Figure 3 Activated polymorphonuclear cells (PMNs) induce a dose- dependent activation of checkpoint components in colon cells. (A) HCT116, HCT116+chr3, HCT116-mlh1-2, Lovo and Lovo+chr2 cells were co-cultured at a ratio of 20:1 for 8 h; cells were harvested and cell lysates were analysed for total levels and phosphorylation status of checkpoint proteins. An increased phosphorylation of Chk1 at Ser317 and p53 Ser15 was detected in HCT116+chr3, HCT116-mlh1-2 and Lovo+chr2 cells and an accumulation of p21waf1/cip1 in HCT116 and HCT116+chr3 cells. Ultraviolet light at an intensity of 25 mJ/m2 was used as positive control. a-tubulin served as loading control. (B) HCT116 cells were co-cultured at ratios from 1:1 to 10:1 with non-activated or activated PMNs for 24 h and cell cycle distribution was measured by flow cytometry. A dose-dependent increase of G2/M arrest was observed upon co-culture with activated PMNs. (C) Total lysates of cells exposed to activated PMNs (ratios of 1:1 to 10:1) for 24 h were analysed by western blotting. A dose-dependent increase of p53 phosphorylation at the site Ser15 and total levels of p21waf1/cip1 was detectable upon co-culture. Co-culture with non-activated PMNs is indicated as (2) and co-culture with activated PMNs is indicated as (+). H2O2 (200 mmol/l) was used as positive control. Tubulin was used as a loading control. Colorectal cancer arrest, independent of hMLH1 expression but dependent on hMSH2 expression. Moreover, co-culture of Lovo(DT40+2)-4-1 cells (a Lovo+chr2-derived cell line lacking hMSH2 expression31) with activated PMNs revealed no G2/M arrest (similar to Lovo and HEC59 cells; fig 2A), consistent with the assumption that the observed G2/M arrest is hMSH2 dependent. Activated polymorphonuclear cells do not change the expression of mismatch repair proteins It was previously suggested that oxidative stress relaxes the MMR system and reduces hMSH6.40 41 As hMSH2 is a potential candidate for installation of the G2/M arrest upon exposure to activated PMNs, we tested for changes in the expression levels of MMR proteins in target cells. However, no changes in MMR protein levels were detectable under these conditions (fig 2C). Activation of the ATM/ATR targets Chk1 and p53 is associated with the PMN-induced G2/M arrest It has been previously established that ATM and ATR are required to activate a p53- and Chk1-dependent G2 arrest upon DNA damage.42 Upon 8 h co-culture with activated PMNs, phosphorylation of Chk1 at Ser317 and of p53 at Ser15 was detected in all cells but HCT116 and Lovo whereas an accumulation of p21 was only seen in HCT116 and HCT116+chr3 cells (fig 3A). However, at 24 h, a dose- dependent phosphorylation of p53 at Ser15 and expression of the p53-downstream CDK-inhibitor p21waf1/cip1 was observed in HCT116 cells, which was paralleled by an increase in G2/M arrest (fig 3B,C). These results indicate the activation of a DNA- damage checkpoint in colon cells independent of hMLH1. To control for a possible role of additional genes transferred through chromosome 3 we also tested HCT116 cells that had been transfected with an hMLH1 construct (HCT116-mlh1-2), expressing wild-type hMLH1 and displaying MMR profi- ciency.33 When co-cultured in the presence of activated PMNs, they exhibited an increase in G2/M, similar to that described for the parental cell line (fig 2A). Western blotting of lysates from HCT116-mlh1-2 cells, co-cultured with activated PMNs, also showed a similar activation of Chk1 and p53 and accumulation of p21waf1/cip1 (fig 3A). Taken together, these results suggest that the presence of hMLH1 accelerates the activation of a checkpoint response but is not essential to achieve such. The PMN-induced G2/M arrest depends on the expression of p53 and p21 Several reports suggest an essential role for p53 and p21waf1/cip1 in installation of the G2/M checkpoint.30 43 Indeed, cells in which the p53 or the p21 gene had been disrupted failed to arrest in G2 following gamma ionising radiations.30 In fact our experiments demonstrated p53 phosphorylation at Ser15 (an ATM and ATR target site) and p21 accumulation (fig 3A,C). In order to investigate the importance of p53 and p21 in our system, the isogenic cell lines HCT116p532/2 and HCT116p212/2, in which the p53 or p21waf1/cip1 genes had been disrupted,30 were co- cultured as described above. Both cell lines failed to undergo a G2/M arrest (fig 4), suggesting that the G2/M arrest caused by activated PMNs depends on the expression of both p53 and p21waf1/cip1. and therefore express hMSH2 (figs 2A and 1B). The same results were observed with dimethylsulfoxide (DMSO)-differentiated neutrophils (d HL60) derived from HL60 cells (fig 2B). Taken together, these results suggest that activated PMNs induce a G2/M Superoxide dismutase and catalase do not inhibit phosphorylation at p53 Ser15 and increased levels of p21waf1/cip1 SOD catalyses the reduction of ?O22 to oxygen and H2O2, whereas catalase (CAT) catalyses the reduction of H2O2 to Gut 2008;57:780–787. doi:10.1136/gut.2007.141556 783