Barrett's Esophagus Cell Research Articles

NADPH Oxidase NOX5-S Mediates Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

G A A b st ra ct s study we examine the role of mPGES1 in the acid-induced increase in PGE2 production and cell proliferation, and studied the role of NADPH oxidase NOX5-S in acid-induced upregulation of mPGES1 in an EA cell line FLO. RT-PCR shows that mPGES1, mPGES2 and cytosolic PGES (cPGES) are present in FLO cells. Pulsed acid treatment increased mPGES1 mRNA by 110% and mPGES2 by 10%, but did not have any effect on cPGES mRNA. Acid treatment significantly increased mPGES1 protein expression. Knockdown of mPGES1 by mPGES1 siRNA blocked acid-induced increase in PGE2 production and thymidine incorporation (an indicator of cell proliferation rate). Knockdown of NOX5-S by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a NF-κB In Vivo activation reporter plasmid pNF-κBLuc. Knockdown of NF-κB1 p50 by p50 siRNA almost abolished acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. In a chromatin immunoprecipitation assay, the promoter region of mPGES1 DNA was detectable in the immunoprecipitated chromatin sample of FLO cell lysate with a p50 antibody, indicating that p50 binds to mPGES1 promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. Supported by NIH NIDDK R01 DK080703.

NADPH Oxidase NOX5-S Mediates Acid-Induced mPGES1 Expression in Barrett's Esophageal Adenocarcinoma Cells

NADPH Oxidase NOX5-S Mediates Acid-Induced mPGES1 Expression in Barrett's Esophageal Adenocarcinoma Cells

Signaling in H 2O 2-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

Signaling in H 2O 2-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

Abstract 4077: Exposure to bile acids alters intracellular expression of MnSOD in Barrett's esophagus

Abstract Introduction: Oxidative stress secondary to bile acid exposure has been associated with metaplastic degeneration of normal esophageal mucosa into Barrett's Esophagus cells and eventually Esophageal Adenocarcinoma. We have previously reported that the macromolecular response of BE cells to this stress is largely regulated by the expression of Manganese Superoxide Dismutase (MnSOD). As the mitochondrion plays a vital role in MnSOD activation, the purpose of this study was to determine the location and activity of MnSOD within the BE cell after exposure to oxidative stress. Methods: BE cells were cultured to 80-90% confluence in 75ml flasks. The cells were then exposed for four hours to 0.4mM concentrations of taurocholic acid (TCA) or a 0.4mM 1:1 mixture of bile salts including sodium cholate (CA), sodium deoxycholate (DCA), sodium glycocholate (GCA), and sodium taurocholate (TCA) to mimic natural biliary refluxate. Proteins were extracted and separated into mitochondrial, nuclear, and cytoplasmic fragments followed by western blot analysis. Enzymatic Activity of MnSOD was determined by silencing of other SOD molecules with potassium cyanate. Lethality testing of biliary exposure was performed with MTT assays. Results: Mitochondrial expression of MnSOD was significantly increased in the cells exposed to a mixture of bile acids vs. TCA and control. This was present on both qualitative and quantitative analyses of Western blots. Cytoplasmic expression of MnSOD was also present and conserved between treated and untreated cells. Control cells lines showed no significant change in baseline MnSOD expression. This expression was confirmed by increased MnSOD activity in the mitochondria of cells exposed to mixed bile. The lethality of mixed bile for Barrett's cells was confirmed by MTT assay, with isolated bile salts and controls having a significantly higher fraction of living cells. Conclusion: This is the first study to evaluate microcellular MnSOD expression which is increased in BE cells in response to the oxidative stress of bile exposure, specifically in the mitochondrion. Mitochondrial MnSOD activity is increased within the BE cell after exposure to bile. Further investigation is required to determine the potential correlation between bile exposure and BE to adenocarcinoma progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2011-4077

Abstract 1684: 2-Methoxyestradiol reverses the epithelial-mesenchymal transition in Barrett's adenocarcinoma cells

