[7][“Molecule K”

Damaged single and doublestranded DNA within the nucleus unwinds and is pulled toward the anode during alkaline electrophoresis, thereby giving a cometlike appearance.Representative photomicrographs of cells subjected to the comet assay are shown. The comet tails seen with cytokine stimulation are further enhanced with incubation with oxidative lesion repair enzyme formamidopyrimidine glycosylase cells showed intact spherical nuclei, indicating that the DNA damage was induced by cytokinestimulated NO via inducible NOS.. NO NO generated in response to inflammatory cytokines, and SNAP inhibited DNA repair in human cholangiocarcinoma cells.To establish that the decrease in overall repair activity of the cholangiocarcinoma cells was in fact a direct effect of NO production by cytokine stimulation of iNOS, LNMMA was added to the culture medium with the cytokines and incubated for h.Inhibition of DNA repair by NO may explain, in part, the accumulation of damaged DNA during cytokine stimulation of cholangiocarcinoma cells.Our findings add human cholangiocarcinomas to this group of inflammationassociated malignancies.Because iNOS was not expressed by cholangiocarcinoma cells in vitro in the absence of cytokines, it would appear that cholangiocarcinomas in vivo elicit a proinflammatory cytokine response sufficient to induce iNOS expression.Indeed, there is histological evidence of tumorassociated inflammatory cells, a potential source of inflammatory cytokines, in most cholangiocarcinoma specimens.The near universal expression of NO by cholangiocarcinoma suggests that it plays an important role in the biology of this cancer.There are many potential mechanisms by which NO may be important in the initiation, promotion, and progression of this cancer.Our data suggest that NO could promote the accumulation of potential oncogenic mutations by inhibiting DNA repair enzymes.Inhibition of proapoptotic effector proteins by protein nitrosylation, such as caspase proteases, may also promote extended survival of malignant cells. Indeed, nitrosylation of caspases would disable apoptotic pathways promoting cell survival despite DNA damage.Finally, NO may confer a survival advantage for cancer by serving as an angiogenesis factor. The induction of iNOS with NO generation could explain, in part, the link between chronic inflammation and cholangiocarcinoma. As demonstrated in our comet assay, NO generated by the induction of iNOS with inflammatory cytokines is sufficient to induce oxidative DNA damage.It is likely that the inhibition of the DNA repair mechanisms contributes to this DNA damage.These observations suggest that NO may play an important role in causing the oncogenic mutations that are important in the development of cholangiocarcinoma.However, the specific potential oncogenic mutations induced by NO remain obscure.Recently, p mutations have been identified in most cholangiocarcinomas associated with primary sclerosing cholangitis. Whether NO can induce genetic alterations in p is unknown, but it would provide a mechanistic link between inflammation, NO formation, and the development of this malignancy.NO may affect proteins by nitrosylation of tyrosine and cysteine residues.Although tyrosine nitrosylation of proteins has been amply documented as a marker of protein oxidation, its effect on the catalytic functions of enzymes is obscure.In contrast, cysteine nitrosylation is known to directly inactivate enzymes. These data demonstrate that the magnitude of NO generated by iNOS in these cells is sufficient to inhibit DNA repair processes.

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