Abstract A17: Colitis-associated tumorigenesis in the Rag2 -/- Helicobacter hepaticus-infected mouse features DNA damage and Retinoblastoma-mediated cell proliferation via nitric oxide but not hypochlorous acid

Colitis-associated cancer (CAC) is characterized by persistent exposure of colonic epithelial cells to inflammatory infiltrates-released free radicals including nitric oxide (NO) and hypochlorous acid (HOCl). These molecules are potential for DNA damage and meanwhile, rapid epithelial tissue renewal also occurs during inflammation, possibly accelerating DNA synthesis and/or limiting the time for DNA repair. In order to identify and elucidate this possible concurring event, the present study was designed to 1.) Quantitatively examine the presence of DNA damage in the proliferating epithelial cells in colitis; 2.) Investigate cell cycle regulation and screen for the highly responsible signaling pathway(s) to explain the increased proliferation; 3.) And differentiate the individual effect of NO and HOCl on DNA damage and cell proliferation. Experiments used 129/SvEv Rag2 −/− mouse, either infected with Helicobacter hepaticus (H. h .) (10-20 weeks) or uninfected as control. NO production was inhibited by addition of N-monomethyl arginine (NMA, an inhibitor of nitric oxide synthase) to the mice drinking water and HOCl production was inhibited by intraperitoneal injection of Gr-1 IgG that neutralizes neutrophils. Colon samples from these mouse experiments were analyzed for DNA damage and cell cycle markers using immunostaining and morphometric methods. The present study found that 1.) Colitis induced by H. h . exhibited a high amount of γH2AX (DNA damage marker) positive cells (~10% significantly increased versus control ~4%) and these cells co-localize with the proliferation marker Ki67 (~55%, significant increased versus control ~23%) in the colonic epithelial crypts. TUNEL assay confirmed that these cells are unlikely apoptotic as the predominant TUNEL positive cells were found in the mesenchyme, but not the epithelia. 2.) For cell cycle regulation, increased expression of cyclin E, but not cyclin D, was observed in the H. h . infection. Concordantly, phosphorylation of Retinoblastoma (RB) gene was found in a high level at the base of the proliferating crypts. Examination of the other proliferation signal transducers including β-catenin, smad4 and HP1γ showed no positive observations. Activation of p38 and ERK was found increased and yet localized in the luminal surface of the colonic crypts. 3.) Importantly, during the H. h . infection when NO production was inhibited by NMA, dramatic decreases of γH2AX (~3%) and Ki67 (~17%) expression levels were observed and NMA treatment also resulted in largely decreased RB phosphorylation. In comparison, inhibition of HOCl by neutrophil Gr1 antibody showed only a small reduction of γH2AX (~8.7%, marginally significant) expression and no changes in Ki67 (~49%) and RB immunostains. In summary, the present study found that the H. h .-induced colitis features co-localized γH2AX and Ki67 positive epithelial cells and mechanistic analysis suggested an essential role of nitric oxide, but not hypochlorous acid, affecting both DNA damage and RB phosphorylation-mediated cell proliferation signaling pathway, with the later possibly releasing high level of cyclin E that facilitates G1/S cell cycle entry. These results enlighten a previously underestimated mechanism for the tumorigenesis of CAC and based on this mechanistic plateau, more detailed investigations are carrying out in the near future. Citation Format: Guanyu Gong, Sureshkumar Muthupalani, Gerald N. Wogan, James G. Fox, Steven R. Tannenbaum. Colitis-associated tumorigenesis in the Rag2 -/- Helicobacter hepaticus-infected mouse features DNA damage and Retinoblastoma-mediated cell proliferation via nitric oxide but not hypochlorous acid. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A17.