title: Halogenated thymidine analogues restore the expression of silenced genes without demethylation.
abstract: Transcriptional silencing of tumor suppressor genes by aberrant DNA methylation is a characteristic frequently observed in cancer cells. Therefore, reversing this process is a therapeutic target against cancer. In this study, we established a screening system for silencing inhibitors with cell lines transfected by a retroviral vector containing a luciferase gene. More than 100 nucleosides were tested for antisilencing activity with a selected clone in which the silenced expression of luciferase could be recovered by 5-aza-2'-deoxycytidine. A group of halogenated thymidine analogues was found to reactivate transcription of not only the reporter retrovirus vector but also endogenous glutathione-S-transferase 1 gene, without influence to DNA hypermethylation. Gel mobility shift assay showed that 5-bromo-2'-deoxyuridine (BrdUrd) or 5-iodo-2'-deoxyuridine incorporation did not affect the binding of the methyl-CpG binding protein motif to methylated DNA. Finally, in the retroviral promoter, BrdUrd treatment increased the acetylated histone H3 level and decreased methylation of histone H3 Lys9 in accordance with recovered transcription. This study shows that halogenated thymidines have an antisilencing effect without changing DNA methylation status by interfering with step(s) between DNA methylation and histone acetylation.
authors.affiliation: Laboratory of Virus Immunology, Institute for Virus Research, Kyoto University, Kyoto, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
authors.forename: Ei-ichi
authors.initials: E
authors.lastname: Kodama
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Nakao
authors.forename: Masao
authors.initials: M
authors.lastname: Matsuoka
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000187:drug effects / Q000235:genetics; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D006657:Histones / Q000187:drug effects / Q000235:genetics; D006801:Humans; D006846:Hydrocarbons, Halogenated / Q000494:pharmacology; D020014:K562 Cells; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000187:drug effects / Q000235:genetics; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D000964:Antimetabolites, Antineoplastic; D006657:Histones; D006846:Hydrocarbons, Halogenated; D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine