No 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.

The production of hydrogen peroxide (H2O2) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H2O2 activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c-Jun N-terminal Kinases (JNK) that induces p21WAF1. Moreover, caspases circumvented the G1/S and intra-S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H2O2 exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H2O2-exposed HCEC cells (C-cell cultures) was associated with (i) increased phospho-p46 JNK, (ii) decreased total JNK and phospho-p54 JNK and (iii) p21WAF1 down-regulation. Altered JNK activation and p21WAF1 down-regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H2O2 exposure. This may cause increased proliferation of C-cell cultures, a defining initiating feature in the inflammation-carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non-apoptotic function of caspases, causing checkpoint adaptation in C-cell cultures. Additionally, loss of cell-contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell-contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H2O2-induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21WAF1, c-Fos, c-Jun/phospho-c-Jun, ATF2/phospho-ATF2, β-catenin/TCF4-signalling, c-Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.

We recently reported that dysregulated c-Jun N-terminal kinases (JNK) activity causes defective cell cycle checkpoint control, inducing neoplastic transformation in a cellular ulcerative colitis (UC) model. In the quiescent chronic phase of UC, p-p54 JNK was down-regulated and p-p46 JNK was up-regulated. Both were up-regulated in the acute phase. Consequently, increased p21WAF1 and γ-H2AX, two JNK-regulated proteins, induced cell cycle arrest. Their down-regulation led to checkpoint override, causing increased proliferation and undetected DNA damage in quiescent chronic phase, all characteristics of tumorigenesis. We investigated expression of p-JNK2, p-JNK1-3, p21WAF1, γ-H2AX and Ki67 by immunohistochemistry in cases of quiescent UC (QUC), active UC (AUC), UC-dysplasia and UC-related colorectal carcinoma (UC-CRC). Comparison was made to normal healthy colorectal mucosa, sporadic adenoma and colorectal carcinoma (CRC), diverticulitis and Crohns disease (CD). We found p-JNK2 up-regulation in AUC and its early down-regulation in UC-CRC and CRC carcinogenesis. With down-regulated p-JNK2, p21WAF1 was also decreased. Ki67 was inversely expressed, showing increased proliferation early in UC-CRC and CRC carcinogenesis. p-JNK1-3 was increased in AUC and QUC. Less increased γ-H2AX in UC-CRC compared to CRC gave evidence that colitis-triggered inflammation masks DNA damage, thus contributing to neoplastic transformation. We hypothesize that JNK-dependent cell cycle arrest is important in AUC, while chronic inflammation causes dysregulated JNK activity in quiescent phase that may contribute to checkpoint override, promoting UC carcinogenesis. We suggest restoring p-JNK2 expression as a novel therapeutic strategy to early prevent the development of UC-related cancer.

No abstract