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. Author manuscript; available in PMC: 2006 Oct 27.
Published in final edited form as: Oncogene. 2005 Jun 9;24(25):4017–4025. doi: 10.1038/sj.onc.1208576

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

Hong S Yoon 1, Amr M Ghaleb 1, Mandayam O Nandan 1, Irfan M Hisamuddin 1, William Brian Dalton 1, Vincent W Yang 1,2,*
PMCID: PMC1626272  NIHMSID: NIHMS12892  PMID: 15806166

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 γ-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following γ-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 γ-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, γ-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 γ-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.

Keywords: cell cycle, GKLF, p53, Cyclin E, Cdk2, small hairpin RNA (shRNA)

Introduction

The centrosome is a microtubule-organizing center that has a crucial function in cell division (Kirschner and Mitchison, 1986). Interphase cells have a single centrosome that is typically located near the nucleus and contains a pair of centrioles that anchor the recruitment of pericentriolar material including the microtubule-nucleating protein γ-tubulin (Joshi et al., 1992). The centrosome is replicated once, and once only, during the cell cycle to give rise to two centrosomes that function as the spindle poles of the dividing cells (Kellogg, 1989). Its key role is to help organize the mitotic spindle, a collection of protein filaments that pull the duplicated chromosomes apart during cell division, thereby ensuring that each daughter cell receives a complete set. Without the centrosome, normal division will not occur.

Recent studies have implicated a role for centrosome amplification in the pathogenesis of human cancers (Lingle et al., 1998; Pihan et al., 1998; Weber et al., 1998; Sato et al., 1999; Kuo et al., 2000). The term ‘centrosome amplification’ is commonly used to describe centrosomes that appear significantly larger than normal (as defined by staining of structural centrosome components, such as γ-tubulin, which exceeds that seen in the corresponding normal tissue or cell type), centrosomes that contain an abnormal number of centrioles, or the presence of more than two centrosomes in a single cell (Lingle and Salisbury, 1999; Schatten et al., 2000). Numerous studies have also implicated centrosome amplification as a potential origin of chromosomal instability in the development of a variety of human tumors (Pihan et al., 1998; Ghadimi et al., 2000; Pihan et al., 2001; Sato et al., 2001; Lingle et al., 2002; Weaver et al., 2002; Al-Romaih et al., 2003; Mayer et al., 2003; Pihan et al., 2003; Bennett et al., 2004). In a study performed in breast cancer, centrosome abnormalities (in size and number) occurred early in the tumorigenic process and were directly correlated with aneuploidy, which can be evidence of chromosomal instability (Lingle et al., 2002). Another study in colorectal cancer cell lines showed that centrosome amplification occurred in aneuploid, but not diploid, cells and was correlated with chromosomal aberrations (Ghadimi et al., 2000).

The tumor suppressor p53 is frequently mutated in human cancers (Hollstein et al., 1991). Its inactivation has been correlated with genetic instability (Carder et al., 1993; Harvey et al., 1993; Lee et al., 1994; Donehower et al., 1996). Importantly, loss of p53 has been implicated in centrosome amplification. Thus, mouse embryonic fibroblasts (MEFs) obtained from mice null for the p53 alleles contained multiple centrosomes, abnormally formed mitotic spindles, and genomic instability as manifested by aneuploidy (Fukasawa et al., 1996; Fukasawa et al., 1997; Tarapore and Fukasawa, 2002). The centrosome amplification in p53-null MEFs was synergistically induced by overexpression of cyclin E (Mussman et al., 2000), which is a key regulator of centrosome duplication in the G1 phase of the cell cycle (Hinchcliffe et al., 1999; Hinchcliffe and Sluder, 2002). Centrosome amplification is also observed in human cancers with mutated p53, either alone (Carroll et al., 1999; Ouyang et al., 2001) or with overexpression of cyclin E (Kawamura et al., 2004).

p53 is a sequence-specific transcription factor that exerts its cellular effects by modulating expression of a host of genes (Vogelstein et al., 2000). An example of a p53 target gene is one that encodes the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (el-Deiry et al., 1993; Waldman et al., 1995). We previously demonstrated that the gene encoding Krüppel-like factor 4 (KLF4, also known as gut-enriched Krüppel-like factor or GKLF) (Garrett-Sinha et al., 1996; Shields et al., 1996) is transcriptionally activated by p53 following DNA damage (Zhang et al., 1998). The induction of KLF4 consequently causes cell cycle arrest at both the G1/S and G2/M boundaries (Yoon et al., 2003; Yoon and Yang, 2004). These results implicate KLF4 as an important factor in mediating the checkpoint function of p53 upon DNA damage. Indeed, KLF4 has been shown to be a potential tumor suppressor in colorectal cancer (Zhao et al., 2004). Here, we present evidence for an equally important role of KLF4 in preventing centrosome amplification after DNA damage caused by γ-irradiation.

