KLF4 deletion alters gastric cell lineage and induces MUC2 expression.

Gastric cancer is one of the most common types of cancer in the world, particularly in underdeveloped countries. The mechanism of gastric cancer is less understood compared with other types of gastrointestinal (GI) cancers. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor and is a potential tumor suppressor in GI cancers. In this study, we have generated two mouse models, Rosa-Cre;Klf4(fl/fl) and Lgr5-Cre;Klf4(fl/fl). KLF4 was deleted by Rosa-Cre in the gastric epithelia cells or by Lgr5-Cre in the antral stem cells in the adult mice. KLF4 deletion resulted in increased proliferating cells and decreased pit mucous cells. Surprisingly, the intestinal goblet cell marker, MUC2, which is not expressed in normal gastric tissues, was strongly induced at the base of the KLF4-deleted antral glands. To understand the clinical relevance of these findings, we analyzed the expression of KLF4 and MUC2 in human gastric cancer. In a subset of human gastric cancer, the expression of KLF4 is negatively associated with MUC2 expression. In conclusion, KLF4 is essential for normal homeostasis of antral stem cells; loss of KLF4 and expression of MUC2 could be important markers for gastric cancer diagnosis.

TSC2/mTORC1 signaling controls Paneth and goblet cell differentiation in the intestinal epithelium.

The intestinal mucosa undergoes a continual process of proliferation, differentiation and apoptosis, which is regulated by multiple signaling pathways. Notch signaling is critical for the control of intestinal stem cell maintenance and differentiation. However, the precise mechanisms involved in the regulation of differentiation are not fully understood. Previously, we have shown that tuberous sclerosis 2 (TSC2) positively regulates the expression of the goblet cell differentiation marker, MUC2, in intestinal cells. Using transgenic mice constitutively expressing a dominant negative TSC2 allele, we observed that TSC2 inactivation increased mTORC1 and Notch activities, and altered differentiation throughout the intestinal epithelium, with a marked decrease in the goblet and Paneth cell lineages. Conversely, treatment of mice with either Notch inhibitor dibenzazepine (DBZ) or mTORC1 inhibitor rapamycin significantly attenuated the reduction of goblet and Paneth cells. Accordingly, knockdown of TSC2 activated, whereas knockdown of mTOR or treatment with rapamycin decreased, the activity of Notch signaling in the intestinal cell line LS174T. Importantly, our findings demonstrate that TSC2/mTORC1 signaling contributes to the maintenance of intestinal epithelium homeostasis by regulating Notch activity.

ERK and AKT signaling cooperate to translationally regulate survivin expression for metastatic progression of colorectal cancer.

The mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eukaryotic translation initiation factor 4E (eIF4E)-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-binding protein 1 (4E-BP1) for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mammalian target of rapamycin complex 1 (mTORC1)-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.

NFAT5 represses canonical Wnt signaling via inhibition of ß-catenin acetylation and participates in regulating intestinal cell differentiation.

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis, which is regulated by multiple signaling pathways. The Wnt/ß-catenin pathway has a critical role in this process. Previously, we have shown that the calcineurin-dependent nuclear factor of activated T cell (NFAT) is involved in the regulation of intestinal cell differentiation, as noted by the alteration of brush-border enzyme intestinal alkaline phosphatase (IAP) activity. Here, we show that calcineurin-independent NFAT5 interacts with ß-catenin to repress Wnt signaling. We found that overexpression of NFAT5 inhibits, whereas knockdown of NFAT5 increases, TOPflash reporter activity and the expression of Wnt/ß-catenin target genes, suggesting that NFAT5 inhibits Wnt signaling. In addition, we demonstrated that NFAT5 directly interacts with the C-terminal transactivation domain (TAD) of ß-catenin, inhibits CBP interaction with ß-catenin, and inhibits CBP-mediated ß-catenin acetylation. Moreover, NFAT5 is expressed in the mucosa of human intestine, with the most pronounced staining in the most differentiated region near the epithelial surface. Knockdown of NFAT5 attenuated sodium butyrate (NaBT)-mediated induction of IAP and sucrase activities; overexpression of NFAT5 induced IAP promoter activity. In summary, we provide evidence showing that NFAT5 is a regulator of Wnt signaling. Importantly, our results suggest that NFAT5 regulation of intestinal cell differentiation may be through inhibition of Wnt/ß-catenin signaling.

