Colorectal cancer Figure 2 Polymorphonuclear cells (PMNs) cause a G2/M arrest in hMSH2-expressing colon epithelial cells without affecting the expression levels or DNA binding activity of mismatch repair (MMR) proteins. (A) HCT116, HCT116+chr3, HCT116-mlh1-2, Lovo, Lovo+chr2, Lovo(DT40+2)-4-1, HEC59 and HEC59+chr2 cells were co-cultured at a ratio of 20:1 with non-activated or activated PMNs for 24 h and cell cycle distribution was analysed by flow cytometry. A significant increase of the G2/M population was observed in all cell lines upon co-culture with activated PMNs except for Lovo, Lovo(DT40+2)-4-1 and HEC59 (cells lacking wild-type hMSH2). Each column presents the mean (SD) of at least three experiments. (B) HCT116 and HCT116+chr3 cells were co-cultured at 20:1 ratios with non-differentiated (nd) or dimethyl sulfoxide (DMSO)-differentiated (d) HL60 cells, both treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA). A significant increase of the G2/M population was observed with DMSO-differentiated HL60 cells. Each column presents the mean (SD) of at least three experiments. (C) HCT116+chr3 and Lovo+chr2 cells were co-cultured in the presence of activated PMNs for 24 h and changes in the total protein levels of MMR proteins (hMre11, hMLH1, hPMS2, hMSH2 and hMSH6) were analysed by western blotting. None of the investigated proteins showed a change in expression levels under these conditions; a-tubulin served as a loading control. A3.7, two plasmid copies)29 were sorted on a FACSVantage SE using CloneCyt Plus sorting technology (Becton Dickinson Immunocytometry Systems), and PMNs were activated with PMA as described above. Cells were cultured at 100:1 ratios for 24 h and PMNs were removed. Target cells were grown for an additional 7 days. The EGFP-positive population with low fluorescence intensity was considered as the ‘‘transiently mutated fraction’’ (M1), and that with high fluorescence intensity as the ‘‘permanently mutated fraction’’ (M2).29 Statistical analysis Experiments were carried out at least in triplicates and repeated twice. Data are represented as mean with the SD and compared by using the Student t test. p-Values ,0.05 were considered to be statistically significant. RESULTS Establishment of an in vitro co-culture system To simulate the carcinogenic environment in ulcerative colitis, we established an in vitro co-culture system in which PMNs were co-cultured with various colon epithelial cells separated by 782 a semi-permeable membrane. Activation of PMNs with PMA is followed by oxidative burst.38 A strong and sustained ?O22 release was observed for at least 2 h after PMA removal (fig 1A). The expression or absence of MMR components, p53 and p21 was tested (fig 1B) in the various cell lines used in this work. Activated polymorphonuclear cells cause an hMHS2-dependent G2/M arrest in colon epithelial cells Oxidative stress induces cellular checkpoints, leading to cell cycle arrest and preventing mitosis of cells with defective DNA replication.39 Activated PMNs were co-cultured with HCT116, HCT116+chr3, Lovo or Lovo+chr2 cells for 24 h. All cell lines, except Lovo, displayed an increase in the G2/M population within 24 h at 20:1 ratios (aPMN:target cells) (fig 2A), consistent with the MMR component hMSH2, but not hMLH1, being involved in the G2/M arrest. No such effect was observed when using non-activated PMNs. A dose effect was observed when HCT116 were cultured at different effector:target ratios (fig 3B). A similar experiment conducted with HEC59 cells or HEC59+chr2, showed a G2/M arrest only in cells, in which an extra chromosome 2 has been introduced, Gut 2008;57:780–787. doi:10.1136/gut.2007.141556