812 Inactivation of Cholecystokinin-2 Receptor Inhibits Progastrin-Dependent Colonic Proliferation and Colorectal Cancer in Mice

of these effects remain largely unknown. CCK-2 receptor (CCK-2R) is the receptor for amidated gastrin, and in some studies has been demonstrated expression in colon. Thus, we examined a possible role for CCK-2R in progastrin-dependent colorectal cancer. Methods: Expression of CCK-2R in the murine colon was examined by RT-PCR. Binding and activation of CCK-2R was studied in AGS-E gastric cancer cells expressing CCK-2R using biotinylated progastrin and FACS. Progastrin-overexpressing hGAS mice were crossed to CCK-2R-KO (knockout) mice to generate four groups of mice in the same genetic (FVB/N) background: hGAS; hGAS/CCK-2R-KO; CCK-2R-KO; WT controls. To assess a colonic cell proliferation, four groups of 6 wk old mice were injected BrdU (10 mg/kg, i.p.) one hour prior to sacrifice, and mouse colons were stained with anti-BrdU antibodies. To evaluate a sensitivity of colonic cells to carcinogenesis, the four groups of 6 wk old mice received 6 weekly doses of azoxymethane (AOM: 12 mg/kg/wk, i.p.) starting at 6 wks of age. Twenty-two (22) weeks after the last injection, mice were euthanized, colons were removed and analyzed for the presence of tumors. Neoplastic and preneoplastic lesions were analyzed for beta-catenin expression by IHC. Results: The expression of CCK-2R in WT murine colon, and absence from the CCK-2R-KO colon, was confirmed by RT-PCR analysis. PG was neither able to bind to nor activate Ca++ fluxes in gastric AGS-E cells expressing CCK-2R. In cell proliferation study, colonic mucosa thickness of hGAS mice (3.69±0.26) was higher than that of WT (3.11±0.21); hGAS/CCK-2R-KO (3.11±0.13) or CCK-2R-KO (2.76±0.6) mice (p<0.05). The percentage of proliferating (BrdU+) crypt cells was significantly higher in hGAS (15.43±2.32) mice than in WT (7.0±2.87); hGAS/CCK-2R-KO (5.75±2.39) or CCK-2R-KO (6.18±2.1) mice (p<0.01). In AOM treated animals, the total number of tumors per colon was significantly higher in hGAS (10.6±2.9) than in WT (6.8±1.9); hGAS/CCK-2R-KO (3.8±1.3) or CCK2R-KO (3.1±0.8) mice (p<0.01). The tumor size/burden per mouse was significantly higher in hGAS mice than in WT, hGAS/CCK-2R-KO or CCK-2R-KO mice (p<0.05). Conclusion: While progastrin does not appear to bind or signal through CCK-2R, our data reveal an unexpected role for CCK-2R signaling in PG-mediated colonic cell proliferation and AOMstimulated carcinogenesis In Vivo.

Kip1

Specific Regulation of Nuclear P27 Expression and Cyclin-Dependent Kinase Inhibitory Activity By Glycogen Synthase Kinase-3β in Intestinal Cell Differentiation Qingding Wang, Yuning Zhou, Xiao-fu Wang, B. M. Evers The cellular mechanisms regulating intestinal differentiation are poorly understood. We have demonstrated that inhibition of PI3-kinase or overexpression of PTEN increases p27Kip1 expression, a cyclin-dependent kinase (CDK) inhibitor, and enhances intestinal cell differentiation. The purpose of this study was to determine the role of glycogen synthase kinase-3β (GSK-3β), a downstream effector of PI3-kinase, in intestinal cell differentiation. METHODS. The human colon cancer cell line, HT29, was treated with sodium butyrate (NaBT), a short chain fatty acid, to induce differentiation; GSK-3β activity was inhibited by either treatment with LiCl or SB216763 or transfection with siRNAs targeting GSK-3β. Cells were infected with a recombinant adenovirus encoding the active form of Akt or GSK-3β. Cell cycle distribution, p27Kip1 protein and mRNA levels were determined. In addition, the activity and expression of intestinal alkaline phosphatase (IAP; a differentiation marker), CDK2, CDK4 and GSK-3β activities, and p27Kip1 binding to CKD2 and CDK4 were measured. RESULTS. Inhibition of GSK-3 by complementary procedures (ie, chemical inhibition or overexpression of active Akt) blocked NaBT-induced G0/G1 arrest and IAP activity and mRNA expression. Moreover, treatment with NaBT increased the nuclear, but not the cytosolic, expression and activity of GSK-3β, suggesting a role for GSK-3β in NaBT-induced cell cycle arrest and differentiation. NaBT decreased CDK2 activity and induced p27Kip1 expression; inhibition of GSK-3β rescued NaBT-inhibited CDK2 activity and blocked NaBTinduced p27Kip1 expression in the nucleus but not in the cytoplasm, suggesting that GSK3β regulation of NaBT-mediated G1 cell cycle arrest may occur through CDK2 or CDK4 inhibition and p27Kip1 induction. In addition, we demonstrate that NaBT decreased the expression of Skp2 protein, which regulates nuclear abundance of p27Kip1; this decrease was attenuated by GSK-3β inhibition or GSK-3β siRNA transfection. Consistently, overexpression of active GSK-3β mutant decreased the expression of Skp2 and increased p27Kip1 expression in the nucleus. Furthermore, NaBT increased p27Kip1 binding to CDK2 which was completely abolished by GSK-3 inhibition. In agreement with these results, overexpression of an active form of GSK-3β increased p27Kip1 binding to CDK2. CONCLUSIONS. We show, for the first time, that GSK-3 regulates nuclear p27Kip1 expression through the downregulation of nuclear Skp2 expression and functions to regulate p27Kip1 assembly with CDK2. Our results indicate that GSK-3β plays a critical role in the G0/G1 arrest associated with intestinal cell differentiation.

