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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 G1 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 G1/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 G1 population of a colon cancer cell line, HCT116, that is null for the p53 alleles (−/−) was abolished following γ irradiation compared with cells with wild-type p53 (+/+). Conditional expression of KLF4 in irradiated HCT116 p53−/− cells restored the G1 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 G1 phase following γ 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 p21WAF1/CIP1. Results of our study indicate that KLF4 is an essential mediator of p53 in controlling G1/S progression of the cell cycle following DNA damage.
The mammalian cell cycle is operationally divided into five distinct phases: gap 1 (G1), DNA synthesis (S), gap 2 (G2), mitosis (M), and growth arrest phase (G0), also called quiescence (1). Complex networks of control mechanisms called “checkpoints” are responsible for the orderly progression of these events within the cell cycle. Defects in checkpoint control increase genetic instability, thereby contributing to uncontrolled proliferation (2). For example, damage to the DNA elicits a series of signal transduction pathways that result in an arrest of the cell cycle at various checkpoints (3). Much of the DNA damage-induced signals are funneled through p53, which directs further downstream actions that lead to inhibition of G1 to S and G2 to M transitions, among other events such as apoptosis (4). Therefore, it is not surprising that p53 is the most frequently mutated tumor suppressor gene in human cancers (5).
The arrest in the transition between the G1 and S phase of the cell cycle elicited by p53 requires in part the transcriptional activation of the gene encoding the cyclin-dependent kinase (Cdk)1 inhibitor p21WAF1/CIP1 (6, 7). p21WAF1/CIP1 binds to several G1 cyclin-Cdk complexes and inhibits phosphorylation of the retinoblastoma susceptibility gene product Rb (8), a step required for the onset of DNA synthesis (9). Recent evidence suggests that p21WAF1/CIP1 is also required to sustain G2 arrest after DNA damage (10). Here, p21WAF1/CIP1 mediates the function of p53 in response to DNA damage by inhibiting Cdc2 (11), a Cdk required for entry into mitosis (12). The proportion of cells that arrests in G1/S or G2/M depends on the cell type and status of checkpoint controls in each cell (13).
Although earlier studies indicate that expression of p21WAF1/CIP1 is the result of direct binding of p53 to its promoter (14), it is now evident that a myriad of transcription factors under various physiologic conditions can also lead to the transcriptional activation of p21WAF1/CIP1 (15). Among these is the zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), also called gut-enriched Krüppel-like factor or GKLF (16, 17). KLF4 is a member of a rapidly expanding family of mammalian Krüppel-like factors that exhibit homology to the Drosophila protein Krüppel (18). Expression of KLF4 is highly enriched in the postmitotic terminally differentiated epithelial cells of the intestine and epidermis (19, 20). In cultured cells expression of KLF4 is associated with growth arrest as a result of serum deprivation or contact inhibition (19, 21). Conversely, enforced expression of KLF4 inhibits DNA synthesis and results in decreased cell proliferation (19, 22, 23). These studies suggest that KLF4 is a negative regulator of cell growth.
Recently, it was demonstrated that expression of KLF4 is also induced by DNA damage and that such induction is dependent on p53 (24). Importantly, KLF4 was shown to physically interact with p53, resulting in a synergistic activation of the p21WAF1/CIP1 promoter. Moreover, antisense inhibition of KLF4 leads to a decreased level of p21WAF1/CIP1 in response to DNA damage (24), suggesting that KLF is a potentially important mediator of p53-induced growth arrest. Indeed, recent studies using an inducible system for KLF4 indicate that its induction leads to arrest in the G1/S transition of the cell cycle (25). In the present study, we further characterize the role of KLF4 in mediating p53-dependent cell cycle arrest. By manipulating KLF4 expression, we show that KLF4 is essential for the G1/S cell cycle arrest that results from DNA damage.
The colon cancer cell lines, wild-type and null for p53, HCT116 p53+/+, and HCT116 p53−/−, respectively, were generous gifts from Dr. Bert Vogelstein of Johns Hopkins University (10). The cells were cultured in McCoy’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. EcR116 p53−/− cells were established by stably transfecting pVgRXR (25), which contains VgEcR and retinoid X receptor that form a receptor for the insect hormone ecdysone, into the parental HCT116 cell line and selecting with 100 μg/ml Zeocin (Invitrogen). The level of retinoid X receptor expression was determined by Western blot analysis.
