An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krü ppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21, suggesting that GKLF may be involved in the induction of p21 Indeed, GKLF activates p21WAF1/Cip1 through a specific Sp1-like cis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated.

Recent advances in molecular cloning have led to the identification of a large number of mammalian zinc finger-containing transcription factors that exhibit homology to the Drosophila melanogaster protein, Krüppel. Although the amino acid sequences in the zinc finger domains of these Krüppel-like factors (KLFs) are closely related to one another, the regions outside the zinc fingers of the proteins are usually unique. KLFs display seemingly different and broad biological properties with each functioning as an activator of transcription, a repressor or both. This review article provides a current phylogenetic classification of the identified KLFs to date. More importantly, the currently known biological activities of the KLFs in regulating transcription, cell proliferation, differentiation and development are summarized and compared. Further characterization of this interesting protein family should provide additional insights into the their respective regulatory role in various important biological processes.

Krü ppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G 1 /S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G 1 /S boundary of the cell cycle.

In vitro and in vivo studies have shown that 1A,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] inhibits angiogenesis in cancer. We now examined whether the antiangiogenic effects of 1,25(OH) 2 D 3 are mediated by the hypoxiainducible factor (HIF)-1 pathway. Our results showed that 1,25(OH) 2 D 3 reduces the protein expression of both the regulated HIF-1A subunit and the vascular endothelial growth factor (VEGF) in various human cancer cells.

Kru¨ppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor, the expression of which is enriched in the postmitotic cells of the intestinal epithelium. KLF4 is a target gene of the tumor suppressor adenomatous polyposis coli (APC). We sought to determine the role of KLF4 in suppressing the tumorigenecity of RKO colon cancer cells, which do not express KLF4. We utilized an established system in RKO cells, in which an inducible promoter controls expression of KLF4. Four independent assays were used to assess the effects of KLF4 induction on tumor cells. We find that KLF4 overexpression reduces colony formation, cell migration and invasion, and in vivo tumorigenecity. The mechanism of action of KLF4 does not involve apoptosis. These findings, along with our previous findings that KLF4 induces G1/S arrest, suggest that KLF4 is a cell cycle checkpoint protein that can reduce tumorigenecity of colon cancer cells.

Hypoxia-inducible factor-1A (HIF-1A) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1A is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1A can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1A is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1A in nonhypoxiamediated cancer cell proliferation remains speculative. We have disrupted HIF-1a by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1A significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1A expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1A promoted cell proliferation and survival under hypoxia in vitro.

BackgroundActivating KRAS mutations are important for cancer initiation and progression; and have recently been shown to cause primary resistance to therapies targeting the epidermal growth factor receptor. Therefore, strategies are currently in development to overcome treatment resistance due to oncogenic KRAS. The hypoxia-inducible factors-1α and -2α (HIF-1α and HIF-2α) are activated in cancer due to dysregulated ras signaling.MethodsTo understand the individual and combined roles of HIF-1α and HIF-2α in cancer metabolism and oncogenic KRAS signaling, we used targeted homologous recombination to disrupt the oncogenic KRAS, HIF-1α, and HIF-2α gene loci in HCT116 colon cancer cells to generate isogenic HCT116WT KRAS, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cell lines.ResultsGlobal gene expression analyses of these cell lines reveal that HIF-1α and HIF-2α work together to modulate cancer metabolism and regulate genes signature overlapping with oncogenic KRAS. Cancer cells with disruption of both HIF-1α and HIF-2α or oncogenic KRAS showed decreased aerobic respiration and ATP production, with increased ROS generation.ConclusionOur findings suggest novel strategies for treating tumors with oncogenic KRAS mutations.

Background & Aims Dual oxidases (DUOX) are conserved NADPH oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOXA (DUOX maturation factor) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1–DUOXA1 and DUOX2–DUOXA2). Expression of these genes is upregulated during bacterial infection and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. Methods Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa−/− mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection—the most common human chronic infection. Results Infection with H felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H felis colonized the mucus layer in the stomachs of Duoxa−/− mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H felis colonization was also increased in Duoxa+/− mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H felis that colonized Duoxa−/− mice, compared with that found in controls (P=.0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H felis failed to prevent a direct bacteriostatic effect, at sustained levels of H2O2 as low as 30 μM. Conclusions Based on studies of Duoxa−/− mice, the DUOX enzyme complex prevents gastric colonization by H felis and the inflammatory response. These findings indicate the non-redundant function of epithelial production of H2O2 in restricting microbial colonization.