3 years ago

Identification of PMN-released Mutagenic Factors in a Co-culture Model for Colitis-associated Cancer.

Theresa Scharl, Siegfried Knasmüller, Vineeta Khare, Christoph Gasche, Christoph Campregher, Nicolas Granofszky, Kristine Jimenez, Gerald Schmid, Michaela Lang, Franziska Ferk
Microsatellite instability (MSI) is present in ulcerative colitis and colitis-associated colorectal cancers. Certain factors released by polymorphonuclear cells (PMN) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116+chr3 and HCEC-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to EGFP to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide, as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges hydrogen peroxide, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as IL-8, IL-6, and TNF-α. In vitro, these cytokines increased MSI in colon epithelial cells and the JAK-inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by DCFDA assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by FPG-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

Publisher URL: http://doi.org/10.1093/carcin/bgx118

DOI: 10.1093/carcin/bgx118

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