title: DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells.
abstract: Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.
authors.affiliation: Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Center Meidling; First Medical Department, Hanusch Hospital, Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Thaler
authors.forename: Silvia
authors.initials: S
authors.lastname: Spitzer
authors.forename: Heidrun
authors.initials: H
authors.lastname: Karlic
authors.forename: Klaus
authors.initials: K
authors.lastname: Klaushofer
authors.forename: Franz
authors.initials: F
authors.lastname: Varga
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D000097584:DNA Methyltransferase 3B; D000097642:GADD45 Proteins
chemical_list: D044503:5-Methylcytosine; D018797:Cell Cycle Proteins; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004121:Dimethyl Sulfoxide; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D000097584:DNA Methyltransferase 3B; C501120:Dlx5 protein, mouse; C485852:Dmap1 protein, mouse; C499072:Gadd45a protein, mouse; D000097642:GADD45 Proteins; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine; C487055:Hells protein, mouse