813 Gastrin Is An Essential Cofactor in Helicobacter-Associated Gastric Carcinogenesis and Metastasis in C57bl/6 Mice Shigeo Takaishi, shuiping Tu, Tomoyuki Okumura, Wataru Shibata, Sheng-Wen Wang, Zinaida Dubeikovskaya, Ramanathan Vigneshwaran, Shanisha A. Gordon, Arlin B. Rogers, Barry H. Rickman, Sureshkumar Muthupalani, Mark T. Whary, James G. Fox, Timothy C. Wang Background & Aims: We have previously described synergistic interaction between hypergastrinemia and Helicobacter felis (H. felis) infection in a mouse (FVB/N) model of gastric cancer. However, spontaneous development of gastric cancer in gastrin deficient mice in different background and housing condition has also been reported. Consequently, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in a uniform C57BL/6 background and SPF housing using hypergastrinemic (INS-GAS), gastrin deficient (GASKO) & C57BL/6 wild type (B6 wt) mice as a control. Methods: INS-GAS, GAS-KO (both backcrossed to B6 more than 10 generations) & B6 wt mice were infected with H. felis for 6, 12 & 18 months. Infection status was assessed by realtime PCR and microscopic evaluation. Proliferating cells, parietal cells and ECL cells were investigated by immunostaining with Ki-67, HK-ATPase-β & chromogranin A antibodies. Expressions of cancer-related genes such as p53, β-catenin, CD44 & TFF2 were also analyzed by IHC. Results: At 12mo postinfection (p.i.), INS-GAS showed severe dysplasia or gastric intramucosal neoplasia (GIN) in corpus. In contrast, B6 wt showed severe gastritis or mild dysplasia and GAS-KO only showed mild to moderate gastritis, respectively. Spasmolytic peptide (TFF2) expressing metaplasia was detected in corpus of all mice from 3 strains. At 18mo p.i., INS-GAS showed GIN or invasive gastric cancer; B6 wt showed severe dysplasia or GIN, while GAS-KO showed severe gastritis but neither dysplasia nor GIN. The numbers of H. felis colonies in antrum were stable over time among 3 strains, while those numbers in corpus decreased at 12 & 18mo p.i. in INS-GAS and B6 wt. At 18mo p.i. the number of Ki-67(+) cells per gland was the highest in INS-GAS and the lowest in GAS-KO. Parietal cell loss was remarkable in INS-GAS and B6 wt, but minimal in GAS-KO. ECL cell density did not differ significantly among 3 strains. CD44 staining was strongly positive in GIN and invasive cancer lesions of INS-GAS and B6 wt, while p53 was negative in these areas. β-catenin membranous staining was strongly positive in the same areas, but nuclear accumulation of β-catenin was not detected. Of note, some 18mo infected INS-GAS showed metastatic cancer in periaortic lymph node and liver or spleen, and these metastatic cells were strongly positive for CD44. Conclusions: Hypergastrinemia and H.felis infection synergistically promoted metastatic gastric cancer in C57BL/6 mice, while gastrin deficiency suppressed H. felis-induced gastric cancer. These results suggest an essential role for gastrin as a cofactor in Helicobacterassociated gastric carcinogenesis.