Interaction with Suv39H1 is critical for Snail-mediated E-cadherin repression in breast cancer.

Expression of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often lost due to promoter DNA methylation in basal-like breast cancer (BLBC), which contributes to the metastatic advantage of this disease; however, the underlying mechanism remains unclear. Here, we identified that Snail interacted with Suv39H1 (suppressor of variegation 3-9 homolog 1), a major methyltransferase responsible for H3K9me3 that intimately links to DNA methylation. We demonstrated that the SNAG domain of Snail and the SET domain of Suv39H1 were required for their mutual interactions. We found that H3K9me3 and DNA methylation on the E-cadherin promoter were higher in BLBC cell lines. We showed that Snail interacted with Suv39H1 and recruited it to the E-cadherin promoter for transcriptional repression. Knockdown of Suv39H1 restored E-cadherin expression by blocking H3K9me3 and DNA methylation and resulted in the inhibition of cell migration, invasion and metastasis of BLBC. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT, but also paves a way for the development of new treatment strategies against this disease.

The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer.

PH domain leucine-rich-repeats protein phosphatase (PHLPP) is a family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt. Our recent studies have demonstrated that the ubiquitin proteasome pathway has an important role in the downregulation of PHLPP in colorectal cancer. In this study, we show that the deubiquitinase USP46 stabilizes the expression of both PHLPP isoforms by reducing the rate of PHLPP degradation. USP46 binds to PHLPP and directly removes the polyubiquitin chains from PHLPP in vitro and in cells. Increased USP46 expression correlates with decreased ubiquitination and upregulation of PHLPP proteins in colon cancer cells, whereas knockdown of USP46 has the opposite effect. Functionally, USP46-mediated stabilization of PHLPP and the subsequent inhibition of Akt result in a decrease in cell proliferation and tumorigenesis of colon cancer cells in vivo. Moreover, reduced USP46 protein level is found associated with poor PHLPP expression in colorectal cancer patient specimens. Taken together, these results indentify a tumor suppressor role of USP46 in promoting PHLPP expression and inhibiting Akt signaling in colon cancer.

Loss of PHLPP expression in colon cancer: role in proliferation and tumorigenesis.

PHLPP (PH domain leucine-rich repeats protein phosphatase) represents a family of novel Ser/Thr protein phosphatases. Two highly related isoforms in this family, PHLPP1 and PHLPP2, have been identified to serve as negative regulators of Akt and protein kinase C by dephosphorylating the kinases directly. In this study, we examined the expression pattern of both PHLPP isoforms in colorectal cancer specimens and the adjacent normal mucosa using immunohistochemical staining. We found that the expression of PHLPP1 or PHLPP2 isoform was lost or decreased in 78 and 86% of tumor tissues, respectively. Stable overexpression of either PHLPP isoform in colon cancer cells decreased the rate of cell proliferation and sensitized the cells to growth inhibition induced by the phosphoinositide-3 kinase inhibitor, LY294002, whereas knockdown of either PHLPP isoform by shRNA promoted the proliferation of DLD1 cells. In addition, we demonstrated that the PHLPP-mediated growth inhibition in colon cancer cells was largely rescued by overexpression of a constitutively active Akt. Moreover, reexpression of either PHLPP isoform in HCT116 cells inhibited tumor growth in vivo. Taken together, our results strongly support a tumor suppressor role of PHLPP in colon cancer.

p27 Kip1 nuclear localization and cyclin-dependent kinase inhibitory activity are regulated by glycogen synthase kinase-3 in human colon cancer cells.

