Colorectal cancer Figure 7 Model of polymorphonuclear (PMN)-induced G2/M cell cycle arrest in colon epithelial cells. An oxidative burst of activated PMNs releases a variety of genotoxic factors [including, but not limited to reactive oxygen species (ROS)] which lead to replication errors [in our system a frameshift at a (CA)13 microsatellite] followed by a G2/M cell cycle arrest. The G2/M checkpoint prevents cells from initiating mitosis when they experience DNA damage. This pathway relies on the presence of functional hMSH2 which is an essential component of the hMutS heterodimer (that also includes hMSH3 or hMSH6; depicted in orange). The hMutS heterodimer acts as a sensor of replication errors and may initiate mismatch repair (by recruiting the hMutL complex). hMSH2 is capable of physically interacting with ATR forming a signalling module. Next, the checkpoint kinase Chk1 undergoes ATR-mediated phosphorylation. Active Chk1 phosphorylates Cdc25c which is inactivated and cells are halted at G2/M. ATR also mediates the phosphorylation of the tumour suppressor p53 (at site serine 15) causing accumulation of the cyclin-dependent kinase inhibitor p21 and cell cycle arrest. The presence of hMSH2, p53 and p21 are essential for the PMN- driven installation of this G2/M checkpoint, a pathway that acts independently of hMLH1 (which is needed for mismatch repair). eg, HOCl).50 However, the HOCl-specific scavenger taurine51 did not compensate the PMN-induced G2/M arrest (data not shown). Further limitations of the model presented are the use of colon cancer cell lines instead of primary colon epithelial cells as well as freshly isolated PMNs which vary from volunteer to volunteer in their composition of released products. Another limitation is the effector:target ratio and time of exposure of colon epithelial cells in our co-culture system as the number of PMNs infiltrating the colon mucosa and the duration of the inflammatory stress are far more dramatic in ulcerative colitis. Direct contact of PMNs with epithelial cells may further increase the observed effects, but is difficult to standardise in vitro. Eukaryotic MMR include two different heterodimeric com- plexes of MutS-related proteins: hMutS-a (hMSH2/hMSH6) and hMutS-b (hMSH2/hMSH3). These complexes have differ- ent mispair recognition properties and abilities to support MMR. hMutS-a senses single-base mismatches and small insertion–deletion loops, whereas hMutS-b recognise inser- tion–deletion loops.17 Our results suggest that the hMutS-a or 786 hMutS-b heterodimer may be responsible for the response induced by oxidative stress during inflammation (fig 2A). In fact, cells lacking hMSH2 (Lovo colon cells and HEC59 endometrial adenocarcinoma cells), but not their nearly isogenic counterparts, in which an extra chromosome 2 has been reintroduced, failed to arrest in G2/M upon co-culture with activated PMNs (fig 2A). As shown in fig 3A, the phosphoryla- tion of Chk1 and p53 was almost absent in Lovo cells after 8 h. In comparison, Lovo+chr2 cells responded much stronger to PMN induced stress. Indeed, it has been reported that hMSH2 physically interacts with Chk1 and Chk2,35 as well as with DNA damage sensors Rad51 and Mre11,52 and that this interaction is required for the installation of cell cycle arrest. However, we can not exclude the possibility that the hMSH2 partners hMSH6 and hMSH3 are also needed for the installation of G2/M arrest. Interestingly, when H2O2 was used in control experiments, the G2/M arrest was also achieved in cells lacking hMSH2 (data not shown). The DNA damage signalling, induced by activated PMNs, seemed to be more pronounced in HCT116+chr3 cells (fig 3A). Here, in fact, phosphorylation of p53 and Chk1 was already visible upon 8 h exposure to activated PMNs (fig 3A), whereas HCT116 showed very low levels of phosphorylation, which increased at 24 h (fig 3C). HCT116+chr3 express wild-type hMLH1, but also other genes that are present on the extra chromosome 3. An interaction between hMHL1 and the checkpoint sensor components hMre1140 and Nbs153 has been reported as well as the co-localisation of MMR proteins and the MRN (Mre11-Rad50-Nbs1) complex to foci of DNA damage.53 However, in our study, HCT116-mlh1-2 cells, expressing almost physiological levels of wild-type hMLH1, have a response more similar to that of the parental HCT116 cells than to HCT116+chr3, suggesting a marginal role for hMLH1 in the response of colon cells to activated PMNs. A number of other proteins have been implicated in MMR, including DNA polymerase delta, single-strand binding protein RPA, clamp PCNA, clamp loader replication factor C (RFC), exonuclease 1 (EXO1), endonuclease FEN1,13 as well as ATR; all these proteins are involved in synthesising DNA and associated with the replication fork.54 We may speculate that the accelerated activation of Chk1 and p53 phosphorylation observed in the HCT116+chr3 upon co-culture with activated PMNs may be a consequence of the introduction of an extra copy of the wild-type ATR gene, which is located on chromosome 3. However, since exposure to ultraviolet light strongly activates this DNA damage pathway in all investigated cell lines (fig 3A) this effect seems to be specific for the exposure to activated PMNs. In ulcerative colitis, p53 and replication errors at repetitive CA sequences are occurring at an early stage within chronic inflamed mucosa independent of dysplasia.55 56 Although the risk of CRC development in ulcerative colitis is increased, the high frequency of these mutations (around 40% of patients) should actually result in far more cancers. One explanation would be that these mutations are of transient nature only. In fact, longitudinal studies of CA repeats on chromosomes 5 (D5S346) and 17 (D17S250) show reversions of mutations over time.57 In our model, PMNs increased the number of replication errors in HCT116+chr3 A3.1 and A3.7 cells (fig 6). However, the observed mutations were more of a transient nature. They are a fingerprint of a PMN-induced increase in replication errors, which have initiated the hMSH2-dependent activation of the G2/M checkpoint. Thus, at its current stage our model reflects the ability of epithelial cells to recover from or Gut 2008;57:780–787. doi:10.1136/gut.2007.141556