Figure 6 Activated polymorphonuclear cells (PMNs) increase replication errors. (A) HCT116+chr3 cells (clones A3.1 and A3.7 harbouring the pIREShyg2-EGFP/(CA)13 construct) were co-cultured with activated PMNs at a ratio of 100:1 for 24 h. After removal of the PMNs, the target cells were expanded for another 7 days and analysed by flow cytometry. Activated PMNs caused a significant increase in the M1 mutant fraction (ie, transient mutations (replication errors) that may be repaired) but not in the M2 mutant fraction (ie, permanent DNA mutations that are transmitted to daughter cells and may expand) in both clones. Colorectal cancer In our study colon cells responded to activated PMNs or granulocyte-like HL60 cells by slowing proliferation and arrest- ing in the G2/M phase of the cell cycle (fig 2A,B). This observation is consistent with previous reports in which exposure of colon epithelial cells to H2O2 and macrophages lead to G2/M arrest.4 28 Cellular damage induces responses that enable the organism either to eliminate or cope with the damage. DNA damage response reactions include removal of damaged DNA and restoration of the continuity of the DNA structure; activation of a DNA damage checkpoint, which arrests cell cycle progression in order to allow repair and transmission of damaged or incompletely replicated chromosomes; or apoptosis, which eliminates seriously damaged cells.39 In most of the cell lines analysed in this study, exposure to activated PMNs caused an arrest of the cell cycle in G2/M, consistent with the activation of a post-replication DNA-damage checkpoint. Evidence for the installation of such a checkpoint,46 apart from the G2/M arrest (fig 2A), includes phosphorylation of Chk1 at the ATM and ATR target sites Ser317 (fig 3A) and Ser345 (data not shown);47 phosphorylation of the p53 tumour suppressor protein at site Ser15 (fig 3A,C);48 increased expression of the cyclin-dependent kinase inhibitor p21waf1/cip1 (fig 3A,C); Gut 2008;57:780–787. doi:10.1136/gut.2007.141556 phosphorylation of the histone isoform c-H2AX at Ser 139 (data not shown) and cleavage of caspase-7 (data not shown). p53 is essential for the maintenance of a G2/M arrest following oxidative stress. In fact, p53 contributes to the inhibition of cdc2, the mitotic cyclin-dependent kinase through Gadd45, p21waf1/cip1, and 14-3-3s. Cyclin B1 is required for cdc2 activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis.49 After disruption of either the p53 or the p21waf1/cip1 gene, gamma-irradiated cells progressed into mitosis in spite of extensive damage.30 In our system, p21waf1/cip1 expression and p53 phosphorylation increased upon exposure to activated PMNs and cells lacking p53 or p21waf1/cip1 failed to undergo a G2/M arrest (fig 4), suggesting that the p53 pathway is required for the response to PMN-induced damage. However, although SOD and CAT scavenge ?O22 and H2O2 (fig 5A), and completely reverse the H2O2 -induced G2/M arrest (fig 5C), they only partially reduced the PMN-induced phosphorylation of p53 (fig 5B) and were unable to reverse the cell cycle arrest caused by PMNs (fig 5C), suggesting that activated PMNs also release additional molec- ules which can induce G2/M arrest. Another potentially harmful species released by PMNs are chlorinating agents (containing an active chloride in a formal +1 oxidation state, 785