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Activated Neutrophils Induce an hMSH2-dependent G2/M Checkpoint Arrest and Replication Errors at a (CA)13-repeat in Colon Epithelial Cells
  1. Christoph Campregher (christoph.campregher{at}meduniwien.ac.at)
  1. Medical University Vienna, Austria
    1. M. Gloria Luciani (maria.luciani{at}meduniwien.ac.at)
    1. Medical University Vienna, Austria
      1. Christoph Gasche (christoph.gasche{at}meduniwien.ac.at)
      1. Medical University Vienna, Austria

        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 analyzed 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 analyzed by western blot.

        Results: Activated neutrophils cause an accumulation of target cells in G2/M, consistent with an install 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 hours. 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.

        • cancer
        • inflammation
        • microsatellite instability
        • neutrophils
        • ulcerative colitis

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