[Methylation quantification of adenomatous polyposis coli (APC) gene promoter in plasma of lung cancer patients].
OBJECTIVE
The protein encoded by adenomatous polyposis coli (APC) gene participates in the signaling transduction pathway. Substantial studies have revealed that hypermethylation of APC gene promoter is closely related to the pathogenesis and development of cancer. This study was to develop a real-time quantitative methylation specific PCR (real-time QMSP) method, and detect the methylation of APC gene promoter in plasma of lung cancer patients.


METHODS
Genomic DNA with methylated APC gene promoter was extracted from the lung cancer cell line NCI-H460 using phenol-chloroform and quantified by spectrophotometric measurements. DNA was added into 200 microL plasma samples of healthy volunteers to make 10-fold serial dilutions. Circulating DNA from simulated plasma samples, 78 lung cancer patients, 31 patients with benign lung diseases and 23 health controls was extracted using magnetic beads and modified by bisulfite. The concentration of cell-free methylated APC gene promoter in the plasma samples was quantified by the external reference method with the standard curve constructed using simulated plasma.


RESULTS
The linear range of the real-time QMSP assay was 1.5x10(2)-1.5x10(5) copies/ mL and its lowest detectability was 1.5x10(2) copies per milliliter plasma. Of 78 lung cancer patients, positive methylation of the APC gene promoter was detected in tumor tissues of 40 cases. Among the 40 lung cancer patients, positive methylation of the APC gene promoter was found in the plasma of 19 patients (47.5%). The concentrations of methylated APC promoter in the 19 lung cancer patients ranged from 1.67x10(2) to 6.78x10(3) copies/mL, with a median concentration of 1.67x10(3) copies/mL. No positive methylation of the APC gene promoter was detected in the plasma of 38 lung cancer patients without APC gene methylation in tissues, 31 benign lung diseases and 23 healthy controls.


CONCLUSIONS
The newly developed real-time QMSP method allows the quantitative measurement of APC gene promoter methylation in plasma. Hypermethylation of the APC gene promoter in plasma is a potential diagnostic marker for lung cancer diagnosis.
Pan
Shi-Yang
SY
Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, PR China. sypan@njmu.edu.cn
Xie
Er-Fu
EF
Shu
Yong-Qian
YQ
Gao
Li
L
Zhang
Li-Xia
LX
Chen
Dan
D
Chen
Jin-Bu
JB
Zhao
Wen-Jun
WJ
Mu
Yuan
Y
Zhang
Ji-Nan
JN
D000230:Adenocarcinoma / Q000097:blood / Q000235:genetics; D025601:Adenomatous Polyposis Coli Protein / Q000097:blood / Q000235:genetics*; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell / Q000097:blood / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D017491:Genes, APC; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic
D025601:Adenomatous Polyposis Coli Protein; D004273:DNA, Neoplasm