Results

γ-Irradiation leads to centrosome amplification in the absence of p53

We first determined whether centrosome amplification occurs in HCT116 cells following γ-irradiation. HCT116 p53+/+ irradiated and p53−/− cells (Bunz et al., 1998) were with 12 Gy γ-ray and stained for centrosomes using a γ-tubulin antibody 2 days later. Figure 1 shows an example of HCT116 p53−/− cells with 1, 2, 3, and > 4 centrosomes after irradiation, with the latter two cells exhibiting evidence of centrosome amplification. After quantifying the numbers of cells with 1, 2, or ≥ 3 centrosomes, it became apparent that HCT116 p53−/−, but not HCT116 p53+/+, cells exhibited centrosome amplification after γ-irradiation (Figure 2b and a, respectively). These results indicate that p53 is required for the maintenance of centrosome stability in HCT116 cells following γ-ray-induced DNA damage.

Figure 1.

Figure 1

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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

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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

Conditional expression of KLF4 reduces centrosome amplification after γ-irradiation in the absence of p53

Previous studies indicated that KLF4 failed to be induced in HCT116 p53−/− cells after γ-irradiation due to the absence of p53 (Yoon et al., 2003; Yoon and Yang, 2004). To determine whether conditional induction of exogenous KLF4 can suppress radiation-induced centrosome amplification, we infected HCT116 p53−/− cells containing stably transfected EcR (EcR116 p53−/− with recombinant adenoviruses designed to express an inducible KLF4, AdEGI-KLF4 (Yoon et al., 2003; Yoon and Yang, 2004). As shown in Figure 2c, there was a significant reduction in the percentage of cells exhibiting centrosome amplification in infected cells that had been irradiated and treated with the inducer, ponasterone A. These results suggest that re-expression of KLF4 can suppress centrosome amplification from γ-irradiation even in the absence of p53.

γ-Irradiation causes centrosome amplification when KLF4 expression is inhibited even in the presence of wild-type p53

To further demonstrate that KLF4 is required for the maintenance of centrosome stability following DNA damage, we established stably transfected HCT116 p53+/+ cells with reduced KLF4 expression using small hairpin RNA (shRNA). Two independent clones of cells were obtained and named KLF4/sh2-1 and KLF4/sh2-2, respectively. As shown in Figure 3, both cell lines exhibited reduced induction in KLF4 following γ-irradiation, with the sh2-2 cell line having a greater degree of inhibition than sh2-1 (lanes 4 and 6). Importantly, the lack of KLF4 induction in the two cells was accompanied by a failure of p21WAF1/CIP1, but not p53, induction (lanes 4 and 6). In contrast, HCT116 p53+/+ cells that had been transfected with the vector alone (sh-vector) exhibited the normal response to γ-irradiation for p53, KLF4, and p21WAF1/CIP1 (lanes 2). These results are consistent with our previous observations that KLF4 is necessary for mediating the effect of p53 in inducing p21WAF1/CIP1.

Figure 3.

Figure 3

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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

To demonstrate that inhibition of KLF4 results in an altered response of cells to DNA damage, we irradiated the KLF4 sh2-2 cells with 12 Gy γ-ray and scored mitotic indices 2 days following irradiation. As seen in Figure 4a, irradiated KLF4 sh2-2 cells contained a higher percentage of cells in mitosis when compared to unirradiated cells. This result is consistent with our previous study that transient inhibition of KLF4 using small interfering RNA (siRNA) led to an increase in mitotic entry following γ-irradiation (Yoon and Yang, 2004). Importantly, inhibition of KLF4 expression due to RNA interference resulted in an increase in centrosome amplification after γ-irradiation (Figure 4b) in a manner similar to that in HCT116 p53−/− cells (Figure 2b).

Figure 4.

Figure 4

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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

Inhibition of KLF4 expression results in increased cyclin E level and Cdk2 activity after γ-irradiation

To determine the mechanism by which KLF4 prevents centrosome amplification following DNA damage, we measured the levels of cyclin E in various cells with or without irradiation. Cyclin E is known to be a crucial factor in regulating centrosome duplication (Tokuyama et al., 2001; Hinchcliffe and Sluder, 2002; Tarapore et al., 2002; Kawamura et al., 2004). As seen in Figure 5a, while the levels of cyclin E remained largely unchanged after irradiation in HCT116 p53+/+ cells and HCT116 p53+/+ cells stably transfected with a sh-vector (lanes 1 and 2, and 5 and 6, respectively), the levels of cyclin E were significantly increased in irradiated HCT116 p53−/− and HCT116 KLF4/sh2-2 cells when compared to unirradiated cells (lanes 3 and 4, and 7 and 8, respectively). Moreover, the Cdk2 activity was also elevated in irradiated HCT116 p53−/− and HCT116 KLF4/sh2-2 cells when compared to unirradiated cells (Figure 5b; lanes 3 and 4, and 7 and 8, respectively). Importantly, the elevated cyclin E level and Cdk2 activity due to irradiation in both cells were correlated with centrosome amplification (Figures 2b and 4b).

