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Epigenetics. 2015;10(1):82-91. doi: 10.1080/15592294.2014.995542. Epub 2015 Jan 23.

Combined analysis of DNA methylation and cell cycle in cancer cells.

Author information

1
a CNRS - Pierre Fabre USR 3388 ETaC; CRDPF BP12562 ; Toulouse , France.

Abstract

DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.

KEYWORDS:

5-azadeoxycytidine; 5AzadC, 5-aza-2′-deoxycytidine; 5mC, 5-methylcytosine; AML, acute myeloid leukemia; CGI, CpG island; DNA methylation; DNMT, DNA methyltransferase; FACS, fluorescence-activated cell sorting; LC-ESI MS/MS, liquid chromatography–electrospray ionization tandem mass spectrometry; MDS, myelodysplastic syndromes; cancer; cell cycle; flow cytometry

PMID:
25531272
PMCID:
PMC4622670
DOI:
10.1080/15592294.2014.995542
[Indexed for MEDLINE]
Free PMC Article

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