Textes juridiques

A602 AGA ABSTRACTS • G2473 INDUCIBLE NITRIC OXIDE SYNTHASE AND NITROTYROSINE IN GASTRIC MUCOSA OF GASTRIC CANCER PATIENTS. T. Goto, K. Haruma, M. Yosihara, Y. Kitadai, M. Ito, M. Mihara, T. Kamada, S. Tanaka, K. Sumii, G. Kajiyama. First Depertment of Internal Medicine, Hiroshima University School of Medicine and Department of Internal Medicine, - .... Hospital, Hiroshima, Japan. AIM: Recent studies have shown that Helicobacter pylori (Hp) infection is etiologically associated with gastric cancer. However, the role of Hp in carcinogenesis is unknown. Hp infection leads to a sustained production of the reactive nitrogen species which cause DNA damage. In this study, we examined the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine in human non-cancerous gastric mucosa. MATERIALS and METHODS: A total of 93 archival gastric biopsy specimens were used. The 34 biopsy specimens from gastric cancer patients were obtained two years or more before diagnosis of gastric cancer (Group A). The other 59 specimens were from the patients who had no evidence of gastric cancer during long term follow-up (Group B). These two groups were age and gender matched. Immunohistochemical analysis was performed to examine the expression of iNOS and nitrotyrosine. RESULTS: iNOS Nitrotyrosie antrum 2.27 -+ O. 14 2.40 -- 0.12 Group A corpus 2.28 -+ 0.14 2.41 -+ 0.13 Group B antrum 0.93 -+ 0.10"* 0.98 + 0.10"* corpus 0.88 -+ 0.08** 0.88 -+ 0.09** ** : p < 0.01 CONCLUSIONS: The level of iNOS and nitrotyrosine were higher in gastric mucosa of Group A as compared with Group B. These results suggest that high production of endogenous reactive intermediates in the gastric mucosa may contribute to carcinogenesis of gastric cancer and may predict the mucosa with high risk for the development of gastric cancer. • G2474 LIPID PEROXIDE-INDUCED MILD OXIDATIVE STRESS ENHANCES PROLIFERATIVE ACTIVITY IN HUMAN COLONIC CaCo-2 CELLS. Y. Goto, T.G. Wang, T.Y. Aw. Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA. The intestinal epithelium has one of the most rapid cell turnover rates of fully differentiated tissue, and organ homeostasis is normally balanced by cell proliferation and cell death. Peroxidized lipids have been implicated in the pathogenesis of intestinal disorders, possibly via oxidant-induced disruption of mucosal turnover. The objective of this study is to determine the effect of subtoxic levels of lipid peroxides (LOOH) on intestinal proliferative activity using a human colonic carcinoma cell line, CaCo-2. Method. CaCo-2 cells were cultured to 80% confluence and incubated with 0 - 5 pM LOOH for 6h or 24h in serum-free DMEM medium. Cell proliferative activity was examined by 3H-thymidine (T) incorporation and ornithine decarboxylase (ODC) activity. LOOH-induced cell cycle changes were analyzed by flow cytometry using propidium iodide staining. Results. Exposure of CaCo-2 cells to LOOH caused dose- and time-dependent changes in ODC activities. At lpM LOOH, ODC activity was significantly increased after 6h and 24h, whereas at 5 laM LOOH, ODC activity increased at 6h, but returned to control level at 24h. Peroxide-induced 3H-T incorporation paralleled the changes in cell ODC. In these studies, treatment with hydroxyurea revealed that DNA replication accounted for all the 3H-T incorporation at 6h at 1 pM LOOH, while 20% of 3H-T incorporation induced by 5pM LOOH was due to DNA repair. By 24h, the ratio of DNA repair to DNA replication increased significantly at both LOOH doses. Initiation of DNA synthesis by LOOH was verified by flow cytometry. Cell cycle analysis showed that LOOH-treated, but not control, cells progressed from G0/G 1 to G2/M after 6 - 9 h oxidant exposure. By 24h, 80% of the peroxide-stressed ceils has progressed through one cell cycle and returned to the resting state. Conclusion. Taken together, the study shows that mild oxidant stress induces intestinal cell proliferation (DNA replication), while oxidative DNA damage is associated with higher oxidant challenge. These findings provide insights into the mechanism of lipid peroxide-mediated disruption of intestinal cell turnover that have important implications for development of gut pathology. This research is supported by NIH grants DK-44510 and DK43785. • G2475 HYPOMETHYLATION OF THE MUC2 GENE PROMOTER IS THE COMMON GENETIC PROPERTY OF THE MUCINOUS CARCINOMA CELLS OF THE COLON AND MAMMA. A. Gratchev, C. B6hm, E.O Riecken, C. Hanski. Universit~itsklinikum Benjamin Franklin der Freien Universit~it Berlin, Berlin, Germany We showed recently that the common property of the mucinous carcinomas of the colon (C), ovary (O), mamma (M) and pancreas (P) is the overexpression (C) or de novo expression (O,M,P) of the MUC2 gene (J. Pathol. GASTROENTEROLOGY Vol. 114, No. 4 182:385,1997). The mncinous phenotype and/or the activation of the normally silent MUC2 gene in the carcinomas of O, M and P correlate with a significantly better prognosis. Aims: To better understand the regulation of a gene whose expression may affect the prognosis, we analysed the methylation of the MUC2 gene promoter. Methods: