Repeated H₂O₂ exposure drives cell cycle progression in an in vitro model of ulcerative colitis

Abstract

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.

Chk1 Promotes DNA Damage Response Bypass following Oxidative Stress in a Model of Hydrogen Peroxide‐Associated Ulcerative Colitis through JNK Inactivation and Chromatin Binding

“…We suggest that the previously found H 2 O 2 -induced ROS generation in C-cell cultures is responsible for this epigenetically regulated signal transduction [5]. Thus, activated Chk1 is not released from chromatin, and H3 T11 phosphorylation is maintained despite the presence of DNA damage, and this process triggers DNA damage response bypass and features of oncogenic transformation, such as increased proliferation and accumulated DNA damage.…”

“…In the experimental model, JNK was activated in the acute phase, while upregulation of p-p46 JNK (C1-C10) and downregulation of p-p54 JNK (C3-C10) were observed in C-cell cultures [4, 5]. Based on the known role of Chk1 as a key regulator in DNA damage response and cell cycle progression, we analyzed its expression and activation in the acute and quiescent chronic phases of our UC cellular model.…”

“…In our recent publication, we reported that G2/M-arrested C10 cells entered mitosis and re-entered the cell cycle in the G1 phase instead of going into apoptosis because of an adapted G2/M and mitotic spindle checkpoint [5]. Cell cycle analysis following H 2 O 2 treatment of Chk1 siRNA-transfected C10 cells showed more cells in the G1 phase after 24 and 72 hrs and fewer cells in G2/M phase after 24 h (Figure 5(b)).…”

JNK1 Deficient Insulin-Producing Cells Are Protected against Interleukin-1β-Induced Apoptosis Associated with Abrogated Myc Expression

“…We believe that this downregulation of JNK1 subtype is not mediated by proteases after cell lysis since we use a broad protease inhibitor cocktail in our lysis buffer. The reduced JNK1 subtype expression may though be a side effect of apoptosis due to caspase 3-mediated cleavage; however, in other stress-induced apoptotic cell lines, JNK protein expression is shown to be caspase-independent [ 37 , 38 ]. Gene expression profiling studies followed by functional characterization of candidate genes have demonstrated that the exposure of insulin-producing cell lines, fluorescence-activated cell-sorted primary rodent β -cells, intact rodent islets, and human islets to proinflammatory cytokines induces not only upregulation of proapoptotic/downregulation of antiapoptotic genes, but also a protective response including downregulation of proapoptotic/upregulation of antiapoptotic genes, with a high degree of concordance between the different model systems [ 39 , 40 ].…”

Role of oxidative stress in facilitating carcinogenesis

“…Similarly, continuous expression of H2O2 in the ulcerative colitis dysregulates cell cycle checkpoints and activates the cell cycle progression and drives cells into tumorigenesis. 32 It is interesting to note that ROS can facilitate post translational modification (phosphorylation) of proteins like MAPK, receptor tyrosine kinase and protein kinase B and etc. and facilitate cell cycle progression.…”