Abstract 2-Methoxyestradiol (2-ME2) is a novel anti-cancer agent because of its ability to potentiate apoptotic cell death and inhibit cancer cell growth and angiogenesis. In our study, we have investigated whether 2-ME2 is able to modulate epithelial-mesenchymal transition (EMT) in Barrett's esophageal adenocarcinoma cell (OE33), which has been postulated as an absolute requirement for tumor invasion and metastasis. Expression of epithelial (E-cadherin, β-catenin, Keratin-19) and mesenchymal markers (Oct-4, Vimentin) and stem cell markers (CD44, CD24) were studied at mRNA and protein levels in OE33 cells that were treated with 5µmolar/L of 2-ME2 for 24 h and 48 h. Our results indicate expression of β-catenin and E-cadherin is activated in a 2-ME2 dependent fashion in OE33 cells. Expression of Vimentin and Oct-4 is significantly down-regulated in 2-ME2 treated OE33 cells. Altogether, these results provide new insights into the role of 2-ME2 in EMT modulation, which may facilitate development of new therapeutic strategies to prevent the tumor invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1684. doi:10.1158/1538-7445.AM2011-1684

Abstract 4205: Regulation of Hif-1α expression by NADPH oxidase 5 (Nox5) in human cancer cell lines

Abstract Reactive oxygen species (ROS) produced by NADPH oxidases (Noxs) participate in signaling cascades regulating proliferation. However, the relative contribution to tumor cell growth by the seven Nox homologues (Nox1-5 and Duox 1 and 2) remains unclear. Several studies have proposed that Nox5 plays a role in promoting cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma cells, and in prostate cancer; however, little is known about the signaling pathways mediated by Nox5. We explored the functional role of Nox5 in a variety of tumor cell lines, including the NCI-60 cell panel; real time RT-PCR revealed that several melanoma lines express Nox4 and Nox5, but no Nox1 mRNA; and that prostate cell lines express Nox5 mRNA. Of the Nox5- expressing NCI-60 human cancer cell lines, we studied UACC-257 melanoma and PC-3 prostate cancer cells. These lines were characterized for variations in their N-terminus Ca2+ binding domains; RT-PCR revealed that both cell lines expressed the Nox5α and Nox5γ forms. The functional role of Nox5 in UACC-257 and PC-3 cells was studied by generating stable Nox5-overexpressing clones, and by transiently silencing Nox5 that was expressed endogenously, as well as that in clones stably overexpressing Nox5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that Nox5 was functional and generated superoxide in UACC-257 and PC-3 clones overexpressing Nox5. Nox5 could be activated by the phorbol ester PMA and by the Ca2+ ionophore ionomycin in overexpressing clones. This enzymatic activity was Ca2+- and flavin dehydrogenase-dependent; the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased Nox5-mediated ROS production. Overexpression of Nox5 in UACC-257 cells resulted in increased normoxic Hif-1α expression that was also Ca2+-, flavin dehydrogenase- and ROS-dependent. Silencing Nox5 by RNAi significantly inhibited both endogenous and PMA-stimulated ROS production, confirming that ROS production was Nox5-mediated. Knockdown of both endogenous and overexpressed Nox5 in both UACC-257 and PC-3 cells resulted in decreased Hif-1α expression. Studies to characterize the mechanisms by which Nox5 modulates cell proliferation through the HIF-1α pathway are ongoing. Our experiments suggest that Nox5 expression may play an important role in the generation of high local concentrations of ROS that could contribute to activation of Hif-1α and promote cell proliferation in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2011-4205

Bile Salts Differentially Sensitize Esophageal Squamous Cells to CD95 (Fas/Apo-1 Receptor) Mediated Apoptosis

