Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry.
Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient's clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2'-deoxycytidine (5mdC) to 2'-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.
Liu
Zhongfa
Z
College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Wu
Jiejun
J
Xie
Zhiliang
Z
Liu
Shujun
S
Fan-Havard
Patty
P
Huang
Tim H-M
TH
Plass
Christoph
C
Marcucci
Guido
G
Chan
Kenneth K
KK
D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D000077209:Decitabine; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D006801:Humans; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003841:Deoxycytidine; D000077209:Decitabine; D004247:DNA; C016569:5-methyldeoxycytidine; D001374:Azacitidine