the G(1) population of a colon cancer cell line, HCT116, that is null for the p53 alleles (-/-) was abolished following gamma irradiation compared with cells with wild-type p53 (+/+). Conditional expression of KLF4 in irradiated HCT116 p53-/- cells restored the G(1) cell population to a level similar to that seen in irradiated HCT116 p53+/+ cells. Conversely, treatment of HCT116 p53+/+ cells with small interfering RNA (siRNA) specific for KLF4 significantly reduced the number of cells in the G(1) phase following gamma irradiation compared with the untreated control or those treated with a nonspecific siRNA. In each case the increase or decrease in KLF4 level because of conditional induction or siRNA inhibition, respectively, was accompanied by an increase or decrease in the level of p21(WAF1/CIP1). Results of our study indicate that KLF4 is an essential mediator of p53 in controlling G(1)/S progression of the cell cycle following DNA damage.

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Figures

Fig. 1
Fig. 1. The effect of γ irradiation on HCT116 p53 +/+ and HCT116 p53−/− cells
In panels A through D flow cytometric analyses of HCT116 p53+/+ and HCT116 p53−/− were performed 24 h after 0 or 12 Gy of γ irradiation. Cells were stained with propidium iodide, and DNA content was analyzed by flow cytometry. The DNA content of haploid and diploid cells is designated 2n and 4n, respectively. Panels E and F show the means and standard deviations of percentage of the G1, S, and G2/M populations from five independent experiments in non-irradiated and irradiated cells, respectively. Open bars represent HCT116 p53+/+, and closed bars represent HCT116 p53−/−. 15,000 cells were analyzed in each experiment. *, p < 0.05 compared with HCT116 p53+/+ cells.
Fig. 2
Fig. 2. Western blot analysis of p53, KLF4, and p21WAF1/CIP1 in response to γ irradiation
The levels of p53, KLF, and p21WAF1/CIP1 were determined by Western blot analysis in HCT116 p53+/+ (lanes 1 and 2) and HCT116 p53−/− (lanes 3 and 4) without irradiation (lanes 1 and 3) and 24 h after 12 Gy of γ irradiation (lanes 2 and 4).
Fig. 3
Fig. 3. The effect of inducible KLF4 expression on cell cycle of p53
−/− cells following γ irradiation. EcR116 p53−/− cells were infected with AdEGI or AdEGI-KLF4 and then irradiated with 12 Gy of γ radiation (panels E, F, G, and H) or not (panels A, B, C, and D). Cells were then treated with vehicle alone (panels A, E, C, and G) or ponasterone A (PA) (panels B, F, D, and H) for 24 h before being harvested for flow cytometric analysis. Panels I–L show the means and standard deviations of percent cells in the three phases of the cell cycle in 5 independent experiments. 15,000 cells were analyzed per experiment. *, p < 0.05 compared with uninduced cells.
Fig. 4
Fig. 4. Western blot analysis of KLF4 and p21WAF1/CIP1 in EcR116 p53
−/− cells. The levels of KLF4 and p21WAF1/CIP1 were determined by Western blot analysis in EcR116 p53−/− cells not infected with any virus (lanes 1–4), infected with AdEGI (lanes 5–8) or with AdEGI-KLF4 (lanes 9–12), followed by 0 (lanes 1, , , , , and 11) or 12 Gy (lanes 2, , , , , and 12) of γ irradiation and induced with ponasterone A (PA) (lanes 3, , , , , and 12) or vehicle alone (lanes 1, , , , , and 10).
Fig. 5
Fig. 5. The effect of KLF4 siRNA on protein levels of p53, KLF4, and p21WAF1/CIP1 in HCT116 p53
+/+ cells following γ irradiation. The levels of p53, KLF4, and p21WAF1/CIP1 were determined in γ-irradiated (even lanes) or non-irradiated (odd lanes) HCT116 p53+/+ cells that were untransfected (lanes 1 and 2), mock-transfected (lanes 3 and 4), transfected with 2 (lanes 5 and 6) or 4 μg (lanes 7 and 8) of KLF4-specific siRNA, or transfected with 2 (lanes 9 and 10) or 4 μg (lanes 11 and 12) of nonspecific siRNA. Cells were harvested 24 h later for Western blot analysis of protein levels.
Fig. 6
Fig. 6. The effect of KLF4 siRNA on the cell cycle in HCT116 p53+/+ cells following γ irradiation
Flow cytometric analyses were performed in HCT116 p53+/+ cells that were non-irradiated (panels A–E) or irradiated (panels F–J) followed by mock transfection (panels A and F), transfection with 2 (panels B and G) or 4 μg (panels C and H) of KLF4-specific siRNA, or transfection with 2 (panels D and I) or 4 μg (panels E and J) of nonspecific siRNA. Cells were studied for 24 h following irradiation. Panels K and L show the means and standard deviations of percent cells in G1 phase for each of the treatments in five independent experiments. 15,000 cells were analyzed in each experiment. *, p < 0.05 compared with mock-transfected cells.
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References

    1. Nurse P, Masui Y, Hartwell L. Nat Med. 1998;4:1103–1106. - PubMed
    1. Malumbres M, Barbacid M. Nat Rev Cancer. 2001;1:222–231. - PubMed
    1. Melo J, Toczyski D. Curr Opin Cell Biol. 2002;14:237–245. - PubMed
    1. Vogelstein B, Lane D, Levine AJ. Nature. 2000;408:307–310. - PubMed
    1. Hollstein M, Hergenhahn M, Yang Q, Bartsch H, Wang ZQ, Hainaut P. Mutat Res. 1999;431:199–209. - PubMed

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