The role of nonacidic reflux contents on the pathophysiology of Barrett's esophagus remains poorly understood. We hypothesized that esophageal squamous epithelium differs from Barrett's columnar epithelium in response to bile salts with respect to subsequent changes in the cell surface expression of CD95 (Fas/Apo-1) and sensitivity to CD95-mediated apoptosis. Immortalized esophageal squamous cells (HET-1A) and Barrett's esophagus cells (BAR-T), and esophageal adenocarcinoma cells (Flo-1) were treated with toxic and nontoxic bile salts at concentrations observed in gastroesophageal refluxate. CD95 cell-surface expression and apoptotic response to activating anti-CD95 antibody treatment was determined by FACScan analysis. Bile salt exposure resulted in a dose-dependent increase in CD95 cell-surface expression in HET-1A cells, but not BAR-T or Flo-1 cells. This response occurred rapidly, within a time-frame inconsistent with de novo protein synthesis and was blocked by protein kinase C (PKC) inhibition. Surprisingly, PKC inhibition in Flo-1 cells resulted in an increase in CD95 cell surface expression. Following bile salt exposure, a corresponding increase in the induction of CD95-mediated apoptosis was observed in HET-1A cells; PKC inhibition sensitized Flo-1 cells to apoptosis. Our findings suggest that esophageal squamous cells are sensitized to CD95-mediated apoptosis following bile salt exposure. This differential response, compared with columnar epithelial cells, could exert a selection pressure that contributes to the pathophysiology of Barrett's esophagus.

S1063 The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate Bile Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

S1063 The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate Bile Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

Abstract 4105: Nitric-oxide-mediated inhibition of Na+/H+ exchange (NHE) is a novel mechanism of bile acid induced damage: Relevance to esophageal carcinogenesis

Abstract Background: Barrett's esophagus (BE) is a premalignant condition that is associated with gastroesophageal reflux disease (GERD). Gastric acid and bile acids are two components of refluxate that induce proliferation, DNA damage and oxidative stress. In combination, the effects of gastric acid and bile acids are amplified and synergistic. The major goal of this study was to evaluate the effect of bile acids alone or in combination with acid on intracellular pH (pHi). A mechanism responsible for bile acid-induced pHi alterations was examined. Methods: CP-A cells (non-dysplastic Barrett's esophagus cells) were treated with medium acidified to pH 5.5 and/or 0.5mM bile acid cocktail in the presence/absence of inhibitors of nitric oxide synthase (NOS) or NHE (sodium-hydrogen exchanger). pHi was measured by BCECF microfluorimetry in perfused cells, nitric oxide by DAF-FM fluorescence and DNA damage by the comet assay. Western blot analysis was used to detect the expression of phosphorylated (activated) eNOS, iNOS and NHE1. Results: A dose-dependent decrease in pHi was observed in CP-A cells exposed to bile acids. Bile acid-induced intracellular acidification was abolished when NO production was inhibited by a nonspecific NOS inhibitor L-NAME or by thea specific iNOS inhibitor 1400W. In contrast the NHE inhibitor DMA, significantly decreased the bile acid-mediated pHi response. Exposure of the cells to acidified medium (pH5.5) containing a 0.5mM bile acid cocktail, markedly decreased pHi pHi to a value below extracellular pH. Importantly, medium acidified to pH5.5 containing 0.5mM bile acids induced a similar decrease in pHi as medium acidified to pH 4.5 without bile acids. This The same treatment resulteds in a 250% increase in acid-mediated DNA damage compared to medium acidified to pH5.5 in CP-A cells. Conclusion: In summary, a new mechanism of bile acid induced damage was identified. Bile acids alone or in combination with acidic pH significantly decrease intracellular pH in esophageal cells. The results suggest the effect ofresponse tpo bile acids is the consequence of NOS activation and that leads to NHE inhibition. The mMarked increase in DNA damage after suggests exposure to bile acids in combination with acid may play a crucial role in esophageal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4105.

