Inhibition ofDNAmethyltransferase leads to increased genomic 5‐hydroxymethylcytosine levels in hematopoietic cells

Abstract

5‐Hydroxymethylcytosine (5hmC) is produced from 5‐methylcytosine (5mC) by Ten‐eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC‐MS/MS‐based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5‐aza‐2′‐deoxycytidine without significantly influencing 5mC levels.

Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers

“…Furthermore, we demonstrate that losses of 5mC within the SPRY2 promoter inversely correlate with increased promoter 5hmC. Indeed, the phenomenon of global epigenetic dysregulation, linking decreased levels of 5mC with increased levels of 5hmC, has been observed in pontine gliomas and hematopoietic cells [44,45]. In other cases, global losses of 5hmC have been linked to increases in intragenic 5mC and subsequently decreased expression of tumor suppressor genes as reported in kidney cancer [46].…”

High-Dose Vitamin C: Preclinical Evidence for Tailoring Treatment in Cancer Patients

“…For combinations therapies, clinical studies should be planned taking into account the different molecular mechanisms involved in vitamin C antitumor activity that can be observed even at lower concentrations, such as induction of DNA demethylation [ 167 , 195 , 196 ]. In particular, the conversion of 5mC to 5hmC deriving from vitamin C-mediated activation of TETs may synergize with passive DNA demethylation (i.e., absence of methylation of newly synthesized DNA strands) obtained by azacytidine or decitabine that are inhibitors of DNMT1 [ 197 , 198 , 199 ].…”

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

“…In this study, we found that Aza could also increase transgene expression levels, stability, and the retention of transgene regardless of selection pressures, which are consistent with the previous studies . As a cytidine analogue, Aza can reduce the methylation level by covalently binding with DNA methyltransferase to decrease the biological activity of DNA methyltransferase . In the previous study, cells were treated by a daily dose of 4 μM of Aza for 3 days and lost productivity was partially restored .…”

“…19 As a cytidine analogue, Aza can reduce the methylation level by covalently binding with DNA methyltransferase to decrease the biological activity of DNA methyltransferase. 34,35 In the previous study, cells were treated by a daily dose of 4 μM of Aza for 3 days and lost productivity was partially restored. 19 However, in our present study, the cytotoxicity of 4 µM Aza concentration is too high to kill the most of the CHO cells, 1 μM of Aza had no significant influence on cell density and viability of stably transfected CHO cells cultured for 60 generations (data not shown).…”