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. Cells were harvested at 24 h after γ irradiation for subsequent assays.
The recombinant adenovirus containing green fluorescence protein and KLF4 (AdEGI-KLF4) or green fluorescence protein alone (AdEGI) were described previously (25, 26). EcR116 p53−/− cells were grown to 40% confluence in 10-cm dishes and replenished with fresh media containing 2% fetal bovine serum 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 medium was changed. Cells were treated with 5 mu;M ponasterone A (Invitrogen) for 24 h and then collected for further analysis.
23-nucleotide single-stranded RNAs were produced by Integrated DNA Technologies (Coralville, IA). The small interfering RNA (siRNA) sequences targeting KLF4 (GenBank™ accession number XM_047517) correspond to the coding region between nucleotides 121–141 from the translation initiation site. The complementary single-stranded RNAs were dissolved in 10 mM Tris-HCl and 1 mM EDTA (pH 7.0) and annealed in 25 mM KoAc, 10 mM Tris-HCl, and 1 mM EDTA (pH 7.0) by briefly heating to 70 °C, then incubating for 20 min each at 37 and 23 °C. A nonspecific double-stranded siRNA with identical length was also generated based on the sequence of an unrelated protein and used as a control.
HCT116 p53+/+ cells were grown to 40% confluence in 10-cm dishes, γ-irradiated for a total of 12 Gy, and transfected with annealed siRNA using DMRIE-C reagent (Invitrogen) for 6 h as recommended by the manufacturer. McCoy’s medium containing 20% fetal bovine serum and 2% penicillin-streptomycin was added to each dish to a final concentration of 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were harvested 24 h later for further assays.
Cell protein extraction and Western blot analyses were performed using standard procedures. Protein samples were mixed with loading buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.01% bromphenol blue, and 10% glycerol), heated at 100 °C for 5 min, and loaded onto a SDS-polyacrylamide gel in electrophoresis buffer containing 25 mM Tris-HCl, pH 8.3, 250 mM glycine, and 0.1% SDS. Protein was then transferred to nitrocellulose membranes using the Trans-Blot semidry system (Bio-Rad). The membranes were immunoblotted with primary antibodies against KLF4 (19), p53, p21WAF1/CIP1, or β-catenin (Santa Cruz Biotechnology). Following incubation with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, 1:10,000 dilution, Santa Cruz Biotechnology), KLF4, p53, p21WAF1/CIP1, or β-catenin was visualized with the Super-Signal West Pico chemiluminescent substrate kit (Pierce).
Cells were rinsed in Dulbecco’s phosphate-buffered saline (Mediatech), trypsinized, resuspended in McCoy’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, collected by centrifugation, washed with Dulbecco’s phosphate-buffered saline, again collected by centrifugation, resuspended in 70% ethanol, and fixed at −20 °C overnight. Cells were pelleted again by centrifugation and re-suspended in a staining solution containing 50 μg/ml propidium iodide, 50 μg/ml RNase A, 0.1% Triton X-100, and 0.1 mM EDTA for 30 min. Flow cytometry was performed on a FACSCalibur (BD Biosciences) cytometer.
Both HCT116 p53+/+ and p53−/− cells exhibited comparable cell cycle profiles before irradiation (Fig. 1, A, B, and E). Following 12 Gy of γ irradiation, HCT116 p53+/+ cells demonstrated a normal cell cycle arrest pattern, with ~15% of cells in G1, ~80% cells in G2, and a significantly reduced S population (Fig. 1, C and F). However, HCT116 p53−/− cells exhibited an abnormal cell cycle pattern after γ irradiation, with ~90% of the cells in G2 and few remaining in either G1 or S (Fig. 1, D and F). Consistent with the effect of γ irradiation on the cell cycle of HCT116 p53+/+ cells, protein levels of KLF4 and p21WAF1/CIP1 were both significantly increased in response to an increase in p53 protein levels (Fig. 2, lanes 1 and 2). In contrast, no induction in the level of either KLF4 or p21WAF1/CIP1 was observed following γ irradiation in HCT116 p53−/− cells (Fig. 2, lanes 3 and 4). These results suggest that at least part of the cell cycle arrest caused by γ irradiation is a result of p53-dependent activation of KLF4 and p21WAF1/CIP1.