Figure 5.

Figure 5

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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

KLF4 overexpression inhibits cyclin E expression and promoter activity

To further determine how KLF4 may influence cyclin E and Cdk2 activity, we performed cotransfection experiments in HCT116 p53−/− cells using a −363 cyclin E-luciferase reporter (Geng et al., 1996) and an expression vector containing KLF4, PMT3-KLF4 (Shields et al., 1996; Shields and Yang, 1997). Figure 6a shows that the reporter activity was significantly reduced in cells transfected with PMT3-KLF4 as compared to those transfected with the PMT3 vector alone (lanes 1 and 2). Similarly, conditional induction of KLF4 in irradiated EcR116 p53−/− cells infected with AdEGI-KLF4 resulted in a reduction in the level of cyclin E (Figure 6b; compare lane 1 and 2). These results indicate that, when overexpressed, KLF4 is an inhibitor of cyclin E transcription.

Figure 6.

Figure 6

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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

Discussion

The duplication of centrosomes is a carefully choreographed event during progression of the cell cycle. In mammalian cells, the centrosome reproduces only once during interphase, thus ensuring a strictly bipolar spindle axis during mitosis (Hinchcliffe and Sluder, 2001). Since there is not a checkpoint in the cell cycle that monitors defects in the number of spindles (Sluder et al., 1997), dysregulated centrosome duplication can lead to genomic instability due to the formation of multipolar mitotic spindles. This is consistent with reports that document a direct correlation between centrosome abnormalities (in size and number) with chromosomal instability in several human cancers (Ghadimi et al., 2000; Pihan et al., 2001; Sato et al., 2001; Lingle et al., 2002).

It is now well established that centrosome duplication is largely controlled by the Cdk2–cyclin E complex (Hinchcliffe and Sluder, 2001; Hinchcliffe and Sluder, 2002). Cdk2–cyclin E activity rises shortly before the onset of S phase, which is the time when evidence of centrosome duplication first appears (Hinchcliffe et al., 1999). This is supported by the finding that inactivation of the Cdk2 inhibitor, p21WAF1/CIP1, results in increased Cdk2 activity and allows the cells to accumulate multiple centrosomes (Mantel et al., 1999). It is also supported by the finding that mutations in the tumor suppressor p53, which directly activates p21WAF1/CIP1, result in centrosome amplification (Fukasawa et al., 1996). However, there appears to be a difference between human and mouse cells with regard to the effect of p53 mutation on centrosome duplication. In MEFs, deletion of p53 alone is sufficient to cause centrosome duplication (Fukasawa et al., 1996). In contrast, inactivation of p53 in normal human fibroblasts does not result in significant centrosome amplification or chromosomal instability (Bunz et al., 2002; Duensing et al., 2000). The reason behind this discrepancy is thought to reside in the activity of cyclin E. In cultured normal human cells, cyclin E expression is strictly controlled and limited to a short period at late G1 phase (Ekholm et al., 2001). In contrast, in cultured mouse cells, cyclin E expression is less stringently controlled, and increased cyclin E activity can often be seen in early G1 phase (Kawamura et al., 2004). A reason for the difference in expression of cyclin E between human and mouse is the divergence in the regulatory sequences between the two cyclin E promoters (Ohtani et al., 1995; Botz et al., 1996). Our finding that under baseline (unirradiated) conditions, both HCT116 p53+/+ and −/− cells had relatively similar and stable number of centrosomes (Figure 2a and b) is consistent with these previous observations.

Following γ-irradiation, HCT116 p53−/− but not p53+/+ cells exhibited evidence of centrosome amplification (Figures 1 and 2a and b). Previous studies also demonstrated that HCT116 p53+/+ cells arrested in G2 phase following irradiation, while p53−/− cells continued into mitosis (Bunz et al., 1998; Yoon and Yang, 2004). These findings highlighted the importance of p53 in maintaining DNA damage-induced checkpoint functions and centrosome duplication. A reason for the increasing number of centrosomes after irradiation of p53-null cells could be due to the inability of these cells to complete cytokinesis (Bunz et al., 1998). However, our study also revealed that irradiated p53−/− cells contained a higher cyclin E level than unirradiated cells (Figure 5a). This could contribute to the observed higher Cdk2 activity in irradiated p53−/− cells (Figure 5b), although another factor could be the previously observed lack of induction in p21WAF1/CIP1 after irradiation of cells lacking p53 (Yoon et al., 2003; Yoon and Yang, 2004). Be that as it may, our study clearly showed that p53 is crucial for preventing centrosome amplification from γ-irradiation-induced DNA damage.

