[Cloning of human telomerase reverse transcriptase promoter for targeted cancer therapy].
OBJECTIVE
An human telomerase reverse transcriptase (hTERT) promoter was cloned to investigate the effect of simplex virus-thymidine kinase (HSV-tk) gene-ganciclovir (GCV) system under control of this promoter on lung cancer cells A549.


METHODS
Specific primers were designed to amplify the core hTERT promoter from HepG2 genome. A set of expression vectors encoding LacZ or tk gene under control of hTERT or hCMV promoter was constructed through standard molecular cloning methods. After the transfection with lipofectamine 2000, reverse transcriptional PCR (RT-PCR) and beta-Gal staining were performed to examine the activity of the hTERT promoter. The cytotoxic effects of GCV/tk on A549 and MRC5 cells transfected with the plasmids encoding tk gene were evaluated by MTT assay.


RESULTS
The hTERT promoter containing -208-+40 bp of the upstream sequence of human telomerase was successfully cloned. beta-Gal staining and RT-PCR were used to detect the expression of Lac Z and the transcription of tk gene under control of the hTERT promoter, respectively, in A549 rather than MRC5 cells. Cyototoxic effect of GCV was observed only in the A549 but not MRC5 cells after the transfection with pDC511 hTERT/tk, and the effect was dose-dependent. The effect, however, was observed in cells transfected with pDC518 hCMV/tk.


CONCLUSIONS
The hTERT promoter cloned in this study can specifically control the target gene expression in telomerase-positive tumor cells but not the normal cells, suggesting that the HSV-tk/GCV system under control of the hTERT promoter is a promising targeted gene therapy for malignant tumors.
Li
Fan
F
Institute of Liver Diseases, General Hospital of Bejing Command, Beijing 100700, China. fantist510@hotmail.com
Tan
Xiao-Hua
XH
Peng
Xiao-Jun
XJ
Cai
Hong-Bing
HB
Hu
Da-Rong
DR
D003001:Cloning, Molecular; D015774:Ganciclovir / Q000494:pharmacology; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology / Q000628:therapy*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D013937:Thymidine Kinase; D019098:Telomerase; D015774:Ganciclovir