SS

AT

Ab

stra

cts

MHC-class I was not. The cell line doubled in 2.46 to 2.83 days. The chemosensitivityprofile of the cell line was analyzed in detail. Conclusions: This newly established and well-characterized, low-passage cell line and its corresponding xenograft of Crohns associatedcarcinoma provide a useful tool for future investigations on the biological characteristics ofa rare and distinct tumor entity. Additionally, matched patient-derived immune cells allowfor comparative genetic studies as well as detailed analysis of immune escape mechanisms.

Mo1791

Colonic Microbiota and Gene Methylation in Colonic CarcinogenesisMarco Scarpa, Melania Scarpa, Luisa Barzon, Giulia Costanzi, Enrico Lavezzo, FrancescaFinotello, Francesca Erroi, Lucia Dallagnese, Silvia Basato, Paola Brun, Stefano Toppo,Barbara Di Camillo, Carlo Castoro, Ignazio Castagliuolo

BACKGROUND: Changes in gut microbiota have been associated to colonic carcinogenesis,although the underlying mechanisms are not clear. DNA methylation of gene promotersmay inhibit gene expression and modulate oncosuppressor activity playing a role in colorectalcarcinogenesis. The aim of this study was to analyze the interplay between colonic microbiotaand gene methylation in colonic carcinogenesis. MATERIALS AND METHODS: In thisprospective study three groups of 8 patients, affected by either hyperplasic polyps, dysplasticadenoma or colonic adenocarcinoma, and 8 healthy subjects were enrolled. Colonic biopsiesof healthy and affected (hyperplasic polyps or dysplastic adenoma or colonic adenocarcinoma)mucosa were obtained. The methylation status of MLH1, MGMT1, CDH13, APC and RUNX1gene promoters was assessed by methylation specific PCR and a methylation score wascreated based on the number of methylated genes. The composition of microbiota adherentto the colonic mucosa was analysed by 454 pyrosequencing of bacterial 16S rRNA gene.Non parametric statistics and correction for multiple testing was used. RESULTS: The analysisof the methylation status of the promoter region of APC, CDH13, MGMT, MLH1 and RUNX3showed that along the colorectal carcinogenesis the promoter region of APC, CDH13,MGMT1 and RUNX3 were more frequently methylated at the invasive cancer step. Therefore,the methylation score resulted significantly higher in patients with cancer (p<0.05). Thepresence of the genus Flavonifractor resulted inversely correlated with the number of methyl-ated genes (r=-0.49, p=0.022). On the contrary, Peptostreptococcus and Schwartzia genusesresulted directly correlated with the number of methylated genes (r=0.45, p=0.047 and r=0.44, p=0.047, respectively). CONCLUSIONS: In our series, Flavonifractor genus resultedinversely correlated to methylation of genes occurring at the first steps of the colorectalcarcinogeneisis. On the contrary, Peptostreptococcus and Schwartzia genuses directly corre-lated with colorectal carcinogenesis-related gene methylation. In vitro test to verify if theseassociation are causal are warranted.

Mo1792

Overexpression of Stem Cell Markers Including Prominin 1/CD133 in CardiacMucosa and Barretts Intestinal MetaplasiaDan Falkenback, Evgenii Borodachev, Yuri V. Bobryshev, Oliver M. Fisher, AngeliqueLevert-Mignon, Sarah J. Lord, Melissa Thomas, Reginald V. Lord

Introduction: It has been hypothesized that intestinal or other stem cells play an importantrole in the etiology of Barretts esophagus, and may influence endotherapy and other treatmentoutcomes. The aim of this preliminary study was to identify potential stem cell markers inthe origin of this disease, focussing on cardiac mucosa and Barretts intestinal metaplasia(IM). Material & Methods: Fresh frozen endoscopic biopsies from 30 patients were investi-gated. The mRNA relative expression levels of the putative stem cell markers PROM1/CD133,ABCG2, MSI1, ATXN1, MYC, KLF4, LGR5 and DCLK1 were measured using standard qRT-PCR methods in normal squamous (n=10), cardiac mucosa (n=10), and Barretts IM (n=10)tissues. The protein expression of Prominin-1/CD133 was measured by immunohistochemis-try. Results: Significant mRNA upregulation at the IM stage was found for ABCG2 and KLF4.An on/off pattern with highest expression in cardiac mucosa was seen for MSI1, ATXN1,LGR5, and DCLK1. PROM1/CD133 was not expressed in normal squamous epithelia butwas progressively and highly overexpressed in cardiac mucosa and IM. Intense PROM1/CD133 protein expression was present in all goblet cells in IM. Conclusions: This studyidentifies novel putative stem cell markers for this disease and provides some support for arole for non-intestinalised cardiac mucosa in the etiology of Barretts esophagus. PROMININ1/CD133 immunohistochemisty selectively stains goblet cells in IM, which may be valuablefor the pathological interpretation of esophageal tissues.