Our study identified KLF4 as an important mediator of p53 in maintaining centrosome stability following γ-irradiation. Thus, in the absence of p53, inducible expression of KLF4 inhibited centrosome amplification in irradiated cells (Figure 2c). Conversely, stable suppression of KLF4 in cells with wild-type p53 was associated with centrosome amplification after γ-irradiation (Figure 4b). Together, these data indicate that KLF4 is both necessary and sufficient for preventing centrosome amplification following DNA damage. As with p53, irradiated cells with reduced KLF4 contained an elevated level of cyclin E and Cdk2 activity (Figure 5). Our study also showed that overexpression of KLF4 reduced the level of cyclin E (Figure 6b), which is in part due to the ability of KLF4 to suppress the cyclin E promoter activity (Figure 6a). In addition, previous studies indicate that KLF4 is required for the induction of p21WAF1/CIP1 expression in a p53-dependent manner (Zhang et al., 2000; Yoon et al., 2003; Yoon and Yang, 2004). This effect must also have contributed to the increase in Cdk2 activity in irradiated cells with reduced KLF4 (Figure 5). Taken together, these findings support a crucial role for KLF4 in regulating centrosome duplication upon DNA damage.

The significance of KLF4 in checkpoint control is further demonstrated by the recent observation that KLF4 is inactivated by either allelic loss or promoter methylation in a subset of human colorectal cancer specimens (Zhao et al., 2004). In tumors with reduced KLF4 expression, KLF4 could be viewed as a tumor suppressor (Zhao et al., 2004). Thus, the lack of crucial checkpoint functions due to the reduction in KLF4 expression may potentially contribute to the tumor phenotype. Although we have not examined tumors with reduced KLF4 expression for evidence of centrosome amplification, we noticed that a particular colorectal cancer cell line, RKO, known to contain exceedingly low level of KLF4 mRNA (Dang et al., 2001; Zhao et al., 2004), exhibited evidence of centrosome amplification after γ-irradiation (Ghaleb and Yang, unpublished observation). As centrosome amplification has been implicated as a factor leading to chromosomal instability in cancer (Pihan et al., 1998; Weber et al., 1998; Ghadimi et al., 2000; Sato et al., 2001; Lingle et al., 2002; Al-Romaih et al., 2003; Mayer et al., 2003; Pihan et al., 2003), it is formally possible that tumors with diminished expression of KLF4 may also exhibit chromosomal instability. Indeed, recent experiments in our lab have shown that shRNA-mediated reduction of KLF4 increases formation of polyploid and subsequent aneuploid cells upon treatment with the microtubule inhibitor nocodazole, thereby suggesting a role for KLF4 in protection against, at the very least exogenously induced, chromosomal instability. Previous studies from our group have also placed KLF4 as an effector downstream from the tumor suppressor APC in colorectal cancer cells, further suggesting the KLF4 may be involved in maintaining genomic stability in an APC-dependent fashion (Dang et al., 2001). Investigations of mechanisms of how KLF4 may affect genomic stability are currently underway.

Materials and methods

Cell lines

The colon cancer cell lines wild type and null for p53, HCT116 p53+/+ and HCT116 p53−/−, respectively, were generous gifts of Dr Bert Vogelstein of Johns Hopkins University (Bunz et al., 1998). The cells were cultured in McCoy's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. EcR116 p53−/− cells were generated by stably transfecting pVgRXR (Chen et al., 2001), which contains VgEcR and RXR that form the receptor for the insect hormone, ecdysone, into the parental HCT116 p53−/− cells and selected with Zeocin (Yoon et al., 2003; Yoon and Yang, 2004).

γ-Irradiation

γ-irradiation of cultured cells was performed using a 137Cs γ-irradiator at 0.8 Gy/min for 15 min, for a total of 12 Gy (Yoon et al., 2003; Yoon and Yang, 2004). Media were changed every 24 h until cells were harvested for analysis.

Centrosome immunostaining

Cells grown on coverslips were cooled on ice and washed with cold phosphate-buffered saline (PBS). They were then fixed with cold 100% methanol and placed at −20°C for 15 min. Afterwards, cells were washed twice with cold PBS and placed in a 0.5% bovine serum albumin (BSA) solution for 1 h at 4°C. FITC-conjugated γ-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was then added to a final concentration of 10 μg/ml and the cells were incubated for 2 h at 4°C. After the incubation, cells were washed twice with cold PBS and counterstained with ToPro3 (Molecular Probes, Eugene, OR, USA) for 5 min at room temperature in the dark. Cells were then washed five times with cold PBS and then with ddH2O. Cells were mounted using ProLong Antifade kit (Molecular Probes), sealed and visualized with a Zeiss 510 confocal microscope. For each experiment, 300 cells with visible centrosomes were counted and grouped based on whether they had 1, 2, or ≥3 centrosomes per cell.

