Activated neutrophils induce an hMSH2-dependent G2/M checkpoint arrest and replication errors at a (CA)13-repeat in colon epithelial cells.
OBJECTIVE
Chronic inflammation in ulcerative colitis is associated with increased risk for colorectal cancer. Its molecular pathway of cancer development is poorly understood. We investigated the role of neutrophil-derived cellular stress in an in vitro model of neutrophils as effectors and colon epithelial cells as targets, and tested for changes in cell cycle distribution and the appearance of replication errors.


METHODS
Colon epithelial cells with different mismatch repair phenotypes were co-cultured with activated neutrophils. Target cells were analysed for cell cycle distribution and replication errors by flow cytometry. Changes in nuclear and DNA-bound levels of mismatch repair- and checkpoint-related proteins were analysed by western blotting.


RESULTS
Activated neutrophils cause an accumulation of target cells in G2/M, consistent with the installation of a DNA-damage checkpoint. Cells that do not express hMSH2, p53 or p21(waf1/cip1) failed to undergo the G2/M arrest. Phosphorylation of p53 at site Ser15 and Chk1 at Ser317, as well as accumulation of p21(waf1/cip1), was observed within 8-24 h. Superoxide dismutase and catalase were unable to overcome this G2/M arrest, possibly indicating that neutrophil products other than superoxide or H(2)O(2) are involved in this cellular response. Finally, exposure to activated neutrophils increased the number of replication errors.


CONCLUSIONS
By using an in vitro co-culture model that mimics intestinal inflammation in ulcerative colitis, we provide molecular evidence for an hMSH2-dependent G2/M checkpoint arrest and for the presence of replication errors.
Campregher
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AKH Wien, Division of Gastroenterology and Hepatology, Währinger Gürtel 18, A-1090 Vienna, Austria.
Luciani
M G
MG
Gasche
C
C
D002374:Catalase / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D000071877:Checkpoint Kinase 1; D018920:Coculture Techniques; D003093:Colitis, Ulcerative / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D003106:Colon / Q000378:metabolism / Q000473:pathology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D053843:DNA Mismatch Repair; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000502:physiology*; D018375:Neutrophil Activation; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016159:Tumor Suppressor Protein p53; D002374:Catalase; D013482:Superoxide Dismutase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein