CtIP promotes G2/M arrest in etoposide-treated HCT116 cells in a p53-independent manner

Acquired resistance to cytotoxic antineoplastic agents is a major clinical challenge in tumor therapy; however, the mechanisms involved are still poorly understood. In this study, we show that knockdown of CtIP, a corepressor of CtBP, promotes cell proliferation and alleviates G2/M phase arrest in etoposide (Eto)-treated HCT116 cells. Although the expression of p21 and growth arrest and DNA damage inducible α (GADD45a), which are important targets of p53, was downregulated in CtIP-deficient HCT116 cells, p53 deletion did not affect G2/M arrest after Eto treatment. In addition, the phosphorylation levels of Ser317 and Ser345 in Chk1 and of Ser216 in CDC25C were lower in CtIP-deficient HCT116 cells than in control cells after Eto treatment. Our results indicate that CtIP may enhance cell sensitivity to Eto by promoting G2/M phase arrest, mainly through the ATR-Chk1-CDC25C pathway rather than the p53-p21/GADD45a pathway. The expression of CtIP may be a useful biomarker for predicting the drug sensitivity of colorectal cancer cells.     

Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis,Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-Flip_L, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21^Cip1 and p27^Kip1), and hypophosphorylation of Rb.     

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

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.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells

Curcumin (CUR), a polyphenol derived from the plant Curcuma longa, displays potential anti-cancer activity. One of the mechanisms stems from its ability to elicit cell cycle arrest followed by suppression of cell proliferation. Herein, we reported that CUR significantly induced DNA damage and mediated S and G2/M phase arrest in colorectal carcinoma HCT116 cells. Unlike etoposide, a classical topoisomerase II inhibitor, CUR-triggered G2/M phase arrest was hardly reversed by caffeine (CAFF) which is an inhibitor of activated ataxia-telangiectasia-mutated (ATM)/ATM- and Rad3-related (ATR), indicating that ATM and ATR signaling pathways may be not involved in CUR-mediated S and G2/M phase arrest in HCT116 cells. Furthermore, we demonstrated that CUR caused mitosis arrest in HCT116 cells by using mitotic protein monoclonal antibody-2 as a mitosis marker and the surface plasmon resonance assay. The findings provide new mechanisms of cell proliferation inhibition triggered by CUR in HCT116 cells.     

Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1

Fucoxanthin, a natural carotenoid, has been reported to have antitumorigenic activity in mouse colon, skin and duodenum models. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against colon cancer using the human colon adenocarcinoma cell lines. Fucoxanthin reduced the viability of WiDr cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G0/G1 phase at 25 microM and apoptosis at 50 microM. Fucoxanthin at 25 microM inhibited the phosphorylation of the retinoblastoma protein (pRb) at Ser780 and Ser807/811 24 h after treatment without changes in the protein levels of the D-types of cyclin and cyclin-dependent kinase (cdk) 4, whose complexes are responsible for the phosphorylation of pRb at these sites. A cdk inhibitory protein, p21WAF1/Cip1 increased 24 h after the treatment with 25 microM of fucoxanthin, but not p27Kip1. In addition, the mRNA of p21WAF1/Cip1 also increased in a dose-dependent manner. According to the experiments using the isogenic human colon adenocarcinoma cell lines, fucoxanthin failed to induce G0/G1 arrest in the p21-deficient HCT116 cells, but not in HCT116 wild-type cells. All of these findings showed that fucoxanthin inhibited proliferation of colon cancer cells. The inhibitory mechanism is due to the cell cycle arrest during the G0/G1 phase mediated through the up-regulation of p21WAF1/Cip1, which may be related to the antitumorigenic activity.     

Pim-2 phosphorylation of p21Cip1/WAF1 enhances its stability and inhibits cell proliferation in HCT116 cells

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21^Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.     

The Effect of GADD45a on Furazolidone‐Induced S‐Phase Cell‐Cycle Arrest in Human Hepatoma G2 Cells

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S‐phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin‐dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone‐induced S‐phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin‐dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone‐induced S‐phase cell cycle arrest. GADD45a influences furazolidone‐induced S‐phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.     

Radiosensitization by Chir-124, a selective Chk1 inhibitor: Effects of p53 and cell cycle checkpoints

Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G2-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR- induced G2-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53-/- and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53-/- cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21-/- HCT116 cells displayed a similar pattern of response as p53-/- cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.     

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G₂/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G₂ phase. G₂ arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G₂ to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G₂ phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (-/-) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.     

Role for cyclin D1 in UVC-induced and p53-mediated apoptosis.

DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA

Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.     

Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Requirement of Krüppel-like factor 4 in preventing entry into mitosis following DNA damage

Previous studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after gamma-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following gamma-irradiation and does so by inhibiting cyclin B1 expression.