Measurement of mitotic indices

Cells grown on coverslips were fixed in 3% formaldehyde for 15 min. Cold 100% methanol was then added and cells were incubated at room temperature for 20 min. Cells were then rinsed three times with Dulbecco's phosphate-buffered saline (DPBS). A Hoechst 33258 solution (10 μg/ml) was added to a final concentration of 0.2 μg/ml and the cells were incubated at room temperature for 15 min. After the incubation, cells were rinsed five times with DPBS and the nuclei were visualized by fluorescence microscopy (Nikon, Melville, NY, USA). A minimum of 300 cells was examined per experiment. Mitotic figures were scored for cells with condensed chromosomes (Yoon and Yang, 2004).

Adenovirus infection

The recombinant adenovirus containing green fluorescence protein (GFP) and KLF4, AdEGI-KLF4, was described previously (Chen et al., 2001). EcR116 p53−/− cells were grown to 40% confluence in 10-cm dishes and replenished with fresh media containing 2% FBS followed by the addition of 108 plaque forming units of recombinant virus per dish. Infected cells were incubated at 37°C for 6 h, at which time cells were γ-irradiated and the media changed. Cells were treated with 5 μM ponasterone A (Invitrogen, Carlsbad, CA, USA) for 48 h and then collected for further analysis.

Preparation of shRNA and stable transfection

Single-stranded DNAs (of 65 nucleotides) were produced by Sigma Genosys (Sigma Genosys, The Woodlands, TX) with the following sequences: 5′-GATCCCGTTGGACCCGGTGTACATTCTTCAAGAGAGAATGTACACCGGGTCCAATTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAATTGGACCCGGTGTACATTCTCTCTTGAAGAATGTACACCGGGTCCAACGG-3′. The single-stranded DNA was dissolved in 10 mm Tris–HCl, pH 7.5, 50 mm NaCl, and 1 mm EDTA. The complementary oligonucleotides were mixed at equimolar concentration and heated to 95°C in a standard heat block for 15 min. Heat block was then removed from the apparatus and allowed to cool to room temperature. The annealed DNA was then inserted to pSilencer 3.1-H1 hygro (Ambion Inc., Austin, TX, USA) between the BamHI and HindIII sites. Positive clones were identified by DNA sequencing and plasmid DNA was amplified and purified. They were then linearized by XmnI restriction enzyme (New England Biolab, Beverly, MA, USA) and transfected into HCT116 p53+/+ cells using Lipofectamine 2000 (Invitrogen). Positive clones were selected by growing the cells in a concentration of 100 μg/ml hygromycin. Cells were also stably transfected with the vector alone, selected with hygromycin and used as a control.

Western blot analysis

Protein extraction and Western blot analyses were performed using standard procedures. The membranes were immunoblotted with primary antibodies against KLF4 (Shields et al., 1996), p53, p21WAF1/CIP1, cyclin E, or β-actin (Santa Cruz). Secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG; 1 : 10000 dilutions (Santa Cruz).

Cyclin-dependent kinase 2 (Cdk2) assay

Cells in plate were washed twice with cold PBS, then 400 μl lysis buffer, PI (0.1% NP40, 50 mm HEPES, pH 7.0, 250 mm NaCl, 0.21% (w/v) NaF, 1 mm PMSF, 1 μg/ml each of pepstatin, leupeptin, and aprotinin) were added. Cells were then scraped of the plate, vortexed for 2 min followed by incubation on ice for 20 min. Cell debris was separated by centrifugation at maximum speed for 10 min at 4°C. The supernatant was collected and the pellet discarded. To new tubes, 200 μg protein per sample was transferred and the volume was brought to 1 ml with lysis buffer PI. In total, 1 μg Cdk2 antibody per 100 μg protein was added and mixed. The mixture was then incubated with thorough gentle mixing for 1 h at 4°C. In all, 25 μl of a 50% EZview Red protein G affinity gel beads slurry (Sigma) was aliquoted to clean tubes and washed twice and equilibrated with 750 μl lysis buffer PI. After the incubation, the mixtures were added to the equilibrated beads, vortexed briefly, and then incubated with thorough gentle mixing for 1 h at 4°C. The tubes were centrifuged for 30 s at 8200 g to pellet the beads and the supernatant was discarded. The pellets were washed twice with lysis buffer PI, then equilibrated in kinase buffer (20 mm HEPES, pH 7.9, 5mm MgCl2, and 10% glycerol). After equilibration, the beads were pelleted again, resuspended in 20 μl of kinase buffer containing 1 mm DTT, 500 μm ATP, and 1 μg histone H1 (Roche Applied Sciences, Indianapolis, IN, USA) and incubated for 30 min at 37°C. The reaction was then stopped by adding 20 μl of 2× loading buffer per sample, and then heated in a boiling water bath for 10 min. Histone H1 phosphorylation was then detected by Western blot using a phospho-specific Histone H1 antibody (Calbiochem, San Diego, CA, USA) at 1.5 μg/ml. Secondary antibody against rabbit IgG was used at 1 : 5000 dilutions (Santa Cruz).

Cotransfection and reporter assays

Co-transfection experiments were performed in HCT116 p53−/− cells with a −363 to +1007 human cyclin E-luciferase reporter, pGL-cyclin E (−363 to +1007) (kindly provided by Dr Robert Weinberg) (Geng et al., 1996), the expression construct containing KLF4, pMT3-KLF4 (Shields et al., 1996; Shields and Yang, 1997), and the internal control Renilla luciferase, pRL-CMV (Promega). Transfection experiments were performed using lipofection (Yoon and Yang, 2004). Luciferase activities were determined 2 days following transfection using the dual Luciferase Reporter Assay System (Promega). All firefly luciferase activities were normalized to the Renilla luciferase internal control.

Acknowledgements

We thank Dr B Vogelstein for providing the HCT116 p53+/+ and HCT116 p53−/− cell line and Dr R Weinberg for providing the cyclin E luciferase reporter plasmid. This work was in part supported by grants from the National Institutes of Health (DK52230, DK64399, and CA84197). VWY is the recipient of a Georgia Cancer Coalition Distinguished Cancer Clinician Scientist award. WBD is a recipient of a Medical Scientist Training Program grant.

Abbreviations

BSA

bovine serum albumin

Cdk2

cyclin-dependent kinase 2

DPBS

Dulbecco's phosphate-buffered saline

FBS

fetal bovine serum

GKLF

gut-enriched Krüppel-like factor

KLF4

Krüppel-like factor 4

MEFs

mouse embryonic fibroblasts

PBS

phosphate-buffered saline

shRNA

small hairpin RNA

References

  1. Al-Romaih K, Bayani J, Vorobyova J, Karaskova J, Park PC, Zielenska M, Squire JA. Cancer Genet. Cytogenet. 2003;144:91–99. doi: 10.1016/s0165-4608(02)00929-9. [DOI] [PubMed] [Google Scholar]
  2. Bennett RA, Izumi H, Fukasawa K. Oncogene. 2004;23:6823–6829. doi: 10.1038/sj.onc.1207561. [DOI] [PubMed] [Google Scholar]
  3. Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B, Eilers M, Hatzigeorgiou A, Jansen-Durr P. Mol. Cell. Biol. 1996;16:3401–3409. doi: 10.1128/mcb.16.7.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B. Science. 1998;282:1497–1501. doi: 10.1126/science.282.5393.1497. [DOI] [PubMed] [Google Scholar]
  5. Bunz F, Fauth C, Speicher MR, Dutriaux A, Sedivy JM, Kinzler KW, Vogelstein B, Lengauer C. Cancer Res. 2002;62:1129–1133. [PubMed] [Google Scholar]
  6. Carder P, Wyllie AH, Purdie CA, Morris RG, White S, Piris J, Bird CC. Oncogene. 1993;8:1397–1401. [PubMed] [Google Scholar]
  7. Carroll PE, Okuda M, Horn HF, Biddinger P, Stambrook PJ, Gleich LL, Li YQ, Tarapore P, Fukasawa K. Oncogene. 1999;18:1935–1944. doi: 10.1038/sj.onc.1202515. [DOI] [PubMed] [Google Scholar]
  8. Chen X, Johns DC, Geiman DE, Marban E, Dang DT, Hamlin G, Sun R, Yang VW. J. Biol. Chem. 2001;276:30423–30428. doi: 10.1074/jbc.M101194200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dang DT, Mahatan CS, Dang LH, Agboola IA, Yang VW. Oncogene. 2001;20:4884–4890. doi: 10.1038/sj.onc.1204645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donehower LA, Godley LA, Aldaz CM, Pyle R, Shi YP, Pinkel D, Gray J, Bradley A, Medina D, Varmus HE. Prog. Clin. Biol. Res. 1996;395:1–11. [PubMed] [Google Scholar]
  11. Duensing S, Lee LY, Duensing A, Basile J, Piboonniyom S, Gonzalez S, Crum CP, Munger K. Proc. Natl. Acad. Sci. USA. 2000;97:10002–10007. doi: 10.1073/pnas.170093297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ekholm SV, Zickert P, Reed SI, Zetterberg A. Mol. Cell. Biol. 2001;21:3256–3265. doi: 10.1128/MCB.21.9.3256-3265.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. Cell. 1993;75:817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]
