Continuous cytokine exposure of colonic epithelial cells induces DNA damage.
OBJECTIVE
Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducible nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells.


METHODS
A colonic cell line (HT29) was stimulated for 1-10 weeks with interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) or both and compared with unstimulated cells or cells stimulated for 48 h. Cells were co-incubated with a selective iNOS inhibitor (N-monomethyl-L-arginine (L-NMMA)) in some experiments. Viability was assessed by the dimethylthiazol diphenyl tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis.


RESULTS
Continuously stimulated colonic cells had increased ROS production, especially those stimulated with TNF-alpha or IFN-gamma/TNF-alpha (P<0.001). The ROS production could be inhibited by L-NMMA co-incubation, indicating that iNOS is responsible for the up-regulation (P<0.05). Continuously stimulated cells had increased DNA instability (P<0.002), whereas short-term stimulated cells did not. The DNA instability was inhibited by L-NMMA co-incubation (P<0.05).


CONCLUSIONS
Continuous cytokine exposure induces an iNOS dependent up-regulation of ROS production and DNA instability. This mechanism could be involved in carcinogenesis in chronic inflammatory diseases of the intestinal tract.
Seidelin
Jakob B
JB
Department of Gastroenterology C, Herlev Hospital, Copenhagen University, Herlev, Denmark. jakse@herlevhosp.kbhamt.dk
Nielsen
Ole Haagen
OH
D000970:Antineoplastic Agents / Q000633:toxicity; D016923:Cell Death / Q000187:drug effects; D003106:Colon / Q000187:drug effects* / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000633:toxicity*; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000633:toxicity; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity; D015854:Up-Regulation / Q000187:drug effects; D019323:omega-N-Methylarginine / Q000633:toxicity
D000970:Antineoplastic Agents; D016207:Cytokines; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D007371:Interferon-gamma; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II