AGA Abstracts pattern was also confirmed via Cyto-ID FACS analysis. To determine a functional role for autophagy in BE pathogenesis, STR and CPA cells were exposed to an acid pulse (pH3.5) followed by incubation in the presence or absence of CQ. Acid exposure increased ROS levels in STR and CPA (n=3; p<0.01) cells at 6 hours. CQ alone had little impact on ROS; however, combination of CQ with acid stress resulted in a synergistic increase in ROS in STR and CPA (n=3; p<0.05) cells. Similarly, while acid alone reduced cell viability 10-15% in STR and CPA cells at 24 hours, CQ synergized with acid treatment to increase cell death to 30-40% of controls at 24 hours (n=3; p<0.001). Conclusions: Our findings establish increased numbers of AVs in human BE compared to normal squamous or EAC. These findings suggest that autophagy functions to reduce oxidative stress and improve cell survival after acid exposure, and thus may play a critical role in BE pathogenesis and progression. Sa1883 High Levels of Dickkopf-1 in Blood Serum and Esophageal Tissue Are Associated With Severe Inflammation and Cellular Senescence in Human Reflux-Esophagitis Orestis Lyros, Parvaneh Rafiee, Rituparna Medda, Linghui Nie, A. C. MacKinnon, Ines Gockel, Reza Shaker Introduction: Overexpression of Dickkopf-1 (Dkk1), the secreted inhibitor of Wnt/β-catenin signaling, has been associated with tissue inflammation and injury. Repression of Wnt/β- catenin signaling has been shown to trigger early onset of senescence program as response to cellular injury. Aims of this study were (a) to evaluate the levels of circulating Dkk1 in patients with human reflux-esophagitis and (b) to explore possible association of Dkk1 tissue expression with induction of cellular senescence, as esophageal mucosal response to reflux-associated injury. Methods: Serum samples were collected from 16 patients with reflux-esophagitis and 11 healthy controls. Dkk1 serum levels were detected by sandwich enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of Dkk1 and p16, a marker of senescence, were examined in esophageal biopsies with reflux-esophagitis and paired normal squamous mucosa by real-time PCR and immunohistochemistry (IHC) respectively. A simple grading system 0-3 for IHC was used. To assess, whether Dkk1 mediates senescence, one normal esophageal squamous cell line (EPC2-hTERT) was treated with recombinant Dkk-1 and cell cycle, senescence-associated β-galactosidase activity (SA- β-gal) and p16 gene expression were examined by flow cytometry, SA-β-gal staining and real- time PCR respectively. Finally, SA-β-gal staining was performed in representative esophageal tissues from both patients groups. Results: Increased levels of Dkk1 in serum were detected in patients with severe reflux-esophagitis compared with healthy controls and patients with mild esophagitis. Gene and protein expression of Dkk1 and p16 were significantly elevated in human reflux-esophagitis tissues compared to paired healthy esophageal mucosa (Mann- Whitney test, p<0.05). Dkk1 gene expression significantly correlated with p16 gene expres- sion in esophagitis patients (Pearson's correlation test, r=0.958, p<0.00001). In vitro, high extracellular levels of human recombinant Dkk1 caused G0/G1 cell cycle arrest and induced p16 gene expression and SA-β-gal activity. Finally, positive SA-β-gal staining was demon- strated in esophageal mucosa with severe reflux-esophagitis in contrast to healthy squamous mucosa. Conclusions: Dkk1 overexpression was correlated with severe inflammation and cellular senescence in human reflux-esophagitis. Dkk1 serum level may serve as serological biomarker for disease severity and evaluation of therapy in patients with reflux-associated esophagitis. Sa1884 BMP4 Induces SOX9 and Columnar Cytokeratin Expression in Esophageal Squamous Cells and Increases Proliferation in Barrett's Cells Through the p38MAPK Pathway: Potential Roles for BMP4-p38MAPK Signaling in Barrett's Pathogenesis and