TY - JOUR

T1 - Continuous cytokine exposure of colonic epithelial cells induces DNA damage

AU - Seidelin, Jakob B

AU - Nielsen, Ole Haagen

PY - 2005/3

Y1 - 2005/3

N2 - 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.

AB - 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.

KW - Antineoplastic Agents

KW - Cell Death

KW - Colon

KW - Colonic Neoplasms

KW - Cytokines

KW - DNA

KW - DNA Damage

KW - Enzyme Inhibitors

KW - Epithelial Cells

KW - HT29 Cells

KW - Humans

KW - Interferon-gamma

KW - Nitric Oxide Synthase

KW - Nitric Oxide Synthase Type II

KW - Reactive Oxygen Species

KW - Tumor Necrosis Factor-alpha

KW - Up-Regulation

KW - omega-N-Methylarginine

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

M3 - Journal article

C2 - 15716663

VL - 17

SP - 363

EP - 369

JO - European Journal of Gastroenterology and Hepatology, Supplement

JF - European Journal of Gastroenterology and Hepatology, Supplement

SN - 0954-691X

IS - 3

ER -