  14. Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Science. 1996;271:1744–1747. doi: 10.1126/science.271.5256.1744. [DOI] [PubMed] [Google Scholar]
  15. Fukasawa K, Wiener F, Vande Woude GF, Mai S. Oncogene. 1997;15:1295–1302. doi: 10.1038/sj.onc.1201482. [DOI] [PubMed] [Google Scholar]
  16. Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B. J. Biol. Chem. 1996;271:31384–31390. doi: 10.1074/jbc.271.49.31384. [DOI] [PubMed] [Google Scholar]
  17. Geng Y, Eaton EN, Picon M, Roberts JM, Lundberg AS, Gifford A, Sardet C, Weinberg RA. Oncogene. 1996;12:1173–1180. [PubMed] [Google Scholar]
  18. Ghadimi BM, Sackett DL, Difilippantonio MJ, Schrock E, Neumann T, Jauho A, Auer G, Ried T. Genes Chromosomes Cancer. 2000;27:183–190. [PMC free article] [PubMed] [Google Scholar]
  19. Harvey M, Sands AT, Weiss RS, Hegi ME, Wiseman RW, Pantazis P, Giovanella BC, Tainsky MA, Bradley A, Donehower LA. Oncogene. 1993;8:2457–2467. [PubMed] [Google Scholar]
  20. Hinchcliffe EH, Li C, Thompson EA, Maller JL, Sluder G. Science. 1999;283:851–854. doi: 10.1126/science.283.5403.851. [DOI] [PubMed] [Google Scholar]
  21. Hinchcliffe EH, Sluder G. Genes Dev. 2001;15:1167–1181. doi: 10.1101/gad.894001. [DOI] [PubMed] [Google Scholar]
  22. Hinchcliffe EH, Sluder G. Oncogene. 2002;21:6154–6160. doi: 10.1038/sj.onc.1205826. [DOI] [PubMed] [Google Scholar]
  23. Hollstein M, Sidransky D, Vogelstein B, Harris CC. Science. 1991;253:49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  24. Joshi HC, Palacios MJ, McNamara L, Cleveland DW. Nature. 1992;356:80–83. doi: 10.1038/356080a0. [DOI] [PubMed] [Google Scholar]
  25. Kawamura K, Izumi H, Ma Z, Ikeda R, Moriyama M, Tanaka T, Nojima T, Levin LS, Fujikawa-Yamamoto K, Suzuki K, Fukasawa K. Cancer Res. 2004;64:4800–4809. doi: 10.1158/0008-5472.CAN-03-3908. [DOI] [PubMed] [Google Scholar]
  26. Kellogg DR. Nature. 1989;340:99–100. doi: 10.1038/340099a0. [DOI] [PubMed] [Google Scholar]
  27. Kirschner MW, Mitchison T. Nature. 1986;324:621. doi: 10.1038/324621a0. [DOI] [PubMed] [Google Scholar]
  28. Kuo KK, Sato N, Mizumoto K, Maehara N, Yonemasu H, Ker CG, Sheen PC, Tanaka M. Hepatology. 2000;31:59–64. doi: 10.1002/hep.510310112. [DOI] [PubMed] [Google Scholar]
  29. Lee JM, Abrahamson JL, Kandel R, Donehower LA, Bernstein A. Oncogene. 1994;9:3731–3736. [PubMed] [Google Scholar]
  30. Lingle WL, Barrett SL, Negron VC, D'Assoro AB, Boeneman K, Liu W, Whitehead CM, Reynolds C, Salisbury JL. Proc. Natl. Acad. Sci. USA. 2002;99:1978–1983. doi: 10.1073/pnas.032479999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lingle WL, Lutz WH, Ingle JN, Maihle NJ, Salisbury JL. Proc. Natl. Acad. Sci. USA. 1998;95:2950–2955. doi: 10.1073/pnas.95.6.2950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lingle WL, Salisbury JL. Am. J. Pathol. 1999;155:1941–1951. doi: 10.1016/S0002-9440(10)65513-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mantel C, Braun SE, Reid S, Henegariu O, Liu L, Hangoc G, Broxmeyer HE. Blood. 1999;93:1390–1398. [PubMed] [Google Scholar]
  34. Mayer F, Stoop H, Sen S, Bokemeyer C, Oosterhuis JW, Looijenga LH. Oncogene. 2003;22:3859–3866. doi: 10.1038/sj.onc.1206469. [DOI] [PubMed] [Google Scholar]
  35. Mussman JG, Horn HF, Carroll PE, Okuda M, Tarapore P, Donehower LA, Fukasawa K. Oncogene. 2000;19:1635–1646. doi: 10.1038/sj.onc.1203460. [DOI] [PubMed] [Google Scholar]
