Agradecimentos: This work was supported by the São Paulo state Research Foundation FAPESP Brazil (Grants No. 2014/23842-0 and 2015/10356-2) and the Brazilian National Council for Research and Development (CNPq) (Grants No. 304668/2014-1 and 421299/2018-5). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. M.A.R received a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) (Finance Code 001). M.L.S.M. received a fellowship from CNPq (grant no. 304668/2014-1). We are thankful to Drs. Shirlei M. Recco-Pimentel, E.R. Pimentel, H.F. Carvalho and J.L. Proença-Módena (Institute of Biology, Unicamp) for equipment facilities and assistance at their laboratories. We also thank Mrs. Camila B.M. de Oliveira for technical assistance in cell culture, and the Obesity and Comorbidities Research Centre (Institute of Biology, Unicamp) for availability of the chemiluminescence imaging equipment Ver menos

Abstract: Sodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phase-arrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxymethylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested Ver menos