The cellular mechanisms regulating intestinal differentiation are poorly understood. Sodium butyrate (NaBT), a short-chain fatty acid, increases p27 Kip1 expression and induces cell cycle arrest associated with intestinal cell differentiation. Here, we show that treatment of intestinal-derived cells with NaBT induced G0/G1 arrest and intestinal alkaline phosphatase, a marker of differentiation, activity and mRNA expression; this induction was attenuated by inhibition of glycogen synthase kinase-3 (GSK-3). Moreover, treatment with NaBT increased the nuclear, but not the cytosolic, expression and activity of GSK-3beta. NaBT decreased cyclin-dependent kinase CDK2 activity and induced p27 Kip1 expression; inhibition of GSK-3 rescued NaBT-inhibited CDK2 activity and blocked NaBT-induced p27 Kip1 expression in the nucleus but not in the cytoplasm. In addition, we demonstrate that NaBT decreased the expression of S-phase kinase-associated protein 2 (Skp2), and this decrease was attenuated by GSK-3 inhibition. Furthermore, NaBT increased p27 Kip1 binding to CDK2, which was completely abolished by GSK-3 inhibition. Overexpression of an active form of GSK-3beta reduced Skp2 expression, increased p27 Kip1 in the nucleus and increased p27 Kip1 binding to CDK2. Our results suggest that GSK-3 not only regulates nuclear p27 Kip1 expression through the downregulation of nuclear Skp2 expression but also functions to regulate p27 Kip1 assembly with CDK2, thereby playing a critical role in the G0/G1 arrest associated with intestinal cell differentiation.

N-myc is a novel regulator of PI3K-mediated VEGF expression in neuroblastoma.

Angiogenesis in neuroblastoma (NB) correlates with increased expression of vascular endothelial growth factor (VEGF) and a worse clinical outcome. Other cellular markers, such as Akt activation and MYCN amplification, are also associated with poor prognosis in NB; therefore, we sought to determine the role of N-myc in the regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt/VEGF pathway. PI3K inhibition, using small-molecule inhibitors or phosphatase and tensin homolog adenovirus, led to decreased levels of VEGF mRNA and/or protein by reducing phosphorylation of Akt and mammalian target of rapamycin (mTOR), and attenuating hypoxia-inducible factor 1alpha expression. Moreover, PI3K inhibition decreased levels of N-myc expression in MYCN-amplified cells. To further clarify the importance of N-myc as a target of PI3K in VEGF regulation, we inhibited N-myc expression by siRNA transfection. MYCN siRNA significantly blocked VEGF secretion, irrespective of serum conditions, in MYCN-amplified NB cells; this effect was enhanced when combined with rapamycin, an mTOR inhibitor. Interestingly, in cells with low-N-myc expression, MYCN siRNA reduction of VEGF secretion was only effective with MYCN overexpression or insulin-like growth factor-1 stimulation. Our results show that N-myc plays an important role in the PI3K-mediated VEGF regulation in NB cells. Targeting MYCN, as a novel effector of PI3K-mediated angiogenesis, has significant potential for the treatment of highly vascularized, malignant NB.

Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase.

Glycogen-synthase kinase-3 (GSK-3) and extracellular signal-regulated kinase (ERK) are critical downstream signaling proteins for the PI3-kinase/Akt and Ras/Raf/MEK-1 pathway, respectively, and regulate diverse cellular processes including embryonic development, cell differentiation and apoptosis. Here, we show that inhibition of GSK-3 using GSK-3 inhibitors or RNA interference (RNAi) significantly induced the phosphorylation of ERK1/2 in human colon cancer cell lines HT29 and Caco-2. Pretreatment with the PKCdelta-selective inhibitor rottlerin or transfection with PKCdelta siRNA attenuated the phosphorylation of ERK1/2 induced by the GSK-3 inhibitor SB-216763 and, furthermore, treatment with SB-216763 or transfection with GSK-3alpha and GSK-3beta siRNA increased PKCdelta activity, thus identifying a role for PKCdelta in the induction of ERK1/2 phosphorylation by GSK-3 inhibition. Treatment with SB-216763 increased expression of cyclooxygenase-2 (COX-2) and IL-8, which are downstream targets of ERK1/2 activation; this induction was abolished by MEK/ERK inhibition, suggesting GSK-3 inhibition induced COX-2 and IL-8 through ERK1/2 activation. The transcriptional induction of COX-2 and IL-8 by GSK-3 inhibition was further demonstrated by the increased COX-2 and IL-8 promoter activity after SB-216763 treatment or transfection with GSK-3alpha or GSK-3beta siRNA. Importantly, our findings identify GSK-3, acting through PKCdelta, as a negative regulator of ERK1/2, thus revealing a novel crosstalk mechanism between these critical signaling pathways.

Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.

BACKGROUND AND AIMS: Determination of intracellular signalling pathways that mediate intestinal epithelial proliferation is fundamental to the understanding of the integrity and function of the intestinal tract under normal and diseased conditions. The phosphoinositide 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in cell proliferation and differentiation. In this study, we assessed the role of PI3K/Akt in transduction of proliferative signals in intestinal epithelial cells. METHODS: A rat intestinal epithelial (RIE) cell line and human colorectal cancer HCA-7 and LS-174 cell lines served as in vitro models. The Balb/cJ mouse was the in vivo model. RESULTS: PI3K activation was critical for G1 cell cycle progression of intestinal epithelial cells. Ectopic expression of either active p110alpha or Akt-1 increased RIE cell proliferation. In vivo experiments demonstrated that PI3K activation was closely associated with the proliferative activity of intestinal mucosa. Treatment of mice with PI3K inhibitors blocked induction of PI3K activity and attenuated intestinal mucosal proliferation associated with oral intake. Epidermal growth factor and transforming growth factor alpha stimulated PI3K activation which was required for growth factor induced expression of cyclin D1. CONCLUSIONS: The PI3K/Akt pathway transduces mitogenic signals from growth factor receptors to the cell cycle machinery and plays a critical role in regulation of intestinal epithelial proliferation.

Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells.

BACKGROUND: The transcription factor encoded by the intestinal Cdx2 homeobox gene and treatment with sodium butyrate (NaB), a byproduct of fibre fermentation by colonic bacteria, exert similar effects on colon cancer cell lines as they both inhibit cell growth and stimulate cell differentiation and apoptosis. AIM: To investigate whether NaB regulates expression of the Cdx2 gene in colon cancer cell lines. METHODS: Human adenocarcinoma cell lines Caco2 and HT29 were grown in the presence or absence of NaB. Cells were analysed for Cdx2 mRNA expression by reverse transcription-polymerase chain reaction, for protein expression by western blotting and electromobility shift assays, and for transcriptional activity of the Cdx2 promoter by transfection with luciferase reporter plasmids. RESULTS: In HT29 and Caco2 cells, NaB stimulated Cdx2 mRNA and protein expression as well as transcriptional activity of the Cdx2 promoter. Stimulation of the activity of the Cdx2 promoter by NaB was dose and time dependent. The Cdx2 promoter contains discrete regions that participate in or inversely that blunt the stimulatory effect exerted by NaB. In addition, NaB stimulated the transcriptional activity of the Cdx2 promoter downregulated by oncogenic ras. CONCLUSION: This study is the first report of an intestine specific transcription factor, Cdx2, stimulated by butyrate. Thus it provides a new mechanism whereby butyrate controls proliferation and differentiation of colon cancer cells.

