A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress

Abstract

The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H2O2−generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2−generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.