title: Combined analysis of DNA methylation and cell cycle in cancer cells.
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.
authors.affiliation: a CNRS - Pierre Fabre USR 3388 ETaC; CRDPF BP12562 ; Toulouse , France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Desjobert
authors.forename: Mounir
authors.initials: M
authors.lastname: El Maï
authors.forename: Tom
authors.initials: T
authors.lastname: Gérard-Hirne
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
authors.forename: Arnaud
authors.initials: A
authors.lastname: Carrier
authors.forename: Cyrielle
authors.initials: C
authors.lastname: Pottier
authors.forename: Paola B
authors.initials: PB
authors.lastname: Arimondo
authors.forename: Joëlle
authors.initials: J
authors.lastname: Riond
mesh_terms: D002453:Cell Cycle; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans
chemical_list: D004273:DNA, Neoplasm
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