increase). NOTCH2, NOTCH4, DLL1, DLL3, DLL4, JAG1, JAG2, NUMB and NUMBL had no statistically significant difference among different cell lines. We also treat CP-A Barrett's cell line with acid (pH 6.5) for 24 hours and measured NOTCH signaling molecules by PCR array. We found that acid treatment significantly decreased NOTCH1 (2 fold decrease) and JAG1 (1.6 fold decrease), and increased NOTCH3 (1.7 fold increase) and NOTCH4 (3.9 fold increase) in CP-A Barrett's cells. Acid treatment did not cause significant changes of DLL1, DLL2, DLL4, JAG2, NOTCH2 and NUMB. In addition, Western blot analysis showed that acid upregulated NOTCH3 and NOTCH4 protein expression. Acid-induced increase in cell proliferation in CP-A Barrett's cells was significantly decreased by knockdown of NOTCH3 (from 285% control to 162% control, p<0.001) and NOTCH4 (from 236% control to 192% control, P<0.001) with their small interfering RNAs. We conclude that downregulation of NOTCH1 and overexpression of NOTCH3 may play an important role in the development of Barrett's associated adenocarcinoma. It is possible that acid reflux present in BE patients may downregulate NOTCH1 and upregulate NOTCH3, increasing cell proliferation and thereby contributing to the progression from Barrett's esophagus to esophageal adenocarcinoma. Supported by NIH NIDDK R01 DK080703. 380 Alcohol Induces ALDH2 As a Novel Cytoprotective Mechanism to Suppress Acetaldehyde-Derived DNA Adduct Formation in Esophageal Epithelial Cells Yusuke Amanuma, Shinya Ohashi, Osamu Kikuchi, Yukie Nakai, Shin-ichi Miyamoto, Koji Tanaka, Kelly A. Whelan, Hiroshi Nakagawa, Tsutomu Chiba, Tomonari Matsuda, Manabu Muto Background: Acetaldehyde is an alcohol-derived major human carcinogen that induces DNA damage. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme, catalyzing oxidation of acetaldehyde, and its deficiency increases the risk for esophageal squamous cell carcinoma (ESCC). ALDH2 is known to be expressed in several tissues including liver, heart and kidney; however, the production and functional roles of ALDH2 in esophageal epithelium remain elusive. Methods: Aldh2-knockout (Aldh2 -/- ) and wild-type ( Aldh2 +/+ ) mice were treated with 10% ethanol in drinking water for 8 weeks, and esophageal tissues were subjected to immunohistochemistry to evaluate ALDH2 levels and liquid chromatography tandem mass spectrometry to determine esophageal N 2 -ethylidene-2'-deoxyguanosine (N 2 -ethylidene-dG), a unique form of DNA adduct induced by acetaldehyde. Next, human esophageal keratino- cytes were directly treated with acetaldehyde, and N 2 -ethylidene-dG and ALDH2 levels were investigated. Moreover, we assessed a protective role of esophageal ALDH2 for acetaldehyde- derived DNA damage using human esophageal keratinocytes that specifically suppress or overexpress ALDH2. Results: Ethanol drinking resulted in an induction of ALDH2 produc- tion in the esophageal squamous epithelium in Aldh2 +/+ mice. By contrast, ethanol drinking induced robustly N 2 -ethylidene-dG in the Aldh2 -/- mouse esophagus, suggesting that acetalde- hyde may induce DNA damage lesions in the absence of ALDH2 expression. In primary mouse and immortalized human esophageal epithelial cell culture, either Aldh2 loss or ALDH2 knockdown by RNA interference resulted in a great enhancement of N 2 -ethylidene- dG induction response to acetaldehyde stimulation. Moreover, ectopically expressed ALDH2 prevented acetaldehyde from inducing N 2 -ethylidene-dG in human esophageal cells, suggest- ing that ALDH2 suppresses the DNA damage mediated by acetaldehyde in esophageal epithelial cells. Conclusions: Our mechanistic study suggests that ALDH2 may be induced in the esophagus to reduce acetaldehyde in response to ethanol drinking and that ALDH2 may have a novel cytoprotective role through suppression of acetaldehyde-induced DNA damage, providing mechanistic insights into the role of ALDH2 in the pathogenesis of ESCC. 381 ALDH2 and Autophagy May Cooperate to Alleviate Acetaldehyde-Mediated DNA Damage and Cytotoxicity in Fission Yeast and Esophageal Epithelial Cells Koji Tanaka, Kelly A. Whelan, Chiaki Noguchi, Jasmine D. Peake, Grant Grothusen, Vinesh Anandarajan, Yusuke Amanuma, Shinya Ohashi, Tsutomu Chiba, Manabu Muto, Eishi Noguchi, Hiroshi Nakagawa Introduction: Acetaldehyde (AA) is a DNA crosslinker and a major human carcinogen that is metabolically produced from alcohol and tobacco smoke, two major risk factors for esophageal squamous cell carcinoma (ESCC). AA is broken down to