  36. Ohtani K, DeGregori J, Nevins JR. Proc. Natl. Acad. Sci. USA. 1995;92:12146–12150. doi: 10.1073/pnas.92.26.12146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ouyang X, Wang X, Xu K, Jin DY, Cheung AL, Tsao SW, Wong YC. Biochim. Biophys. Acta. 2001;1541:212–220. doi: 10.1016/s0167-4889(01)00157-4. [DOI] [PubMed] [Google Scholar]
  38. Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, Doxsey SJ. Cancer Res. 1998;58:3974–3985. [PubMed] [Google Scholar]
  39. Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Cancer Res. 2001;61:2212–2219. [PubMed] [Google Scholar]
  40. Pihan GA, Wallace J, Zhou Y, Doxsey SJ. Cancer Res. 2003;63:1398–1404. [PubMed] [Google Scholar]
  41. Sato N, Mizumoto K, Nakamura M, Maehara N, Minamishima YA, Nishio S, Nagai E, Tanaka M. Cancer Genet. Cytogenet. 2001;126:13–19. doi: 10.1016/s0165-4608(00)00384-8. [DOI] [PubMed] [Google Scholar]
  42. Sato N, Mizumoto K, Nakamura M, Nakamura K, Kusumoto M, Niiyama H, Ogawa T, Tanaka M. Clin. Cancer Res. 1999;5:963–970. [PubMed] [Google Scholar]
  43. Schatten H, Wiedemeier AM, Taylor M, Lubahn DB, Greenberg NM, Besch-Williford C, Rosenfeld CS, Day JK, Ripple M. Biol. Cell. 2000;92:331–340. doi: 10.1016/s0248-4900(00)01079-0. [DOI] [PubMed] [Google Scholar]
  44. Shields JM, Christy RJ, Yang VW. J. Biol. Chem. 1996;271:20009–20017. doi: 10.1074/jbc.271.33.20009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shields JM, Yang VW. J. Biol. Chem. 1997;272:18504–18507. doi: 10.1074/jbc.272.29.18504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sluder G, Thompson EA, Miller FJ, Hayes J, Rieder CL. J. Cell Sci. 1997;110(Part 4):421–429. doi: 10.1242/jcs.110.4.421. [DOI] [PubMed] [Google Scholar]
  47. Tarapore P, Fukasawa K. Oncogene. 2002;21:6234–6240. doi: 10.1038/sj.onc.1205707. [DOI] [PubMed] [Google Scholar]
  48. Tarapore P, Okuda M, Fukasawa K. Cell Cycle. 2002;1:75–81. [PubMed] [Google Scholar]
  49. Tokuyama Y, Horn HF, Kawamura K, Tarapore P, Fukasawa K. J. Biol. Chem. 2001;276:21529–21537. doi: 10.1074/jbc.M100014200. [DOI] [PubMed] [Google Scholar]
  50. Vogelstein B, Lane D, Levine AJ. Nature. 2000;408:307–310. doi: 10.1038/35042675. [DOI] [PubMed] [Google Scholar]
  51. Waldman T, Kinzler KW, Vogelstein B. Cancer Res. 1995;55:5187–5190. [PubMed] [Google Scholar]
  52. Weaver Z, Montagna C, Xu X, Howard T, Gadina M, Brodie SG, Deng CX, Ried T. Oncogene. 2002;21:5097–5107. doi: 10.1038/sj.onc.1205636. [DOI] [PubMed] [Google Scholar]
  53. Weber RG, Bridger JM, Benner A, Weisenberger D, Ehemann V, Reifenberger G, Lichter P. Cytogenet. Cell Genet. 1998;83:266–269. doi: 10.1159/000015168. [DOI] [PubMed] [Google Scholar]
  54. Yoon HS, Chen X, Yang VW. J. Biol. Chem. 2003;278:2101–2105. doi: 10.1074/jbc.M211027200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yoon HS, Yang VW. J. Biol. Chem. 2004;279:5035–5041. doi: 10.1074/jbc.M307631200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhang W, Geiman DE, Shields JM, Dang DT, Mahatan CS, Kaestner KH, Biggs JR, Kraft AS, Yang VW. J. Biol. Chem. 2000;275:18391–18398. doi: 10.1074/jbc.C000062200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang W, Shields JM, Sogawa K, Fujii-Kuriyama Y, Yang VW. J. Biol. Chem. 1998;273:17917–17925. doi: 10.1074/jbc.273.28.17917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Oncogene. 2004;23:395–402. doi: 10.1038/sj.onc.1207067. [DOI] [PMC free article] [PubMed] [Google Scholar]

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