Colorectal cancer Figure 4 G2/M arrest induced by polymorphonuclear cells (PMNs) is dependent on the expression of p53 and p21waf1/cip1. HCT116, HCT116p212/2 and HCT116p532/2 cell lines were cultured with activated PMNs at s ratio of 20:1 for 24 h and analysed for cell cycle distribution by flow cytometry. HCT116p212/2 and HCT116p532/2 cells failed to undergo a G2/M arrest under these conditions. Each column represents the mean (SD) of three independent experiments. water and oxygen. ?O22 release by activated PMNs was measured by lucigenin-amplified chemiluminescence in the presence of SOD, CAT or both enzymes. SOD but not CAT showed a strong ?O22 scavenging effect (fig 5A). To test the effect of these enzymes on the G2/M arrest, total cell lysates of HCT116+chr3 cells were analysed following co-culture in the presence of both CAT and SOD. Although the addition of CAT and SOD during co-culture with activated PMNs reduced the phosphorylation of p53 at site Ser15 and accumulation of p21waf1/cip1 (fig 5B), it had no effect on the G2/M arrest (fig 5C), suggesting that PMN products in addition to ?O22 or H2O2 activate the p53/p21 pathway and are sufficient for the installment of the cell cycle arrest. Activated polymorphonuclear cells cause replication errors in colon epithelial cells Recruitment of the MMR complex following DNA replication errors leads to cell cycle arrest.44 Our experiments so far show an hMSH2-dependent G2/M arrest, which may be a consequence of an increase in replication errors upon exposure to activated PMNs (fig 2A). HCT116+chr3 (clones A3.1 and A3.7) bearing a GFP-expressing plasmid in which the EGFP sequence is kept out of frame by a (CA)13 repeat,29 were exposed to activated PMNs for 24 h, and then expanded for 7 days. Analysis of the fluorescent fraction by flow cytometry revealed a significant rise in the number of transiently mutated (M1) fraction whereas the increase in the highly fluorescent population (M2) was not significant (fig 6). No changes in the mutant fraction were observed with non-activated PMNs. This result could suggest that the PMN-induced G2/M arrest is a consequence of increased replication errors. As PMNs did not increase the number of permanent mutations (M2 cells), it is likely that the DNA repair system was functional. DISCUSSION Ulcerative colitis is a chronic inflammatory disease of the large intestine which is associated with increased CRC risk.1 45 The mucosal injury of active ulcerative colitis is characterised by enhanced transepithelial migration of activated PMNs forming crypt abscesses.3 In this study, we have developed an in vitro co- culture model in which primary PMNs acted as effectors and colon cell lines as targets of inflammation-driven carcinogenesis. 784 Figure 5 Superoxide dismutase (SOD) and catalase (CAT) do not prevent G2/M arrest but reduce p53 phosphorylation and p21waf1/cip1 accumulation. (A) Polymorphonuclear cells (PMNs) were activated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) for 30 min. After removal of PMA by washing the cells twice with Hanks’ balanced salt solution (HBSS), ?O22 release was measured by lucigenin-amplified chemoluminescence in the presence or absence of SOD (1000 U/ml) or CAT (1000 U/ml). A strong ?O22 scavenging effect was observed in the presence of SOD and, to a lower extent, of CAT. Very low ?O22 production was measurable in the absence of PMA. (B) HCT116+chr3 cells were co-cultured with activated PMNs in the presence of SOD and CAT as in (A) and the total levels of p21 and p53 phosphorylation at Ser15 were analysed by western blotting. The addition of SOD and CAT considerably reduced both p53 phosphorylation and accumulation of p21waf1/cip1 within 8 h. b-Actin served as a loading control. (C) HCT116+chr3 cells were co-cultured as in (A) or exposed to 200 mmol/l H2O2, in the presence of SOD and CAT, and cell cycle distribution was measured by flow cytometry. Under these conditions, SOD and CAT blocked H2O2-dependent, but not PMN-induced, G2/M arrest after 24 h. RLU, relative light units. Gut 2008;57:780–787. doi:10.1136/gut.2007.141556