Modelling Barrett's oesophagus

Barrett's oesophagus is the replacement of normal squamous oesophageal epithelium with an intestinalized columnar epithelium. Although some insight has been gained as to what Barrett's oesophagus is, how this columnar epithelium emerges from within a stratified squamous epithelium remains an unanswered question. We have sought to determine whether oesophageal keratinocytes can be trans-differentiated into Barrett's oesophagus cells. Using an Affymetrix microarray, we found unexpectedly that gene-expression patterns in the Barrett's oesophagus were only slightly more similar to the normal small intestine than they were to the normal oesophagus. Thus gene-expression patterns suggest significant molecular similarities remain between Barrett's oesophagus cells and normal squamous oesophageal epithelium, despite their histological resemblance with intestine. We next determined whether directed expression of intestine-specific transcription factors could induce intestinalization of keratinocytes. Retroviral-mediated Cdx2 (Caudal-type homeobox 2) expression in immortalized human oesophageal keratinocytes engineered with human telomerase reverse transcriptase (EPC2-hTERT cells) could be established transiently, but not maintained, and was associated with a reduction in cell proliferation. Co-expression of cyclin D1 rescued proliferation in the Cdx2-expressing cells, but co-expression of dominant-negative p53 did not. Cdx2 expression in the EPC2-hTERT.D1 cells did not induce intestinalization. However, when combined with treatments that induce chromatin remodelling, there was a significant induction of Barrett's oesophagus-associated genes. Studies are ongoing to determine whether other intestinal transcription factors, either alone or in combination, can provoke greater intestinalization of oesophageal keratinocytes. We conclude that, on the basis of gene-expression patterns, Barrett's oesophagus epithelial cells may represent an intermediate between oesophageal keratinocytes and intestinal epithelial cells. Moreover, our findings suggest that it may be possible to induce Barrett's oesophagus epithelial cells from oesophageal keratinocytes by altering the expression of certain critical genes.

Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo

BackgroundBarrett's oesophagus is a common premalignant lesion caused partly by acid reflux. Although the requisite therapy, proton pump inhibitors (PPIs), have been implicated in the progression of Barrett's oesophagus in...

Protective effects of glycoursodeoxycholic acid in Barrett's esophagus cells

Barrett's esophagus (BE) is a premalignant condition associated with the development of esophageal adenocarcinoma (EAC). Previous studies have implicated hydrophobic bile acids and gastric acid in BE and EAC pathogenesis. In this study, we tested the hypothesis that DNA damage, cytotoxicity and oxidative stress induced by bile acids and gastric acid can be attenuated by the cytoprotective, hydrophilic bile acid glycoursodeoxycholic acid (GUDCA). Non-dysplastic BE cells were exposed for 10 min to pH 4 and/or bile acid cocktail or to pH 4 and a modified cocktail consisting of a mixture of bile acids and GUDCA. DNA damage was evaluated by the comet assay; cell viability and proliferation were measured by trypan blue staining and the MTS assay; reactive oxygen species (ROS) were measured using hydroethidium staining; oxidative DNA/RNA damage was detected by immunostaining with antibody against 8-OH-dG; thiol levels were measured by 5-chloromethylfluorescein diacetate (CMFDA) staining; and the expression of antioxidant proteins was evaluated by western blotting. DNA damage and oxidative stress were significantly increased, while thiol levels were decreased in BE cells treated with pH 4 and bile acid cocktail compared with cells treated with pH 4 alone or untreated cells. Bile acids and low pH also significantly decreased cell proliferation. Expression of the antioxidant enzymes, MnSOD and CuZnSOD, was elevated in the cells treated with bile acids and low pH. When GUDCA was included in the medium, all these effects of pH 4 and bile acids were markedly reduced. In conclusion, treatment of BE cells with acidified medium and a bile acid cocktail at physiologically relevant concentrations induces DNA damage, cytotoxicity, and ROS. The cytoprotective bile acid, GUDCA, inhibits these deleterious effects by inhibiting oxidative stress.