Cells lines from carcinomas of C (N=7), M (N=5) and P (N=3) were tested in Northern blotting and RT-PCR for their MUC2 expression. Then the genomic DNA was isolated from the MUC2 expressing and nonexpressing carcinoma cell lines from C, M and P and digested with the methylation-sensitive enzyme Hpa II. Radiolabelled amplimer from the MUC2 gene promoter region was used as a probe in Southern blots. The methylation was further analysed by sequencing after bisulfite treatment of the DNA. Cells nonexpressing MUC2 (Colo 205) were grown in 5 mM methylation inhibitor 5-aza-2'-deoxycytidine for 6 weeks and then cloned by limiting dilution. The morphology and the methylation of the resulting clones were then analysed. Results: MUC2-expression was detected in carcinoma cell lines of C (4), M (1) and P (2). Five methylation sites (M1-M3) were detectable within the promoter region by Southern blotting. The methylation of M1, M2 and M3 coincided with the lack of MUC2 expression in C and M but not in P. The methylation status of the first intron had no effect on MUC2 expression. The methylation inhibition in MUC2-nonexpressing ceils induced cell clones expressing MUC2 and exhibiting mucinous morphology. In these clones the lack of methylation of the MUC2 gene promoter correlated with the mucinous morphology of the cells. Conclusion: These results are the first indication that the lack of methylation of MUC2 gene promoter may contribute to the development of the mucinous phenotype. The methylation pattern of the MUC2 gene promoter may indicate a selective genetic lesion characteristic for mucinous carcinomas of the colon and mamma. Supported by the DFG Project. No. 1520/5-4 and Wilhelm Sander Stiftung, Project No. 97.017. I. • G2476 CHANGES IN SIGNAL TRANSDUCTION RESULTING FROM FOCAL ADHESION KINASE (FAK) CLEAVAGE DURING APOPTOSIS OF HUMAN INTESTINAL EPITHELIAL CELLS (IEC). J. Grossmann, A.W. Grasso, C. Fiocchi, A.D. Levine. Case Western Reserve University School of Medicine, Cleveland, OH. FAK is a non-receptor tyrosine kinase which associates with the integrin- linked signaling complex formed at sites of cell-substratum contact, referred to as focal adhesions. FAK participates in integrin-mediated signal transduction by directly phosphorylating various tyrosine kinases of the src family and the lipid kinase phosphatidylinositol 3-kinase (PI3-K). PI3-K is considered to be a critical mediator of mitogenic "survival" signals since its inhibition renders cells more susceptible to apoptosis. Furthermore, PI3-K plays an important role in the maintenance of cell-cell contact and cytoskeletal integrity. Freshly isolated human IEC undergo detachment- induced apoptosis within three hours of separation from the basement membrane, a process which involves the sequential, caspase-mediated cleavage of FAK into 94 and 84 kDa fragments. We investigated the changes in FAK catalytic activity and signal transduction subsequent to cleavage of FAK. IEC were isolated from colonic surgical specimens using a new protocol specifically designed to assess early events of the apoptotic cascade. The catalytic activity of FAK and its fragments was determined by an in vitro kinase assay. The p94 and the p84 fragments incorporated ['[-32P]-ATP in vitro, demonstrating that the catalytic domain of FAK remained intact despite cleavage. Several other proteins at 54, 56, 68, 72, 85 and 102 kDa co-immunoprecipitated with FAK and incorporated [T-32P]-ATP. However, no signal could be detected for the FAK substrates pp60 sre and ppl30 c~. The signal at 85 kDa, which is the molecular weight of the regulatory subunit of PI3-K, disappeared as p94FAI<and p84 FAK were detected. The association of PI3-K with both uncleaved FAK and FAK fragments was confirmed by immunoprecipitation for both FAK and PI3-K. Phosphotyrosine analysis for FAK showed a marked decrease in phosphorylation of FAK, p94 yAK, p84 FAK and associated proteins following detachment. Cleavage of FAK during apoptosis may not only disrupt the architecture of the integrin-signaling complex, but also leads to the formation of proteolytic fragments which exhibit substrate specificities different from uncleaved FAK. The loss of PI3-K activity, presumably through direct interaction with FAK and caspase-generated FAK fragments, may link caspase activation to the dramatic deterioration of cytoskeletal integrity and loss of mitogenic signaling observed during programmed cell death. • G2477 SEQUENTIAL AND SELECTIVE ACTIVATION OF CASPASES DURING DETACHMENT-INDUCED APOPTOSIS OF HUMAN INTESTINAL EPITHELIAL CELLS (IEC). J. Grossmann, C. Fiocchi, A.D. Levine. Case Western Reserve University School of Medicine, Cleveland, OH. Caspases, a family of cysteine proteases, play a pivotal role in the execution of apoptosis. The death signal is amplified in a proteolytic cascade involving caspases, which are activated in a hierarchical fashion by cleavage of a proenzyme into two distinct subunits which form the active protease. However, the molecular order of caspase activation in response to different

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