[Study on cloning of hTERT promoter and its targeting transcriptional activities in telomerase-positive lung cancer cells].
OBJECTIVE
To clone sequence of hTERT promoter and study its transcriptional activity and its relationship with hTERT mRNA expression and telomerase activity in various kinds of human lung cancer cells and normal cells, and to investigate the targeting transcriptional activity of hTERT promoter in tumor cells.


METHODS
About 1.1 kb promoter of the 5'flanking sequence of the hTERT was amplified from genomic DNA isolated from 293 cells by polymerase chain reaction (PCR). After being confirmed by DNA sequencing, the hTERT promoter was inserted into luciferase reporter vectors (pGL3-basic) to reconstruct a recombinant named pGL3-hTERTp. Then pGL3-hTERTp was transiently transfected into lung cancer cell A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC-9, GLC-82, 95D, A2, and normal cell of MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities. hTERT mRNA expression and telomerase activity were determined by RT-PCR and TRAP ELISA.


RESULTS
Eelectrophoresis demonstrated that the hTERT promoter amplified by PCR was about 1.1 kb long, and DNA sequencing showed a sequence the same as the hTERT promoter registered in GenBank being 1084 bp in length. The recombinant of plasmid pGL3-hTERTp was confirmed by double digestion and PCR methods with correct results. hTERT mRNA and telomerase activity were expressed in all of eight lung cancer cell lines at varied levels, but not expressed in normal cell. Transient transfection assay and Luciferase assay also revealed that hTERT promoter had different transcriptional activities in various lung cancer cells, but no transcriptional activity was shown in normal cells.


CONCLUSIONS
1084 bp hTERT promoter cloned has specific transcriptional activities in various telomerase-positive lung cancer cells, and it may act as control element in tumor-targeting gene therapy.
Wang
Yan-ping
YP
Laboratory of Lung Cancer Molecular Biology, West China Hospital, Sichuan University, Chengdu 610041, China.
Tang
Xiao-jun
XJ
Chen
Xiao-he
XH
Che
Guo-wei
GW
Zhu
Da-xing
DX
Sun
Zhi-lin
ZL
Zhou
Qing-hua
QH
D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D012333:RNA, Messenger; C509186:TERT protein, human; D019098:Telomerase