Carcinogenesis Yuji Tamagawa, Peiguo Ding, Qiuyang Zhang, Thai H. Pham, Edaire Cheng, Norihisa Ishimura, Yoshikazu Kinoshita, Stuart J. Spechler, Rhonda F. Souza, David H. Wang Introduction: Barrett's esophagus is a squamous-to-columnar epithelial metaplasia caused by GERD through molecular mechanisms that remain unknown. In earlier studies, we showed that: 1) acid and bile salts activate Sonic hedgehog (Shh) signaling in esophageal squamous epithelium, 2) Shh released by epithelial cells induces BMP4 expression by esophageal stromal cells, 3) stromal BMP4 signals back to the epithelium, upregulating its expression of the transcription factor SOX9, and 4) SOX9 induces columnar features in squamous cells including expression of columnar cytokeratins (CK 8, CK18). BMP4 can signal through Smads (canonical pathway) or through p38MAPK in a non-canonical pathway. We previously found that acid and bile salts increase proliferation in Barrett's metaplasia by activating the p38MAPK pathway. Based on these findings, we hypothesized that BMP4 S-346 AGA Abstracts could regulate esophageal expression of SOX9, CK8 and CK18 through the p38MAPK pathway, and that BMP4 could also regulate proliferation in Barrett's metaplasia through the p38MAPK pathway. Methods: Telomerase-immortalized Barrett's columnar (BAR-T and BAR-10T) and esophageal squamous (NES-B3T and NES-B10T) epithelial cell lines (all derived from patients with Barrett's esophagus) were treated with recombinant BMP4 or were transfected with constitutively active BMPRIA, in the presence or absence of a specific p38MAPK pharmacologic inhibitor (SB203580). We performed: a) real-time PCR for SOX9, CK8 and CK18; and b) Western blot for total p38MAPK, phospho-p38MAPK, SOX9, CK8 and CK18. We evaluated effects of BMP4-p38MAPK signaling on cell viability and proliferation by MTS and BrdU assays. Results: In both squamous and Barrett's cell lines, treatment with BMP4 or overexpression of BMPRIA significantly increased expression of SOX9, CK8 and CK18; this was associated with increased levels of phospho-p38MAPK. Treatment with SB203580 decreased BMP-induced expression of SOX9, CK8, and CK18. BMP4 treatment or BMPRIA overexpression caused a significant increase in viability and proliferation of Barrett's epithelial cells, effects that were blocked by SB203580. Conclusions: In both esophageal squamous cells and Barrett's columnar epithelial cells, BMP4 induces the expres- sion of SOX9, CK8 and CK18 through the p38MAPK pathway. BMP4 also increased the proliferation of Barrett's cells through the p38MAPK pathway. These findings suggest that esophageal BMP4 signaling through the non-canonical (p38MAPK) pathway might cause squamous cells to acquire columnar features, contributing to the pathogenesis of Barrett's metaplasia. Our observation that BMP4 signaling through this pathway causes increased proliferation in Barrett's cells also suggests a potential mechanism whereby the reflux of acid and bile (which induce esophageal BMP4 expression) might promote carcinogenesis in Barrett's esophagus. Sa1885 ΔNp63 Upregulates Expression of Squamous Cytokeratins and Increases Proliferation in Squamous Cells, but Decreases Proliferation in Barrett's Cells: A Potential Role for Downregulation of ΔNp63 in Barrett's Pathogenesis Yuji Tamagawa, Peiguo Ding, Qiuyang Zhang, Thai H. Pham, Edaire Cheng, Norihisa Ishimura, Yoshikazu Kinoshita, Stuart J. Spechler, Rhonda F. Souza, David H. Wang Introduction: Expression of the transcription factor p63, a homolog of tumor suppressor gene p53, is required for development and maintenance of stratified epithelia in organs such as the cervix, skin, and esophagus. The p63 gene encodes two major, functionally-distinctive protein isoforms, TAp63 and ΔNp63, whose expression depends on which p63 gene promoter is activated. ΔNp63, which lacks the N-terminal transactivation domain of TAp63, is the predominant isoform expressed in esophageal squamous cells. In contrast, ΔNp63 