578 MEK1 Kinase and Rho Kinase ROCK2 Mediate Acid-Induced NOX5-S Expression and H 2O 2 Production in Barrett's Esophageal Adenocarcinoma Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We therefore investigated the role of NADPH oxidases in acid-induced changes in Barrett's EA cell line FLO. RT-PCR and 5'-RACE showed that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression was significantly greater in EA cell lines (FLO, and OE33) than in esophageal squamous epithelial cell line HET-1A. The levels of NOX5 mRNA were also markedly increased in human Barrett's mucosal biopsies with moderate dysplasia and in EA tissues, when compared with normal esophageal mucosa or BE mucosa. Immunohistochemical studies showed that NOX5-S was present in the cytosol of FLO cells. This result was further confirmed by transfection of FLO cells with GFP-tagged NOX5-S plasmid. In FLOEA cells, knockdown of NOX5-S by NOX5 siRNA significantly decreased cell proliferation at basal condition and inhibited acid-induced increase in cell proliferation, suggesting that NOX5-S may play a role in growth and proliferation of EA cells. Acid treatment significantly increased H2O2 production in both FLO cells and BE mucosa. Acid treatment significantly increased NOX5-S mRNA expression and H2O2 production, and knockdown of NOX5-S with NOX5 siRNA abolished acid-induced H2O2 production in FLO cells, indicating that NOX5-S mediates acid-induced H2O2 production. Acid-induced NOX5-S expression and H2O2 production were significantly decreased by knockdown of Rho kinase ROCK2 and MEK1 kinase with their siRNAs. Overexpression of constitutively active ROCK2 Rho kinase and constitutively active MEK1 kinase significantly increased NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased Rho kinase activity, an increase which was not affected by knockdown of MEK1 kinase. Overexpression of constitutively active ROCK2 significantly increased MEK1 phosphorylation. These data suggest that acidinduced NOX5-S expression depends on sequential activation of Rho kinase ROCK2 and MEK1 kinase. In conclusion, in EA cells acid induces H2O2 production by activation of NADPH oxidase NOX5-S and causes upregulation of NOX5-S expression through sequential activation of Rho kinase ROCK2 and MEK1 kinase. In these cells NOX5-S contributes to increased cell proliferation. Supported by NIH NIDDK R21 DK073327-01.

Keyhole limpet hemocyanin, an immunological adjuvant, inhibits locomotion and egg laying in C. elegans

Keyhole limpet hemocyanin (KLH) is the copper‐containing respiratory pigment of the keyhole limpet mollusk. It has been used as an adjuvant in many vaccines. It is not known how KLH stimulates the immune system. KLH can also induce apoptosis and necrosis in Barrett's esophageal adenocarcinoma cells (McFadden et al. 2003). To understand the mechanism of KLH function, we tested its effects on C. elegans, a small, free‐living nematode worm frequently used as a genetic and developmental model system. In C. elegans treated with 300 mg KLH/mL, there was decreased feeding behavior, locomotion, and increased egg‐retention. All three of these behaviors are regulated by serotonin signaling. Using locomotion and egg laying assays, we screened for the effect of KLH on 13 mutant lines that modulate serotonin signaling in C. elegans. Loss of function mutants in the SER‐4 pathway showed a significant decrease in egg laying, while loss of function mutants in the SER‐1 pathway did not. SER‐1 mutants showed a significant decrease in locomotion indicating that SER‐1 is not affected by KLH. These results suggests that KLH functions primarily through the activating SER‐1 signaling pathway downstream of SER‐1. Supported by NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence

Attomole protein analysis by CIEF with LIF detection

We have coupled CIEF with an LIF detector that is based on a post-column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the epsilon-amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503-labeled beta-lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.

Sorafenib inhibits MAPK-mediated proliferation in a Barrett's esophageal adenocarcinoma cell line