nontoxic acetic acid by aldehyde dehydrogenases (ALDHs). ALDH2 deficiency is common in East Asians with increased ESCC susceptibility. It remains elusive as to (i) how ALDHs differentially influence AA-mediated cytotoxicity, and (ii) which cytoprotective mechanisms may be activated upon AA exposure. Methods: We analyzed fission yeast ( S. pombe) strains defective for the mammalian ALDH ortholog Atds; Aldh2 -/- and Aldh2 +/+ (wild-type) murine esophageal cells; and normal human esophageal cells (EPC2-hTERT) and derivatives with or without p53 R175H . Cells were treated with AA to determine DNA damage, reactive oxygen species (ROS) production, autophagy, cell growth and viability. DNA damage foci were visualized in yeast strains carrying a yellow fluorescent protein reporter fused to Rad52 which localizes at sites of DNA damage. 2',7'-dichlorodihydrofluorescein diacetate dye was used to determine ROS. Autophagy was determined by flow cytometry detecting autophagosome formation with cyto-ID fluorescent dye and Western blotting for LC3B. Autophagy was pharmacologically inhibited by chloroquine (CQ). Results: Three mutant yeast strains ( atd1, atd2 and atd3) displayed hypersensitivity to AA with significantly decreased cell growth and viability with a reciprocal increase in the DNA damage foci ( P<0.05, n=3) within 2 h after AA exposure. Most sensitive to AA was atd1, whose molecular structure is closest to ALDH2, consistent with its AA detoxification activity in mammals. >1 mM AA increased lethality in Aldh2 -/- esophageal cells compared to Aldh2 +/+ cells. In addition, p53 R175H limited AA-mediated cell death in EPC2-hTERT cells, implying activation of the p53-dependent cell-cycle checkpoint functions in response to AA. Both human and mouse esophageal cells displayed >90% viability in the presence of 1 mM or less AA exposure where AA stimulated autophagy in a dose-dependent manner, suggesting that autophagy may serve as a cytoprotective mechanism. Such a premise was corroborated by increased cell death with robust ROS induction upon treatment with CQ in the presence of AA. Moreover, Aldh2 -/- cells displayed higher basal S-79 AGA Abstracts autophagy. Conclusions: ALDH2 may have an evolutionarily conserved role in AA detoxifica- tion. While excessive acetaldehyde causes DNA damage and p53-dependent cell death, autophagy and ALDH2 may cooperate to alleviate AA-induced oxidative stress and cytotoxic- ity, thereby, serving as a failsafe mechanism to limit AA-mediated carcinogenesis. These findings provide novel mechanistic insights into the protective role of ALDH2 against malig- nant transformation in alcohol and AA-related malignancies including ESCC. 382 The Polycomb Histone Lysine Methyltransferase Ezh2 Reversibly Regulates the Fate of Interstitial Cells of Cajal (ICC) In Vivo Sabriya A. Syed, Gabriella B.Gajdos, Srdjan Milosavljevic, Yujiro Hayashi, Raul A. Urrutia, Sabine Klein, Dieter Saur, Tamas Ordog Background & Aims: Kit + ICC, pacemakers and neuromodulators of the GI tract, are reduced in several GI motor disorders and aging. ICC depletion can be reproduced by inhibiting Kit signaling and may reflect phenotypic changes including loss of Kit rather than cell death. Using clonally-derived, conditionally immortalized ICC we previously showed that this transition may be due to reversible, mitotically heritable, transcriptional repression of Kit and other genes by the polycomb histone lysine methyltransferase Ezh2 (DDW 2013, 2014). Here, we used genetic lineage tracing and genome editing to obtain in-vivo evidence of ICC survival beyond the loss of Kit and the role of Ezh2 in establishing this "post-ICC" phenotype. Methods: In postnatal day (PD) 7-10 Kit CreERT2/+ ;R26 mT-mG/mT-mG mice, Kit- transcribing cells were indelibly labeled by inducing Cre-mediated expression of membrane- targeted green fluorescent protein (mG) with tamoxifen (75 μg/g bwt/day ip for 3 days). The distribution of mG + cells among cells lacking hematopoietic markers (HP - ) and expressing high, low or no Kit on their surface was analyzed by flow cytometry in the gastric corpus+antrum tunica muscularis on PD11 (n=7) and between PD107-163 (n=5). Genomic deletion of Ezh2 in Kit CreERT2/+ ;Ezh2 fl/fl mice was induced by the same tamoxifen regimen between PD46-127 (n=6). The ratio of Kit low to Kit + cells in the HP - Cd34 - populations of the gastric, ileal and colonic tunica muscularis was analyzed by flow cytometry 60 days post- injection. Age- and genotype-matched mice injected with vehicle were