811 Aging Gastric Mucosa Exhibits Impaired Angiogenesis, Arrested Vasculogenesis and Delayed Healing. Reduced VEGF, VEGFR2, cAMP, Increased Endostatin and Dysfunction of Bone Marrow-Derived Endothelial Precursors Are the Underlying Mechanisms. Andrzej Tarnawski, Amrita Ahluwalia, Xiaoming Deng, Wanjun Bae, Zsuzsanna Sandor, Sandor Szabo Background: Aging gastric mucosa has increased susceptibility to injury and delayed healing, but the cellular targets and the mechanisms are not fully defined. Angiogenesis - sprouting of new blood vessels - is essential for delivery of oxygen and nutrients to the healing site and thus is critical for healing. Angiogenesis in aging (vs. young) gastric mucosa in response to injury has not been examined before. Methods & Studies: In 84 Fisher F344 rats, 3 month (young) and 24 months old (aging), at baseline and 2, 8 and 24 hr after intragastric ethanol administration (8 ml/kg of 50%) we studied: 1) mucosal structural changes by light and transmission electron microscopy (TEM); 2) mucosal blood flow with a laser-Doppler; 3) expression of VEGF, VEGFR-2, cAMP and endostatin; 4) the extent of mucosal injury and 4) angiogenesis following injury by: quantification of sprouting microvessels (immunostained for factor VIIIRA) and TEM. 5) A contribution of bone marrow-derived endothelial precursors (BMDEP) to neovascularization was assessed by CD34 immunostaining. To determine human relevance, we examined in human microvascular endothelial cells (HMVEC), neonatal (Neo) and from aging individuals, in-vitro angiogenesis (ability to form microvascular tubes and lumina on matrigel) at baseline and in response to VEGF treatment. Results: Aging gastric mucosa at baseline exhibits reduced blood flow (~60%; p<0.002), decreased expression of VEGF and nuclear VEGFR2 and increased endostatin (all p<0.01 vs. young rats). Ethanol-induced gastric mucosal injury in aging rats was increased by 350% (p<0.001 vs. young rats); injury of gastric endothelial cells preceded epithelial damage. At 24 hr after injury in aging rats, angiogenesis in gastric mucosa bordering necrosis was reduced 12-fold (p<0.001 vs. young rats) and mucosal healing was significantly delayed. In contrast to young, in aging gastric mucosa at the injury border there was absence of BMDEP cells indicating impaired chemotaxis and/or function of these cells and thus ineffective vasculogenesis. HMVEC from aging individuals exhibit decreased expression of VEGF, impaired VEGFR2 nuclear translocation, reduced cAMP, impaired in-vitro angiogenesis(reduced >2.8-fold; p<0.001), and defective response to VEGF. Conclusions: 1) In aging gastric mucosa angiogenesis and vasculogenesis in response to injury are reduced 12-fold and mucosal healing is delayed. 2) Aging HMVEC display significantly reduced angiogenesis. 3) Reduced expression of VEGF, VEGFR2 and cAMP, and increased endostatin in aging gastric mucosa and aging HMVEC are the key mechanisms responsible for impaired angiogenesis and delayed healing.

814 Octreotide Stimulates Menin in Somatostatin-Expressing Cells of the Duodenum Edith Mensah-Osman, Juanita L. Merchant Background: Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by neuroendocrine (NE) tumors including duodenal gastrinomas. Menin is the ubiquitous 67kDa nuclear protein product of the MEN 1 locus, which causes hypergastrinemia and interacts with a number of transcription factors as well as the nuclear matrix to recruit histone deacetylase complexes. Although menin plays a role in critical cellular functions and development of NE tumors, the expression of menin in the duodenum where MEN1 gastrinomas develop has not been examined. Despite the presence of several hormoneexpressing cell types, e.g., CCK, secretin in the proximal duodenum, only gastrin and to a lesser extent somatostatin (SST) peptides are overexpressed in subjects carrying mutations in MEN1 alleles (Anlauf et al, 2005, 2006). Objective: We therefore tested the hypothesis that the distribution and regulation of menin-expressing cells might differ in duodenum compared to the antrum explaining why gastrin and SST-expressing cells preferentially

812 Inactivation of Cholecystokinin-2 Receptor Inhibits Progastrin-Dependent Colonic Proliferation and Colorectal Cancer in Mice Guangchun Jin, Alexander N. Dubeykovskiy, David M. Pritchard, Kelly S. Betz, Andrea Varro, Timothy C. Wang Background & Aims: Many human colorectal cancer cells abundantly express progastrin(PG), unprocessed product of the gastrin gene. Overexpression of progastrin has been shown to predispose colonic hyperproliferation and colorectal cancer in transgenic mice, but mediators

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AGA Abstracts

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