Mo1793

Analysis of the Cyclooxygenase-2 (COX-2) and MethylenetetrahydrofolateReductase (MTHFR) Gene Polymorphisms in Esophageal CancerEvelise Pelegrinelli-Zaidan, Michele T. Tomitão, Márcia Kubrusly, Adriana V. Safatle-Ribeiro, Evandro S. de Mello, Rubens A. Sallum, Ivan Cecconello, Ulysses Ribeiro

Background: Cyclooxygenase-2 (COX-2) is induced in response to growth factors andcytokines, expressed in inflammatory diseases, premalignant and esophageal tumors. Theproduct of folate metabolism by the enzyme methylenetetrahydrofolate reductase (MTHFR)acts in DNA synthesis, alteration or inhibition of this enzyme increases the susceptibility tomutations, damage and altered DNA methylation, gene expression of transforming the tumorsuppressor and proto- ontogenesis, potential risk factors for esophageal cancer. Cox-2 andMTHFR polymorphisms might modify the levels of protein expression and may have aconsiderable influence on disease phenotype, which may have important clinical/genomicimplications. Aims: To evaluate single nucleotide polymorphisms (SNPs) in the Cox-2 andMTHFR genes and their prognostic values for patients operated on for esophageal cancer;and to investigate possible interactions between these genetic variations and clinicopathologiccharacteristics in esophageal cancer. Methods: Cox-2 and MTHFR SNPs were analyzed in114 prospective patients who underwent surgical resection, and had a minimum of 5 yearsfollow-up. DNA was isolated from leukocyte using extraction and purification kit, followed

S-1072SSAT Abstracts

by amplification by polymerase chain reaction (PCR). Real-time analysis was used for genotyp-ing Cox-2 and MTHFR SNPs through the TaqMan ® SNP Genotyping Assay. Results: Wedetermined frequencies of three COX-2 polymorphisms (1195A>G/ -1329A>G, 8437T>C,1759G>A), with nine haplotypes; and two MTHFR biallelic polymorphisms (1298 A>C,677 C>T), with six haplotypes. A high frequency of the wild genotype Cox-2-1195GGwas detected in adenocarcinoma tumors. Homozygous Cox-2-8437CC was associated toCaucasians and younger patients at diagnosis. Polymorphic wild genotype MTHFR-1298AAwas increased in squamous cell carcinoma, and younger patients. Wild homozigous MTHFR-677CC was associated to improved survivalship, and to heavy active tobacco smokingpatients. Wild homozygous genotype of Cox-2 and MTFHR were significantly correlated toa worst progression-free survival and overall survival when compared to the combinedheterozygous or recessive genotypes in a multivariate analysis. Conclusions: 1. Wild homozy-gous Cox-2 and MTHFR SNPs were associated to disease progression and survival in patientswith advanced esophageal cancer; 2. Cox-2 and MTFHR SNPs may be useful markers ofaggressiveness in these patients, and may orientate the appropriate target therapy in novelclinical trials.

Mo1794

Heparin Fragments Effects in Liver Injury Secondary to Liver Ischemia/Reperfusion (I/R)Ênio R. Vasques, José Eduardo M. Cunha, Ana Maria M. Coelho, Emílio E. Abdo, SandraN. Sampietre, Helena B. Nader, Ivarne S. Tersariol, Eleazar Chaib, Luiz Augusto C.D'Albuquerque