is expressed only weakly in columnar cells of Barrett's esophagus, the condition in which metaplastic cells with malignant predisposition replace esophageal squamous cells damaged by GERD. In certain non-esophageal epithelial cells, p63 has been shown to regulate prolifera- tion and to induce expression of squamous cytokeratins (CK5 and CK14). To explore a potential role for ΔNp63 in the pathogenesis of Barrett's metaplasia, we sought to determine whether ΔNp63 regulates proliferation and the expression of CK5 and CK14 in human esophageal squamous and metaplastic Barrett's cells. Methods: We used telomerase-immor- talized esophageal squamous (NES-B3T and NES-B10T) and Barrett's columnar (BAR-T and BAR-10T) epithelial cell lines; all cell lines were derived from patients with Barrett's esopha- gus. The expression of Sox2 (an upstream regulator of p63), total p63, ΔNp63, CK5 and CK14 was examined using quantitative real-time PCR and Western blot under basal conditions, and following transfection with TAp63- or ΔNp63-expressing plasmids or siRNAs targeting total p63. We evaluated effects of p63 on cell viability and proliferation by MTS and BrdU assays. Results: Under basal conditions, mRNA and protein levels of Sox2, ΔNp63, CK5 and CK14 were higher in the squamous (NES) cell lines than in the Barrett's (BAR) cell lines. In NES-B3T and NES-B10T cells, overexpression of ΔNp63 increased CK5 and CK14 expression and significantly increased cell growth. As in squamous cells, ΔNp63-overexpres- sion also increased CK5 and CK14 expression In BAR-T and BAR-10T cells but, unlike NES cells, ΔNp63-overexpression significantly decreased cell growth in the Barrett's cells. Furthermore, NES-B3T and NES-B10T cells transfected with total p63 siRNAs showed decreased CK5 and CK14 expression and a significant decrease in cell growth. Conclusion: In esophageal squamous cells and in Barrett's metaplastic cells, ΔNp63 upregulates expression of squamous cytokeratins, supporting a role for ΔNp63 in squamous cell differentiation in the esophagus. Our observation that ΔNp63 increases proliferation in squamous cells and decreases proliferation in Barrett's cells suggests that loss of ΔNp63 in the esophagus could facilitate the development of Barrett's metaplasia. These findings support a potential role for downregulation of ΔNp63 in Barrett's pathogenesis. Sa1886 Esophageal Squamous Cells Exposed Repeatedly to Acid and Bile Salts In Vitro Exhibit Columnar Differentiation With Features of Barrett's Metaplasia Carlos D. Minacapelli, Christina L. Cheng, Manisha Bajpai, Abhishek A. Chouthai, Rhonda F. Souza, Stuart J. Spechler, Kiron M. Das Barrett's esophagus (BE) is a squamous-to-columnar metaplasia that results from the gastro- esophageal reflux of acid and bile through mechanisms that remain unclear. One hypothesis holds that reflux alters the expression of key developmental transcription factors in native esophageal cells, causing them to differentiate into columnar cells (transdifferentiation or transcommitment). Others suggest that reflux merely destroys esophageal cells, enabling columnar progenitor cells in the proximal stomach to expand into the esophagus and replace its squamous lining. To explore the former hypothesis, we studied the effects of repeated exposures of acid and bile on esophageal squamous cell lines in vitro. Methods: We used two non-neoplastic, telomerase-immortalized esophageal squamous cell lines: one established from a patient with Barrett's esophagus (NESB-10T), the other from a patient who had GERD without Barrett's esophagus (NES-G2T). Cells were exposed to a solution of acid and bile salts (A+B) containing HCl (pH 4) and glycochenodeoxycholic acid (200 μM) for 5 minutes/day for 30 weeks. Morphological changes were examined weekly. Real-time q PCR was used to determine the expression of genes involved in columnar differentiation (CDX2, SOX9, Villin, and Cox-2). Indirect immunofluorescence was performed for mAb DAS-1 and