Hereditary Genetics: Current Research

Valproic acid effects on DNA methylation during the cell cycle of HeLa cells

4^th International Congress on Epigenetics & Chromatin

September 03-05, 2018 | London, UK

Marina Amorim Rocha, Veronezi G M B, Gatti M S V and Mello M L S

University of Campinas, Brazil

Posters & Accepted Abstracts: Hereditary Genet Curr Res

Abstract:

Valproic acid (VPA), an anticonvulsant drug that is a histone deacetylase inhibitor, also affects DNA methylation levels. In HeLa cells, the VPA - induced DNA demethylation process involving modified cytosine is a matter of discussion, if by an active pathway, with the participation of ten-eleven-translocation (TET) enzymes, or by a passive DNA replication-dependent pathway. Here, this process was investigated throughout the cell cycle of synchronized and non-synchronized HeLa cells treated with 1 mM and 20 mM VPA for 4 h and compared to the cell cycle of cells treated with 5-aza-CdR, an agent generally used as a passive DNA demethylation control. The methodology used involved flow cytometry, immunofluorescence, ELISA assay and real-time quantitative PCR. The drugs used did not induce cytotoxicity but affected the cell cycle progression. VPA was found to induce over expression of TET1 and TET2 genes, decrease in 5mC abundance and increase in 5hmC abundance, either in G1 cells or in proliferative cells. These findings indicate an active pathway performed by VPA in the DNA demethylation process. However, because VPA also affected the DNMT1 expression, it may also act in the passive DNA demethylation pathway. Although 5-aza-CdR reduced the expression of the DNMT1 gene, depending on cell proliferation and without affecting the TET1 and TET2 gene expression at any stage of the cell cycle, it induced an increase in 5hmC abundance. 5-aza-CdR may thus also act on the active DNA demethylation pathway, because VPA induces reduction in the DNA methylation patterns of nonreplicating HeLa cells, a significant potential implication of its use may arise for a therapeutic reversal of DNA methylation in tissues where no further involvement of the cell division process occurs. ls Recent Publications: 1. Veronezi G M B, Felisbino M B, Gatti M S V, Mello M L S and Vidal B C (2017) DNA methylation changes in valproic acid-treated HeLa cells as assessed by image analysis, immunofluorescence and vibrational microspectroscopy. PLoS One 12(1):e0170740. 2. Han B R, You B R and Park W H (2013) Valproic acid inhibits the growth of HeLa cervical cancer cells via caspasedependent apoptosis. Oncology Reports 30(6):2999-3005. 3. Detich N, Bovenzi V and Szyf M (2003) Valproate induces replication-independent active DNA demethylation. Journal of Biological Chemistry 278(30):27586-27592. 4. Desjobert C, E L Maï M, Gérard-Hirne T, Guianvarc'h D, Carrier A, Pottier C, Arimondo P B and Riond J (2015) Combined analysis of DNA methylation and cell cycle in cancer cells. Epigenetics 10(1):82-91. 5. Sajadian S O, Ehnert E, Vakilian H, Koutsouraki E, Damm G, Seehofer D, Thasler W, Dooley S, Baharvand, Sipos B and Nussler A K (2015) Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma. Clinical Epigenetics 7(98):1-14.

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