Butyrate sensitizes human colon cancer cells to TRAIL-mediated apoptosis.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor family, induces apoptosis in TRAIL-sensitive tumors through the activation of the caspase pathway. Sodium butyrate (NaBT) induces differentiation and apoptosis in certain colorectal cancers; the molecular mechanisms for these effects have not been clearly defined. The purpose of our study was to determine whether NaBT sensitizes TRAIL-resistant human colon cancer cells to the effects of TRAIL. METHODS: Human colon cancer cells (KM12C, KML4A, and KM20) that are resistant to TRAIL treatment alone were treated with TRAIL (100 ng/mL), NaBT (5 mmol/L), or a combination of these agents and harvested for total RNA and protein. Western blots were performed to assess intracellular expression of Flice-like inhibitory protein (FLIP), a caspase inhibitor. Percent-specific apoptosis, relative caspase-3 activity, and Annexin-V immunofluorescence were determined at 24 and 48 hours. Cell cycle--related gene expression was assessed by RNase protection. RESULTS: Treatment with NaBT for 24 and 48 hours decreased FLIP protein expression in all cell lines. Furthermore, NaBT sensitized these resistant cancer cells to the effects of TRAIL with significant increases noted in cell death, caspase-3 activity, and Annexin-V staining compared with NaBT alone. CONCLUSIONS: Our findings suggest that the reduction of FLIP protein levels by NaBT renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL with agents (such as NaBT, which target proteins that prevent cell death) may provide a more effective and less toxic regimen for the treatment of resistant colon cancers.

Alterations of MAPK activities associated with intestinal cell differentiation.

Three distinct groups of mitogen-activated protein kinases (MAPKs) have been identified in mammalian cells (i.e., ERK, JNK, and p38) which play an important role in the differentiation and apoptosis of various cells. The purpose of our present study was to determine MAPK activity and levels associated with sodium butyrate (NaBT)-mediated differentiation and apoptosis in the human colon cancer cell lines Caco-2 and HT29. Intestinal alkaline phosphatase (IAP) activity, a marker of intestinal differentiation, was increased at 48 h after NaBT treatment followed by cell death at 72 h. ERK activity was decreased in differentiated Caco-2 cells either induced with NaBT or allowed to differentiate spontaneously and in HT29 cells treated with NaBT. The combination of the MEK inhibitor, PD98059, with NaBT further increased IAP activity and cell death compared with NaBT alone. In contrast to ERK, JNK1 activity and c-Jun phosphorylation was increased 8 h after NaBT treatment suggesting a role for the JNK pathway in intestinal cell differentiation and apoptosis. p38 activity was increased at 24 and 48 h after NaBT treatment. Taken together, our results suggest that alterations in MAPKs (i.e., ERK inhibition and JNK induction) contribute to the differentiation and apoptotic pathways in intestinal cells.

Molecular mechanisms contributing to necrotizing enterocolitis.

OBJECTIVE: To examine the cellular mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC). SUMMARY BACKGROUND DATA: Necrotizing enterocolitis is a major cause of death and complications in neonates; the cellular mechanisms responsible for NEC are unknown. The inducible form of cyclooxygenase (i.e., COX-2) is activated by the transcription factor nuclear factor (NF)-kappaB and is thought to play a role in inflammation. METHODS: Segments of perforated and adjacent uninvolved small intestine from neonates with NEC were analyzed for COX-2 expression by immunohistochemistry. NEC was induced in weanling (18 days old) rats by occlusion of superior mesenteric vessels for 1 hour and intraluminal injection of platelet activating factor (50 micro/kg). Small intestine was harvested for protein extraction. Western immunoblot was performed to determine expression of COX-2. Gel shift assays were performed to assess NF-kappaB binding activity. RESULTS: Immunohistochemical analysis showed increased COX-2 protein expression in the perforated intestinal sections of all 36 neonates but not in adjacent normal intestine. Increased expression of COX-2 protein and NF-kappaB binding activity was noted in the small intestine of weanling rats at 0 and 3 hours after induction of NEC. CONCLUSIONS: Increased COX-2 expression was identified in all neonatal intestinal segments resected for perforated NEC. In addition, a coordinate induction of COX-2 expression and NF-kappaB binding was noted in a rodent model of NEC. These findings suggest that the COX-2/NF-kappaB pathway may play a role in the pathogenesis of NEC. Therapeutic agents that target this pathway may prove useful in the treatment or possible prevention of NEC.