The failure of γ irradiation to induce expression of KLF4 and p21WAF1/CIP1 in HCT116 p53−/− cells correlated with the reduction in G1 and S populations (Figs. 1 and and2).2). This suggests that activation of KLF4, with consequent activation of p21WAF1/CIP1, may be necessary for the accumulation of cells in G1. To test this hypothesis, we established a stable HCT116 p53−/− cell line that expressed the receptors for the insect nuclear hormone, ecdysone, and its partner, retinoid X receptor (25). This cell line, called EcR116 p53−/−, was infected with the recombinant adenovirus AdEGI or AdEGI-KLF4 (25) that contained enhanced green fluorescence protein as a control or enhanced green fluorescence protein plus KLF4, respectively. Following infection, cells were γ-irradiated or not and then treated with the inducer, ponasterone A, or vehicle alone for 24 h before being harvested for cell cycle analysis. As seen in Fig. 3, treatment of AdEGI-KLF4-infected cells with ponasterone A without irradiation resulted in a statistically significant increase in the G1 population and a decrease in the G2/M population (Fig. 3, C, D, and J), whereas AdEGI-infected cells without irradiation and treated with ponasterone A had no effect on the cell cycle when compared with untreated cells (Fig. 3, A, B, and I). Cells infected with AdEGI followed by irradiation showed G2/M arrest in the absence or presence of ponasterone A (Fig. 3, E, F, and K) as did cells infected with AdEGI-KLF4 and irradiated without any ponasterone A treatment (Fig. 3, G and L). In contrast, upon the addition of ponasterone A, AdEGI-KLF4-infected and irradiated cells had a statistically significant increase in the G1 population (Fig. 3, H and L). This finding is reminiscent of the G1/S arrest seen in HCT116 p53+/+ cells following irradiation (compare Figs. 3H and and1C1C).
Fig. 4 shows that only cells infected by AdEGI-KLF4 and induced with ponasterone A (lanes 11 and 12) had appreciable amounts of KLF4. The increase in the KLF4 level correlated with an increase in the p21WAF1/CIP1 level, a finding consistent with our previous observation that KLF4 is an activator of p21WAF1/CIP1 expression (25). The combined results of Figs. 3 and and44 indicate that the inducible expression of KLF4 in irradiated cells lacking p53 restores the characteristic G1/S arrest in cells with wild type p53 following irradiation. This finding indicates that KLF4 is necessary and sufficient in mediating the G1 cell cycle effect of p53 following DNA damage.
Recently, Tuschl and co-workers (27, 28) demonstrated that RNA interference can be provoked in mammalian cell lines through the introduction of siRNA. The mediators of sequence-specific mRNA degradation are 21–23-nucleotide siRNA duplexes that trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response (27–29). To determine whether we could “knock down” KLF4 expression using siRNA, we synthesized a 23-nucleotide siRNA duplex specific for KLF4 to transfect HCT116 p53+/+ cells with or without irradiation. As seen in Fig. 5, siRNA for KLF4 significantly reduced the level of KLF4 in response to γ irradiation when compared with untransfected or mock-transfected cells (lanes 2, 4, 6, and 8). In contrast, a control nonspecific siRNA failed to abrogate the DNA damage-induced synthesis of KLF4 (lanes 10 and 12). Again, there was a corresponding reduction in the level of p21WAF1/CIP1 in response to γ irradiation in KLF4 siRNA-treated cells (lanes 6 and 8). Importantly, KLF4 siRNA but not nonspecific siRNA abolished the G1 population in cells upon γ irradiation (Fig. 6, lanes F, G, H, I, and J). These results complement those from the preceding sections and provide strong evidence that KLF4 is an essential factor in mediating p53-dependent G1 arrest in response to DNA damage.
Cell cycle progression is regulated by checkpoint controls, which function to safeguard the integrity of the genome. Activation of DNA integrity checkpoints occurs through the detection of damaged or unreplicated DNA and is in effect until DNA damage has been repaired (30). The checkpoint that arises after DNA damage can activate during G1, S, or G2 (3, 31). Arrest in G1 permits repair prior to replication, whereas arrest in S or G2 permits repair of the genome before mitotic segregation. The p53 tumor suppressor has been shown to be integral to both the G1 (32, 33) and G2 (34, 35) DNA damage machinery. This was supported by the results in Fig. 1, which showed that HCT116 p53+/+ cells arrested at either G1 or G2/M after γ irradiation as expected for cells with intact checkpoint function. The resultant activation of p53 because of γ irradiation was accompanied by a significant increase in the level of KLF4 and p21WAF1/CIP1 (Fig. 2) in a manner similar to the previously observed response of fibroblasts subjected to DNA damage caused by methyl methanesulfonate (24). HCT116 p53−/− cells, in contrast, showed no induction of either KLF4 or p21WAF1/CIP1 by γ irradiation and arrested only in G2/M (Fig. 1). The latter result was consistent with that from a previous study, which also demonstrated that p53 was necessary to sustain G2 arrest (10).
