[Cloning of human telomerase catalytic subunit gene promoter and studying on its specific transcriptional activities in human lung cancer cells].
BACKGROUND
To clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell.


METHODS
hTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.


RESULTS
Electrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line.


CONCLUSIONS
The hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.
Key Laboratory of Lung Cancer Molecular Biology of Sichuan Province, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R.China.
Xiaojun
X
Tang
Yanping
Y
Wang
Qinghua
Q
Zhou
Wen
W
Zhu
Guowei
G
Che
Xiaohe
X
Chen
Daxing
D
Zhu
Zhilin
Z
Sun