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| Author(s): |
Giovana Maria Breda Veronezi
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2017-02-24 |
| Examining board members: |
Maria Luiza Silveira Mello;
Alberto da Silva Moraes;
Daniel Pacheco Bruschi
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| Advisor: | Maria Luiza Silveira Mello; Benedicto de Campos Vidal |
| Abstract | |
Valproic acid (VPA) is a well-established antiepileptic drug and also an inhibitor of histone deacetylases (HDAC), leading to hyperacetylation of histones H3 and H4. Moreover, VPA has been suggested to induce DNA demethylation in several cell types, possibly through an active and replication-independent way. In the present work, the effects of VPA over the 5-methylcytosine (5mC) abundance and active DNA demethylation elements were studied in HeLa cells, compared to 5-aza-2¿-deoxycytidine (5-aza-CdR), a known DNA demethylator. In addition, the contribution of the VPA-induced DNA demethylation on the chromatin remodeling, previously attributed to HDAC inhibition, was investigated in this cell type. The effect of VPA on DNA 5mC marks was also studied analyzing DNA infrared spectral profiles. In both VPA- and 5-aza-CdR treated cells, immunocytochemistry and ELISA techniques were used to evaluate DNA methylation levels. The cytosine derivatives 5-hydroxymethylcytosine (5hmC), 5-carboxylcytosine (5caC) and 5-formylcytosine (5-fC) were investigated by immunocytochemistry. The gene expression of DNA methyltransferase 1 (DNMT1) and of enzymes of the ten-eleven translocation family (TET), TET1 and TET2 was assessed by quantitative real time PCR, and protein levels of DNMT1 and TET2 were obtained using Western blotting. Scanning microspectrophotometer assays allowed the analysis of changes in chromatin supraorganization of HeLa cells treated with VPA and spectral profiles of DNA were obtained by Fourier-transform infrared (FT-IR) microspectroscopy. Induction of DNA hypomethylation was found to be induced by both VPA and 5-aza-CdR treatments. The reduction of 5mC levels concomitant to a chromatin decondensation status induced by VPA in these cells persisted for 24 h in the absence of the drug, although not for 48 h, demonstrating a role of DNA methylation on chromatin remodeling events, with long-standing effects. The demethylation observed in response to VPA also influenced the DNA infrared spectral profiles, affecting more significantly the peaks related to symmetric and antisymmetric stretching vibrations of the methyl group from 5mC. No changes in 5caC and 5fC were observed in VPA- or 5-aza-CdR treatments, although both drugs increased 5hmC levels and differentially affected the enzymes studied here (DNMT1, TET1 and TET2). Thereby, the influence of VPA and 5-aza-CdR on active DNA demethylation pathways was reinforced and it was demonstrated that these drugs act by different mechanisms. Taken these results together, more information regarding the effects of VPA on DNA methylation in HeLa cells was obtained, allowing further comparisons between the effects of this drug on other tumor cell lines as aggressive as HeLa cells. Furthermore, a parallel was established between the epigenetic action of VPA and that of another compound, 5-aza-CdR, thus contributing with additional and new data on the mechanisms of action of these widely used drugs (AU) | |
| FAPESP's process: | 14/23842-0 - DNA methylation in HeLa cells after treatment with valproic acid |
| Grantee: | Giovana Maria Breda Veronezi |
| Support Opportunities: | Scholarships in Brazil - Master |