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. DESIGN 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.