used as controls (n= 5). Results: At D11, recombination efficiency in gastric ICC (HP - Kit + Cd34 - cells) was 83±5% (mean±SEM), with 75±4% of HP - mG + cells coinciding with ICC. By D107-163, the number of ICC identified by immunolabeling decreased by 57%, whereas mG + ICC only decreased by 50%, reflecting a relative gain of ~1400 mG + ICC possibly from mG + Kit low precursors. The net age-related ICC loss was paralleled by a shift of mG + cells away from Kit + ICC, leaving only 42±2% of HP - mG + cells in the ICC fraction. Concomitantly, mG + cells in the HP - Kit low/- CD34 - fraction containing presumed post-ICC increased >2.8-fold, accounting for 49% of the loss of mG + ICC. In mice showing no overt phenotypic change 60 days after inducing genomic deletion of Ezh2, the ratio of Kit low to Kit + cells in the HP - Cd34 - populations of the gastric, ileal and colonic muscles decreased by 20% ( P=0.04), 16.5% ( P>0.05) and 52% (P=0.01), respectively. Conclusions: During the first 3-5 months of life, ~25% of murine gastric ICC likely die while another ~25% lose Kit but survive as Kit low/- Cd34 - cells. Age-related ICC loss is slightly mitigated by the differentiation of Kit low/- precursors. Ezh2 reversibly regulates aging-related phenotypic transition in the ICC lineage by inhibiting Kit expression. Support: NIH DK58185. 383 Modulation of Ca 2+ Waves in Human Enteric Glial Cells Andromeda Linan Rico, Fabio Turco, Suren Soghomonyan, Iveta Grants, Josh Enneking, Daniel R. Tso, Bradley J. Needleman, Alan Harzman, Rosario Cuomo, Fernando Ochoa- Cortes, Fievos L. Christofi Background and Significance: Purinergic signaling is involved in enteric neuron-to-glial communication and motility in rodent gut. We sought to investigate glial function and purinergic signaling in cultured human enteric glial cells (hEGC) or glia in ganglia of the intact submucous plexus (hSMP). Experimental Design: LSM fluo-4/Ca 2+ imaging was used to study Ca 2+ responses. Effects on membrane potential (V m ) in hEGCs were determined with whole-cell patch recordings. Surgical specimens were from jejunum (N=23), sigmoid colon (N=5) or ileum (N=3). Cultures of purified hEGCs were prepared as before (Gut, 2013) from 7 surgical cases. EGCs grown to confluency were used at passage 3-6. Results: Spontaneous Ca 2+ waves lasting > 15 min occur in hEGC. The rate of Ca 2+ oscillations is 3.5±0.2/5min in colon myenteric glia (n=72) and 3.2±0.0.4 / 5 min (n=40) in submucous glia. In jejunum the rate of oscillations is 2.9±0.13/5min. Absence of extracellular Ca 2+ abolished Ca 2+ waves; the ryanodine Ca 2+ channel inhibitor ruthidium-red (10μM) mimicked the effect in 70% cells (n=120). In colon the PLC inhibitor U73122 blocked the Ca 2+ wave and induced a prolonged-Ca 2+ transient (80%cells; n=100). Ca 2+ waves were interrupted by a Cx-43 gap junction inhibitor carbenoxolone (50μM,Carbx), and it caused a robust and prolonged increase in [Ca 2+ ] i (n=60). In hEGCs without Ca 2+ waves, Carbx caused Ca 2+ waves in 80% of cells. There was strong Cx-43-ir in hEGC. No effects were observed in jejunum. The gap junction mimetic peptide 43Gap26 (10μM) modulated Ca 2+ waves in a different manner. ADPßS, ATP or apyrase (2U/ml) enhanced peak Ca 2+ responses during each oscilla- tion. Adenosine caused a large-Ca 2+ transient (>3 x peak responses) that desensitized allowing Ca 2+ oscillations to resume. Effects occurred in glia in both nerve plexuses. Patch-clamp studies revealed oscillations 1-3mV in membrane potential (V m ) at a rate of 6/min. ATP caused biphasic depolarization of V m (fast+slow delayed depolarization) suggestive fast-P2X channels and P2Y-receptors. Glial Ca 2+ waves are rare in glia of intact hSMP. ATP or ADPßS caused a Ca 2+ transient in glia of hSMP. At low 10μM ATP only glia (not neurons) responded in both plexuses (p<0.0009,n=43). The EC 50 for elevating [Ca 2+ ]I=96μM in glia of hSMP like hEGC (s100-ir). In hSMP neural-to-glial responses evoked by fiber tract stimulation (FTS,25Hz) was blocked by apyrase. Conclusions: Human enteric neural-glial communica- tion and glial-to-glial communication involves purinergic signaling. The hEGC is a suitable model to study modulation of Ca 2+ waves and Vm unlike hSMP. Ca 2+ waves involve Ca 2+ influx and ryanodine-sensitive channels and the G q /PLC/IP 3 -Ca 2+ pathway. Communication between hEGCs may involve ‘excitatory or inhibitory signals' via Cx-43 hemichannels. Data suggest a modulatory role of hEGCs in the gut (NIH DK093499; NSSP funds). AGA Abstracts