Krüppel-like factor 4 prevents centrosome amplification following gamma-irradiation-induced DNA damage - PubMed (original) (raw)

Krüppel-like factor 4 prevents centrosome amplification following gamma-irradiation-induced DNA damage

Hong S Yoon et al. Oncogene. 2005.

Abstract

Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by gamma-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following gamma-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53-/-) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following gamma-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53-/- cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53+/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, gamma-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following gamma-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.

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Figures

Figure 1

Figure 1

Centrosome immunostaining in HCT116 _p53_−/− cells following γ-irradiation. HCT116 _p53_−/− cells were irradiated with 12 Gy γ-ray and stained for centrosomes using a γ-tubulin antibody 2 days later. Examples of a cell with 1, 2, 3, or >4 centrosomes are shown. Nucleus was counterstained with Topro3

Figure 2

Figure 2

Centrosome profiles of HCT116 p53+/+ and _p53_−/− cells with and without γ-irradiation and of irradiated EcR116 _p53_−/− cells infected with AdEGI-KLF4. HCT116 p53+/+ (a) and _p53_−/− (b) cells were irradiated (+γ) or not (−γ) on day 0 and maintained in culture for 2 days before stained for centrosomes. A total of 200 cells with visible centrosomes were scored in each experiment and the experiments were conducted three additional times for a total N of 4. *P<0.001. (c) EcR116 _p53_−/− cells were infected with recombinant adenovirus containing KLF4, AdEGI-KLF4 (Chen et al., 2001), and irradiated with 12 Gy of γ-ray. Cells were then treated (+PA) or not (−PA) with ponasterone A (PA) to induce KLF4 expression. Centrosomes were scored 2 days later. A total of 200 cells were counted in each experiment and the experiments were repeated three additional times for a total N of 4. *P<0.05

Figure 3

Figure 3

Establishment of stable cell lines with reduced KLF4 expression by RNA interference. HCT116 p53+/+ cells were stably transfected with a plasmid containing small hairpin RNA (shRNA) directed against KLF4 or vector alone. Two independent clones were derived and named KLF4/sh2-1 and KLF4/sh2-2. Cells were irradiated or not on day 0 and maintained in culture for 2 days before being harvested for Western blot analysis for p53, KLF4, p21WAF1/CIP1, and β-actin. Sh-vector indicates cells transfected with vector alone

Figure 4

Figure 4

Mitotic indices and centrosome profiles of HCT116 KLF4/sh2-2 cells with and without irradiation. HCT116 KLF4/sh2-2 cells were irradiated (+γ) or not (−γ) (a) on day 0 and maintained in culture for 2 days before being examined for the presence of mitotic figures. N = 4. *P<0.01. In (b), the cells were irradiated (+γ) or not (−γ) on day 0 and maintained in culture for 2 days before staining for centrosomes. A total of 300 cells were examined in each experiment and the experiments were repeated two additional times for a total N of 3. *P<0.01

Figure 5

Figure 5

Western blot analysis of cyclin E and Cdk2 kinase activity in irradiated and unirradiated cells. HCT116 p53+/+ (lanes 1 and 2), _p53_−/− (lanes 3 and 4), HCT116 p53+/+ transfected with sh-vector (lanes 5 and 6) and HCT116 p53+/+ transfected with KLF4/sh2-2 vector (lanes 7 and 8) were irradiated with 12 Gy (+), or not (−). After 2 days, extracts were prepared for Western blot analysis of cyclin E and β-actin (a) or Cdk2 kinase assay (b). Phospho-H1 is detected with a phospho-specific antibody against histone H1. Immunoglobulin G (IgG) is used as a loading control

Figure 6

Figure 6

KLF4 suppresses cyclin E expression. (a)HCT116 _p53_−/− cells were cotransfected with the −363 cyclin E-luciferase reporter, Renilla luciferase internal control, and PMT3 (lane 1) or PMT3-KLF4 (lane 2). Luciferase activity was determined 2 days following transfection and normalized to the internal control. The means of six independent experiments are shown. *P<0.005. (b)EcR116 _p53_−/− cells were infected with AdEGI-KLF4, irradiated with 12 Gy, and treated (lane 2) or not (lane 1) with the inducer, ponasterone A (PA). Cell extracts were prepared 2 days later and examined for the content of cyclin E and β-actin by Western blotting

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