The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells.

Basudev Chowdhury,Joseph Irudayaraj,Bruce Cooper,Timothy Chevassut,Il-Hoon Cho,Amy C Lossie,Andrew Mcgovern,Samrat Roy Choudhury,Yi Cui

doi:10.1038/srep09281

Abstract

The USFDA approved “epigenetic drug”, Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC.

Highlights

The USFDA approved ‘‘epigenetic drug’’, Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology
Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels
We sought to determine the changes in the patterns of methyl-CpG-binding domain proteins (MBDs) in response to DAC, which led us to the unexpected observation that the 5hmC content in HL-60 cells increased in response to DAC

Summary

Introduction

The USFDA approved ‘‘epigenetic drug’’, Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. DNMT3A and 3B are traditionally recognized for their role as de novo DNA methyltransferases during early development and differentiation, recently it has been proposed that DNMT3A and 3B may be involved in the DNMT1-mediated methylation process[19]. DNMT1 recognizes these Decitabine-Guanine dinucleotides as a natural substrate and initiates a methyltransferase reaction, but is trapped in the process[23]. This leads to depletion of DNMT1 from the cell, loss of maintenance methylation and ‘passive demethylation’ of genomic DNA following cell division[16,17,18]. TET proteins have been demonstrated to be capable of catalyzing both hemi-methylated and fully-methylated CpGs10, it is not clear if the TET proteins display a selective preference for hemimethylated or fully-methylated CpG dinucleotides in cells

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