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Keywords:

  • hydrogen peroxide-associated colitis;
  • DNA-damage checkpoints;
  • non-apoptotic caspase function;
  • JNK-dependent cell cycle arrests;
  • inflammation;
  • neoplastic transformation;
  • ATM degradation;
  • γ-H2AX

Abstract

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)-associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non-tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H2O2). A population of HCEC survived H2O2-induced oxidative stress via JNK-dependent cell cycle arrests. Caspases, p21WAF1 and γ-H2AX were identified as JNK-regulated proteins. Up-regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan-caspase inhibitor Z-VAD-FMK caused up-regulation of γ-H2AX, a DNA-damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G1-phase as first response to oxidative stress and increased S-phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non-apoptotic function by promoting cells through G1- and S-phase by overriding the G1/S- and intra-S checkpoints despite DNA-damage. This led to the accumulation of cells in the G2/M-phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ-H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase-dependent proteolytic degradation of the DNA-damage checkpoint protein ATM that is upstream of γ-H2AX. As a consequence, undetected DNA-damage and increased proliferation were found in repeatedly H2O2-exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non-apoptotic function of caspases. Overexpression of upstream p-JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.