Detection of telomerase activity in cultured cells and tumor tissue of lung carcinoma by modified telomeric repeat amplification protocol.
Telomerase activity is found in various cell types including stem cells, neoplastic cells, and immortalized cells, suggesting a close association with their proliferation capacity. The telomeric repeat amplification protocol (TRAP) has been traditionally used to detect semi-quantitatively the telomerase activity by polyacrylamide gel electrophoresis (PAGE), which is difficult to apply for large scale analysis because of laborious post-PCR manipulation and potential carryover contamination. In the present study, a specific reverse primer was designed and the TRAP protocol was adapted to either PAGE or real-time PCR assay. Using cultured cell lines, the real-time TRAP showed a dramatic improvement in the reliability and accuracy of quantitation of telomerase activity and was able to discriminate the A549 cells from hundreds-fold human embryonic lung cells. Using clinical samples of 60 lung cancers and 8 inflammatory lesions, the real-time TRAP was also superior in quantitation, high-throughput capability and standardization. Our modified real-time TRAP should be applicable for the detection of telomerase activity for the initial screening and progression monitoring of lung cancer patients. Our approach is particularly useful when only limited clinical specimen is available, such as fine needle aspiration or other cytological specimens that may contain only a small number of tumor cells.
Department of Pathology, Peking University Health Science Center, Beijing, China.
Yan
Y
Liu
Bing-quan
BQ
Wu
Hao-hao
HH
Zhong
Mei-lin
ML
Xu
Wei-gang
WG
Fang
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology*; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics
D019098:Telomerase