Figure 1 Characterisation and analysis of colon epithelial cell lines and ?O22 release by activated polymorphonuclear cells (PMNs). (A) Freshly isolated PMNs were activated with 50 ng/ml phorbol 12-myristate 13- acetate (PMA) for 30 min. Cells were washed twice and ?O22 release was measured by lucigenin-amplified chemiluminescence. A strong induction of ?O22 release was observed upon activated PMNs (solid line) that lasted for more than 2 h. Data are given as relative light units (RLUs) per 16106 cells. Non-activated cells (dotted line) were subjected to the same procedures (isolation and washing) and served as a control. A slightly increased level of ?O22 release by non-activated cells was observed. Each data point presents the mean (SD) of three independent measurements. (B) Western blotting of hMLH1, hMSH2, p21waf1/cip1 and p53 expression in target cell lines. The western blot confirmed the expected loss or reinstalled expression of protein expression of these cells (HCT116hMLH12/2, HCT116+chr3hMLH1+/2, LovohMSH22/2, Lovo+chr2hMSH2+/2, HEC59hMSH22/2, HEC59+chr2hMSH2+/2, HCT116p212/2, HCT116p532/2 and HCT116-mlh1-2). In this work, we hypothesise that the chronic exposure of the intestinal mucosa to activated PMNs leads to DNA damage, which may activate checkpoint kinases and initiate MMR, or if this is inefficient, may drive colon carcinogenesis. In order to simulate the carcinogenic environment in ulcerative colitis, we established an in vitro co-culture system with primary PMNs as effector cells and various colon cell lines as targets. Our results show that exposure of colon cells to activated PMNs install a G2/ M cell cycle checkpoint, indicative of DNA damage, through a mechanism that does not require hMLH1, but rather p53/p21 and hMSH2. This G2/M arrest is associated with an increase in replication errors. Colorectal cancer HCT116+chr3 A3.7,29 HCT116-mlh1-2hMLH1+/2, HCT116p532/2, HCT116p212/230 and LovohMSH22/2 and their derivatives Lovo+chr2hMSH2+/2 Lovo(DT40.2)-4-1hMSH22/2,31 as well as the human endometrial adenocarcinoma cell line HEC59hMSH22/2 and HEC59+chr2hMSH2+/232 were grown in IMDM (Gibco/ Invitrogen, Lofer, Austria) containing 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany). The medium for HCT116+chr3 contained 400 mg/ml and for HEC59+chr2 and Lovo+chr2 cells 700 mg/ml G418 (Gibco), respectively. The medium for HCT116-mlh1-2 cells33 contained 100 mg/ml hygro- mycin B (Invitrogen). The clones HCT116+chr3 A3.1 and HCT116+chr3 A3.7 were grown with 150 mg/ml hygromycin B and 400 mg/ml G418. The promyelocytic leukaemia cells line HL60 (ATCC CCL-240) was cultured in RPMI (Gibco/ Invitrogen) supplemented with 10% FBS. Isolation and activation of polymorphonuclear cells PMNs were freshly isolated from heparinised blood of healthy volunteers by dextran T500 (Pharmacia, Uppsala, Sweden) sedimentation followed by density gradient centrifugation through Ficoll-Paque (Amersham, Uppsala, Sweden) or HL60 cells were derived to granulocyte-like neutrophils by differentia- tion as described previously;34 erythrocytes were lysed in NaCl (0.2%) followed by NaCl (1.6%) and cells were washed in Ca/Mg-free Hanks’ balanced salt solution (HBSS) (Gibco). CD66b mAb (BD 55572) was used to confirm the purity of isolated PMNs by flow cytometry.35 PMNs were activated in HBSS containing 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma, St Louis, Missouri, USA) at 37uC and 5% CO2 for 30 min. PMA was removed by washing the PMNs twice with HBSS. The production of ?O22 was determined by lucigenin- enhanced chemiluminescence as described.36 37 Superoxide dis- mutase (SOD, 1000 U/ml) (Sigma) and catalase (CAT, 1000 U/ ml) (Sigma) were used as scavengers for ?O22 and H2O2, respectively. Co-culture and cell cycle analysis Target cells at a concentration of 76104 were seeded onto 6-well plates. Twenty-four hours later, PMNs were added to the upper chamber of a Transwell 0.45 mm microporous insert preventing cell-to-cell contact. PMNs and target cells were co-cultured at effector:target ratios of 0:1 to 100:1 for up to 24 h. Target cells were harvested using accutase (PAA Laboratories, Linz, Austria), fixed and analysed for cell cycle distribution as described.36 Western blot analysis Cell lysates and DNA-bound fractions were obtained as described.36 For western blotting, 50–150 mg of lysates were used with the following antibodies: rabbit polyclonal antibody (pAb) anti-phospho-p53 Ser15 (Cell Signaling Technology, Danvers, MA, USA); pAb anti-cleaved caspase-7 (Cell Signaling); mAb anti-p21waf1/cip1 (Cell Signaling); mAb anti- hMSH2 (Becton-Dickinson, San Jose, California, USA), mAb anti-hMHS6 (Becton-Dickinson), mAb anti-hPMS2 (Becton- Dickinson), mAb anti-hMre11 (Becton-Dickinson) and mAb anti-hMLH1 (Becton-Dickinson); mAb anti-tubulin (Abcam); mAb anti-actin (Sigma); pAb anti-phospho-Chk1 Ser317 (Cell Signaling). MATERIALS AND METHODS Cell lines The human colorectal carcinoma cell lines HCT116hMLH12/2 and their derivatives HCT116+chr3hMLH1+/2,26 HCT116+chr3 A3.1, Analysis of replication errors Five thousand non-fluorescent HCT116+ch3 cells, bearing the enhanced green fluorescent protein (EGFP)-based plasmid pIREShyg2-EGFP/CA13 (clones A3.1, one plasmid copy, and Gut 2008;57:780–787. doi:10.1136/gut.2007.141556 781