Abstract 2543: A dual-fluorescent reporter system facilitates identification of thiol compounds that suppress oxidative frameshift mutations

Abstract

Abstract DNA mismatch repair (MMR) system corrects replicative errors, especially in microsatellites, and minimizes mutations caused by DNA-damaging agents. Patients with chronic and cancer-associated inflammation display microsatellite instability (MSI), indicative of an inactivated MMR system. MMR-deficient patients also display frameshift mutations in coding microsatellites of many genes, including oncogenes and tumor suppressors. We previously reported that oxidative stress, which occurs in the inflammatory settings, not only inactivates the MMR function but also increases accumulation of frameshift mutations. However, it remains unclear whether oxidative frameshift mutations are preventable due to the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing RFP harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogeneic human colorectal cancer cell lines. By flow cytometry, the new reporter sensitively detected a low level of H2O2-induced frameshift mutations in MMR-proficient cells. Without a functional MMR system, 0.5 mM H2O2 increased frameshift mutations by approximately 10 folds in the cells. In MMR-deficient cells, our reporter revealed that 5 mM glutathione or N-acetylcysteine suppressed oxidative frameshift mutations by approximately 75% or 90% respectively, without affecting cell viability. In contrast, 0.5 mM ascorbic acid augmented H2O2-induced frameshift mutations by two folds, whereas 1 mM aspirin had no effects. Glutathione and N-acetylcysteine also partially suppressed oxidative frameshift mutations in coding microsatellites of endogenous hMSH6 and CHK1 genes, determined by a fluorescinated PCR-based assay. The new utility of these thiol compounds is potentially valuable for preventing oxidative frameshift mutations in the inflammatory settings. The dual-fluorescent reporter system with improved sensitivity will facilitate identification of additional compounds that modulate frameshift mutations relevant to cancer initiation and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2543. doi:1538-7445.AM2012-2543