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Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

World Journal of Gastroenterology 2012年第47期18卷 6935-6942页

作者： Kazuya Shinmura Masanori Goto Hong Tao Shun Matsuura Tomonari Matsuda Haruhiko Sugimura Department of Tumor Pathology Hamamatsu University School of MedicineShizuoka 431-3192Japan Research Center for Environmental Quality Management Kyoto UniversityShiga 520-0811Japan

AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human ***:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this *** H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH *** expression was examined after cumate induction using Western blotting analysis and immunofluorescence *** intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently ***,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation ***:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with *** of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged *** mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of *** mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2

关键词： 8-hydroxyguanine Mutation MUTYH MUTYH-associated polyposis Oxidative mutagenesis supF forward mutation assay piggyBac transposon colorectal polyposis

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