Colorectal cancer Medical University of Vienna, Department of Internal Medicine III, Division of Gastroenterology and Hepatology and Christian Doppler Laboratory on Molecular Cancer Chemoprevention, Vienna, Austria Correspondence to: Professor Christoph Gasche, AKH Wien, Division of Gastroenterology and Hepatology, W¨ahringer G¨urtel 18, A-1090 Vienna, Austria; christoph.gasche@meduniwien. ac.at Revised 8 January 2008 Accepted 2 February 2008 Published Online First 13 February 2008 This paper is freely available online under the BMJ Journals unlocked scheme, see http:// gut.bmj.com/info/unlocked.dtl 780 Activated neutrophils induce an hMSH2-dependent G2/M checkpoint arrest and replication errors at a (CA)13-repeat in colon epithelial cells C Campregher, M G Luciani, C Gasche ABSTRACT Objective: Chronic inflammation in ulcerative colitis is associated with increased risk for colorectal cancer. Its molecular pathway of cancer development is poorly understood. We investigated the role of neutrophil-derived cellular stress in an in vitro model of neutrophils as effectors and colon epithelial cells as targets, and tested for changes in cell cycle distribution and the appearance of replication errors. Design: Colon epithelial cells with different mismatch repair phenotypes were co-cultured with activated neutrophils. Target cells were analysed for cell cycle distribution and replication errors by flow cytometry. Changes in nuclear and DNA-bound levels of mismatch repair- and checkpoint-related proteins were analysed by western blotting. Results: Activated neutrophils cause an accumulation of target cells in G2/M, consistent with the installation of a DNA-damage checkpoint. Cells that do not express hMSH2, p53 or p21waf1/cip1 failed to undergo the G2/M arrest. Phosphorylation of p53 at site Ser15 and Chk1 at Ser317, as well as accumulation of p21waf1/cip1, was observed within 8–24 h. Superoxide dismutase and catalase were unable to overcome this G2/M arrest, possibly indicating that neutrophil products other than superoxide or H2O2 are involved in this cellular response. Finally, exposure to activated neutrophils increased the number of replication errors. Conclusions: By using an in vitro co-culture model that mimics intestinal inflammation in ulcerative colitis, we provide molecular evidence for an hMSH2-dependent G2/ M checkpoint arrest and for the presence of replication errors. Chronic inflammation leads to tumour develop- ment.1 Ulcerative colitis is associated with an increased risk of development of colorectal carci- noma (CRC). One of the key features of ulcerative colitis is the presence of crypt abscesses, which are accumulations of polymorphonuclear cells (PMNs) within colonic crypts.2 3 It has been suggested that reactive oxygen species (ROS) released by PMNs are one of the main contributing factors to colon carcinogenesis.1 Oxidative stress can alter cellular components including proteins, mRNAs and DNA.4–6 It is unclear, however, whether oxidative stress on its own may cause mutations in cells.7 8 Activated PMNs not only produce ROS, but also excrete lactoferrin9 and other proteins including several cytokines.10 11 Thus, previous in vitro studies that focused on H2O2-induced mutagen- esis8 12 only partially reflected the pathophysiolo- gical condition of colon carcinogenesis. The mismatch repair (MMR) system plays a central role in promoting genetic stability by correcting DNA replication errors. Homologs of the bacterial MutS and MutL MMR proteins in eukaryotes form heterodimers with discrete roles in MMR-related processes. The discovery of a link between human cancer and MMR defects has led to an increased interest in eukaryotic MMR.13 Frameshift mutations of short-tandem repetitive sequences indicate instability of these sequences [microsatellite instability (MSI)] and represent a hallmark of MMR deficiency in human cancers.14 15 Since MSI can be detected in colitis tissue without dysplasia, inactivation of the MMR system must be an early event in colon carcinogenesis in ulcerative colitis. However, the nature of inflam- mation-induced microsatellite mutations is still obscure. The MMR system can be activated after replication to repair DNA errors. Evidence sug- gested that the proliferating cell nuclear antigen (PCNA) is required for MMR recruitment prior to DNA repair synthesis,16 leading to the hypothesis that replication and MMR may be coupled and that the replication fork provides the strand discrimination signal for repair.17 Exposure of eukaryotic cells to agents that alter the DNA structure results in transient arrest of the progression through the cell cycle. Ataxia telangiec- tasia mutated kinase (ATM) acts as a sensor of oxidative damage, coordinating stress responses with cell cycle checkpoint control and repair of such damage.18 Cell cycle checkpoints give the cell the opportunity to either mend the DNA damage or undergo apoptosis. In particular, the G2/M check- point allows cells to overcome replication errors before entering mitosis, thereby ensuring genomic integrity. Apart from ATM, key components of the G2/M cell cycle checkpoint include the ATM-and- Rad3-related kinase (ATR), the downstream check- point kinases Chk1 and Chk219 20 and the tumour suppressor protein p53,21 which is stabilised by phosphorylation at ATM and ATR sites.22 23 Phosphorylation of p53 correlates with enhanced transcription of the cyclin-dependent kinase inhibi- tor p21waf1/cip1.24 25 DNA-alkylating agents induce phosphorylation and activation of p53, leading to an increased expression of p21waf1/cip1. Cell lines with MMR deficiency are resistant to these alkylating agents and bypass the cell cycle arrest, indicating that the MMR has a role in post-replication checkpoints.26 27 However, nitric oxide (NO) and H2O2 are capable of arresting hMLH1 mutant cells in G2/M.4 28 No information exists on the role of hMSH2 in mediating such a cell cycle arrest. Gut 2008;57:780–787. doi:10.1136/gut.2007.141556