Esophageal adenocarcinoma continues to rise in incidence. Despite recognition of Barrett's metaplasia as the histological precursor, prognosis remains poor. The mitogen-activated protein kinases (MAPK) pathway is activated in Barrett's-associated dysplasia and adenocarcinoma and this activation is, in part, due to acid and bile acid reflux. We investigated the effects of sorafenib, an orally active Raf-inhibitor, on acid and bile acid-stimulated growth and signaling in SEG-1 cells, derived from a Barrett's esophageal cancer. SEG-1 cells were pretreated with sorafenib or vehicle and subsequently stimulated with acid or bile acid. MAPK signals, including phospho-ERK and phospho-p38, as well as cyclin D1 expression were assessed by Western blotting. Cell proliferation was measured by WST-1 colorimetric assay. Acid (pH 3.0-4.0) and bile acid (taurocholate 50-100 micromol/L) activated ERK and p38. Acid and bile acid exposure also increased levels of cyclin D1, a G1 to S cell cycle regulator. Furthermore, acid and taurocholate exposure increased cell proliferation. Sorafenib abrogated MAPK activation and cyclin D1 up-regulation and significantly inhibited cell growth. In summary, sorafenib inhibits acid or bile acid-stimulated Barrett's esophageal cancer cell proliferation by a mechanism involving the MAPK pathway. Our results suggest that sorafenib might be useful in the management of Barrett's-associated dysplasia and adenocarcinoma. These findings provide a foundation for in vivo studies to assess the efficacy of sorafenib in Barrett's-related neoplasia.

Reaction of fluorogenic reagents with proteins: III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503

The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were determined by continually infusing Chromeo P503-labeled α-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concentration than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis analysis of Chromeo P503-labeled α-lactalbumin produced concentration detection limits (3σ) of 12 pM and mass detection limits of 0.7 zmol, but with modest theoretical plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis analysis of a homogenate prepared from a Barrett's esophagus cell line; the separation quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.

M1965 Phosphatidylinositol-Specific Phospholipase C (PI-PLC) Mediates Bile Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Esophageal Adenocarcinoma Cells

We have shown that NADPH oxidase NOX5-S (S = short, i.e. lacking EF-hand motifs at its N-terminal) is overexpressed in SEG1 esophageal adenocarcinoma cells associated with Barrett's esophagus (BE) and in BE with high-grade dysplasia. NOX5-S also contributes to increased cell proliferation and decreased apoptosis in SEG1 cells. We have also shown that low dose of bile acid increases cell proliferation through upregulation of NOX5-S. In the present study we examined the role of PI-PLC in bile acid-induced NOX5-S expression in SEG1-esophageal adenocarcinoma cells. In SEG1 cells, low dose of taurodeoxycholic acid (TDCA 10-11M, 24h) significantly increased NOX5-S expression and hydrogen peroxide (H2O2) production. TDCA-induced increase in NOX5-S expression and H2O2 production was significantly reduced by the PI-PLC inhibitor U73122, and the MAP kinase kinase inhibitor PD98059. In SEG1 cells transfected with NOX5-S luciferase reporter plasmid NOX5LP containing NOX5-S promoter fragment (-2501 to -1 from ATG), TDCA significantly increased the luciferase activity, an increase which was significantly decreased by U73122. In addition, TDCA significantly increased ERK2 phosporylation, which was significantly reduced by U73122. The data suggest that TDCA-induced NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Isoforms of PI-PLC β1, β3, γ1 and γ2 were detectable by Western blot analysis, but not β2, β4, δ1 and δ2. Knockdown of PI-PLC γ2 with siRNA significantly decreased TDCA-induced NOX5-S protein expression and H2O2 production. The data suggest that PI-PLC γ2 may be involved in TDCA-induced NOX5-S expression. We conclude that in SEG1 cells low dose of TDCA increases NOX5S expression and H2O2 production. This increase in NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Supported by NIDDK R21 DK073327-01.

M1966 NADPH Oxidase NOX5-S Mediates Cyclin D1 Expression in Barrett's Esophageal Adenocarcinoma Cells

M1966 NADPH Oxidase NOX5-S Mediates Cyclin D1 Expression in Barrett's Esophageal Adenocarcinoma Cells

W1954 Sulforaphane, An Antioxidant Derived from Broccoli, Induces Apoptotic Cell Death in Barrett's Esophageal Adenocarcinoma Cells

W1954 Sulforaphane, An Antioxidant Derived from Broccoli, Induces Apoptotic Cell Death in Barrett's Esophageal Adenocarcinoma Cells