Background: Ischemia and reperfusion (I/R) is an essential phenomena in tissue damagein pathological conditions such as acute myocardial infarction, liver surgery and organtransplantation. Intracellular calcium concentrations are determining factors in cell deathinduced by I/R. It is estimated that 60% of the calcium entering into the cell is regulatedby the sodium calcium exchanger (Na-Ca exg). In calcium overload situations the exchangercan operates extrusion of intracellular calcium excess. Therefore, if the intracellular calciumhomeostasis is related to cell death in the presence of calcium overload, drugs acting onthe Na-Ca exg could be a strategy to minimize hepatocellular injury. Some heparin fragmentssuch as Trissulfated Disaccharide (TD) act on the Na-Ca exg inhibiting the exchange inhibitorypeptide (XIP), which inhibits the cytoplasmic calcium extrusion, and consequently decreasesthe intracellular calcium overload. XIP inhibition is applicable as a possible new strategy tominimize hepatotoxicity secondary to the ischemia/reperfusion injury due to calcium over-load. Objective: To evaluate the effects of TD administration in liver injury secondary to theischemia/reperfusion in rats. Methods: Wistar male rats, kept under mechanical ventilation,underwent partial liver ischemia induced by 60 min. pedicle clamping of medium and leftanterior lateral segments. Animals were divided into 2 groups: Control Group (n=12): ratsreceiving saline solution 10 minutes before reperfusion, TD Group (n=12): rats receivingTD in a dosage of 0.2 mg/kg IV, 10 minutes before reperfusion The volume administeredof each drug was 0.4 ml. Four hours after reperfusion, blood was collected for determinationsof AST and ALT. Liver tissue samples were assembled for mitochondrial oxidation andphosphorylation and malondialdehyde (MDA) content. Pulmonary vascular permeabilitywas also determined. Results: Four hours after liver reperfusion AST and ALT serum levelincreases in TD Group animals were significantly lower than in Control Group (p<0.05).Significant reductions of liver MDA content and ofmitochondrial dysfunction were observedin TD Group when compared to Control Group (p<0.05). No differences in pulmonaryvascular permeability were observed between the two groups. Conclusion: TD maintainsliver mitochondrial function and increases liver tolerance to I/R injury. These effects requirefurther evaluation before the introduction of this drug in the clinical setting.

Mo1795

PTK6 Increases Apoptosis With Gemcitabine Treatment in Pancreatic CancerCells by Enhancing DNA DamageHiroaki Ono, Marc D. Basson, Hiromichi Ito

Background: Protein Tyrosine Kinase 6 (PTK6) is a non-receptor type tyrosine kinase knownto be aberrantly expressed in various cancers including pancreatic cancer. The role of PTK6in cancer chemo-resistance remains unknown. We tested our hypothesis that PTK6 regulatesgemcitabine (GEM) resistance in pancreatic cancer, and explored its mechanism. Methods:We studied 2 human pancreatic cancer cell lines, Panc1 and MIAPaCa2. For some studies,GEM resistant clones of Panc1 and MIAPaCa2 were isolated by culture with GEM for 2months. Cell survival was measured by WST-8 assay. GEM-induced apoptosis and DNAdamage were evaluated by Western blotting. The effect of PTK6 overexpression on theefficacy of GEM therapy for pancreatic cancer in vivo was assayed using a xenograft mousemodel. Results: Endogenous PTK6 expression was increased at 24-48 hours with GEMtreatment in Panc1 and MIAPaCa2 cell lines. PTK6 gene-silencing increased cell survivalafter GEM treatment and decreased cleaved Caspase3 and PARP, indicating decreasedapoptosis, while PTK6 overexpression decreased cell survival and increased apoptosis. BasalPTK6 expression was significantly reduced in the GEM resistant clones compared with theparental lines (0.29 fold decrease in MIAPaCa2 and 0.60 fold decrease in Panc1, respectively,p<0.05). Restoration of PTK6 expression using an over-expression vector made the resistantMIAPaCa2 clone cells sensitive to GEM (0.77 fold decreased survival compared to theresistant clone, p<0.05). To explore the mechanism in which PTK6 regulates the cytotoxiceffect of GEM on pancreatic cancers, we tested the effect of altered PTK6 expression onDNA damage induced by GEM. GEM-induced H2AX phosphorylation ( γ-H2AX), which isa specific marker for DNA double-strand breaks, was significantly reduced by PTK6 gene-silencing, while GEM-induced γ-H2AX was increased by PTK6 overexpression. Furthermore,PTK6 gene silencing also inhibited the GEM-induced activation of ataxia-telangiectasiamutated (ATM) protein kinase, a central initiator of key signal responses to DNA damagesin both cell lines. Conversely, ATM kinase activation was enhanced by PTK6 overexpression(Figure). In the mouse xenograft model, subcutaneous tumors with PTK6-overexpressingMIAPaCa2 showed significant growth reduction compared with tumors with control MIA-PaCa2 after 5 weeks treatment with GEM (150 mg/kg IP, twice a week) (606 mm3 vs 252mm3 in size, 609 mg vs 128 mg in weight, p<0.01, respectively) . Conclusion: PTK6 is