Sensitization of human colon cancer cells to TRAIL-mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor (TNF) family, is thought to induce apoptosis preferentially in cancer cells; however, increasing evidence suggests that a number of cancers are resistant to TRAIL treatment. FLICE-like inhibitory protein (FLIP), which structurally resembles caspase-8, can act as an inhibitor of apoptosis when expressed at high levels in certain cancer cells. The purpose of our present study was to determine whether human colon cancer cells are sensitive to TRAIL treatment and, if not, to identify potential mechanisms of resistance. Colon cancer cells of different metastatic potential (KM12C, KML4A, and KM20) were found to be resistant to the effects of TRAIL when used as a single agent. FLIP expression levels were increased in all three KM cell lines. Treatment with either actinomycin D (Act D;10 :g/ml) or cycloheximide (CHX; 10 :g/ml) decreased FLIP expression levels in all three cell lines. The decrease in cellular levels of FLIP was associated with sensitization to TRAIL-mediated apoptosis, as demonstrated by enhanced cell death and caspase-3 activity compared with either Act D or CHX alone. Our findings suggest that reduction of FLIP levels by Act D or CHX renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL along with agents such as Act D or CHX, which target proteins that prevent cell death, may provide a more effective and less toxic regimen for treatment of resistant colon cancers.

Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation.

BACKGROUND & AIMS: Phosphatidylinositol 3-kinase (PI3K), an important mediator of intracellular signal transduction, has been shown to affect proliferation, differentiation, and apoptosis in a number of cells; the role of PI3K in intestinal cell differentiation is not known. METHODS: The effect of PI3K inhibition on enterocyte-like differentiation of the human colon cancer cells, HT29 and Caco-2, was assessed using complementary approaches (i.e., chemical inhibition with wortmannin, transfection with a dominant negative p85 mutant, or overexpression of the tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 [PTEN]). Brush-border enzyme (intestinal alkaline phosphatase [IAP] and sucrase) activities, IAP messenger RNA levels, and IAP promoter induction were measured. RESULTS: The PI3K inhibitor, wortmannin, in combination with sodium butyrate, synergistically induced IAP and sucrase enzyme activities and IAP messenger RNA levels in a time- and dose-dependent fashion. Consistent with these results, cotransfection using the dominant negative mutant of p85 (Deltap85) induced IAP promoter activity. Moreover, overexpression of PTEN, which antagonizes PI3K, significantly augmented the induction of IAP enzyme activity in HT29 and Caco-2 cells treated with sodium butyrate and in spontaneously differentiated Caco-2 cells. CONCLUSIONS: Our results show that inhibition of PI3K significantly enhances enterocyte-like differentiation of HT29 and Caco-2 cells. Taken together, our findings suggest a contributory role for the PI3K/PTEN pathway in intestinal cell differentiation.

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers.

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.

Isolation and molecular characterization of the 5'-upstream region of the human TRAIL gene.

TRAIL, a novel member of the TNF family, acts through membrane receptors to induce apoptosis of activated T lymphocytes and may represent a mechanism for the "immune escape" of certain cancers. Various cytokines appear to increase expression of other TNF family members; however, the regulation of TRAIL has not been defined. The purpose of this study was to assess molecular mechanisms regulating TRAIL gene expression in human colon cancers. In this study, we have cloned the human TRAIL (hTRAIL) promoter ( approximately 1.6 kb) and identified a number of putative transcription factor binding sites such as NFAT, AP-1 and Sp1 sequences which are important for the expression of other TNF family members. Transient transfections of 5'-deletion promoter constructs into either Caco-2 or HT29 colon cancer cells identified TRAIL promoter regions critical for both basal and interferon-gamma (IFN-gamma)-mediated induction. Furthermore, induction of TRAIL mRNA levels was demonstrated in HT29 and Caco-2 cells with IFN-gamma treatment suggesting an important role for this cytokine in TRAIL expression.

Acute pancreatitis results in induction of heat shock proteins 70 and 27 and heat shock factor-1.

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.