title: Targeted TET oxidase activity through methyl-CpG-binding domain extensively suppresses cancer cell proliferation.
abstract: DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl-CpG-binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD-TET1-CDwt were also reactivated after treatment with a DNMT inhibitor, 5-azacytidine (Aza-CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD-TET1-CDwt in a prostate cancer cell line, LNCaP. The induction of MBD-TET1-CDwt demethylated and upregulated glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.
authors.affiliation: Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Mizuguchi
authors.affiliation: Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Saiki
authors.affiliation: Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Horii
authors.affiliation: Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan. fukushige@med.tohoku.ac.jp.|Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi, Japan. fukushige@med.tohoku.ac.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Fukushige
mesh_terms: D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D051549:Glutathione S-Transferase pi / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000070593:Methyl CpG Binding Domain / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D024363:Transcription Initiation Site
chemical_list: D006899:Mixed Function Oxygenases; D051549:Glutathione S-Transferase pi
keywords: DNA demethylation; DNA methyltransferase inhibitor; hypermethylated genes; methyl-CpG-binding domain; ten- eleven translocation proteins