Recent studies indicate that the G1 checkpoint control after DNA damage consisted of two steps (31). The first step is a rapid and p53-independent induction of the G1 checkpoint. It is a result of rapid redistribution of p21WAF1/CIP1 from cyclin D1-Cdk4/6 complexes to cyclin E-Cdk2 complexes, which are inhibited by p21WAF1/CIP1 (36, 37). The second step involves the post-translational modifications of p53 by upstream protein kinases, including ataxia telangiectasia mutated/ataxia telangiectasia and Rad3 related and Chk1/Chk2 (38, 39), which results in p53 activation and subsequent transcriptional induction of p21WAF1/CIP1 (31). Several lines of evidence suggest that KLF4 is involved in the p53-dependent induction of p21WAF1/CIP1. First, p53 mediates the transcriptional induction of KLF4 in response to DNA damage (24). Second, the induction in KLF4 precedes that in p21WAF1/CIP1 following DNA damage (24). Third, KLF4 binds to a specific cis-element in the proximal promoter of the p21WAF1/CIP1 gene and activates the promoter (24). Fourth, p53 and KLF4 physically interact and cause a synergistic induction in p21WAF1/CIP1 gene expression (24). The importance of KLF4 in mediating the transcriptional induction of p21WAF1/CIP1 is further demonstrated by the observation that p53 fails to activate the p21WAF1/CIP1 promoter if the KLF4 response element in the promoter is mutated (24).
In addition to the biochemical evidence supporting a crucial role for KLF4 in mediating the transcriptional induction of p21WAF1/CIP1 by p53, the present study provides the genetic evidence to further substantiate the significance of KLF4 in p53-mediated G1 arrest caused by DNA damage. Specifically, the conditional induction of KLF4 in γ-irradiated HCT116 p53−/− cells restored the G1 population of cells that are normally present in irradiated HCT116 p53+/+ cells (Fig. 3). Conversely, inhibition of KLF4 expression in irradiated HCT116 p53−/− cells resulted in an abolishment of the G1 peak in a manner that resembles the consequence of γ irradiation of HCT116 p53−/− cells (Fig. 6). In each case, the induction or inhibition of KLF4 expression was accompanied by a corresponding increase or decrease, respectively, in the level of p21WAF1/CIP1 (Figs. 4 and and5).5). Coupled with the findings from our previous study, which demonstrated that inducible expression of KLF4 causes a G1/S cell cycle arrest (25), it is highly likely that KLF4 serves a pivotal role in mediating the G1 checkpoint function of p53 in response to DNA damage.
In addition to its effect on the G1/S checkpoint, p53 also regulates the G2/M transition in response to DNA damage (11). Part of the mechanism by which p53 inhibits the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis (12). Binding of Cdc2 to cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 contributes to the blocking of entry into mitosis (40, 41). p53 also represses expression of the Cdc2 gene (42, 43) to help ensure that cells do not escape from the initial block. Moreover, several of the transcriptional targets of p53 can inhibit Cdc2, including p21WAF1/CIP1, 14-3-3σ, and Gadd45 (44–46). Therefore, it is of great interest to note that a recent analysis of KLF4 target genes by cDNA microarrays showed that KLF4 activates expression of 14-3-3σ, in addition to p21WAF1/CIP1, and represses expression of Cdc2 (47). Whether KLF4 is also involved in mediating the G2 checkpoint function of p53 in response to DNA damage is currently being determined.
We thank Dr. Bert Vogelstein for kindly providing the HCT116 p53+/+ and −/− cell lines.
*This work was supported in part by Grants DK52230 and CA84197 from the National Institutes of Health.
1The abbreviations used are: Cdk, cyclin-dependent kinase; EcR, ecdysone receptor; KLF4, Krüppel-like factor 4; siRNA, small interfering RNA; Gy, gray; AdEGI, recombinant adenovirus containing green fluorescence protein.