High-performance capillary electrophoretic method for the quantification of global DNA methylation: application to methotrexate-resistant cells.
Global DNA hypomethylation in tumor tissue is a common characteristic in a variety of malignancies such as breast, colon, oral, lung, and blood cancers. A rapid and sensitive method has been developed for the determination of global DNA methylation in cells. Five substances--2'-deoxycytidine (dC), 5-methyl 2'-deoxycytidine (mdC), 2'-deoxyadenosine (dA), 2'-deoxythymidine (dT), and 2'-deoxyguanosine (dG)--were completely separated by high-performance capillary electrophoresis in 10 min. Intraday coefficient of variation was less than 1%, and interday coefficient of variation was less than 2%. The minimal detection limit was 1 microM. Acquired drug resistance to methotrexate (MTX) is one of the most serious problems in cancer chemotherapy. Under optimal conditions, we analyzed global DNA methylation levels in A549 and A549/MTX cells, and only 10(5) cells are needed to obtain reliable results. The percentage of 5-methyl-2'-deoxycytidine (5-mC) was 4.80+/-0.52% in A549 cells, and this decreased to 4.20+/-0.44% in A549/MTX cells. It was considered as statistically significant. This demonstrated that the mechanisms of acquired drug resistance to MTX might be concerned with DNA methylation.
Li
Ming
M
Anhui Provincial Center for Clinical Laboratories, Anhui Provincial Hospital Affilated to Anhui Medical University, Hefei, China.
Hu
Shi-lian
SL
Shen
Zuo-jun
ZJ
He
Xiao-dong
XD
Tao
Shao-neng
SN
Dong
Lin
L
Zhu
Yuan-yuan
YY
D019175:DNA Methylation; D003839:Deoxyadenosines / Q000302:isolation & purification; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000302:isolation & purification; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D015203:Reproducibility of Results; D013936:Thymidine / Q000302:isolation & purification
D003839:Deoxyadenosines; D003841:Deoxycytidine; C016569:5-methyldeoxycytidine; D003849:Deoxyguanosine; C058118:2'-deoxyadenosine; D013936:Thymidine; D008727:Methotrexate