[An experimental study on targeting suicide gene therapy for lung cancer with HSV-TK driven by hTERT promoter].
OBJECTIVE
To study the approach of targeting expression of suicide gene HSV-TK driven by human telomerase catalytic subunit (hTERT) promoter in lung cancer cells, and to investigate inhibitory effect of HSV-TK/GCV driven by hTERT promoter on proliferation of lung cancer cell line A549 in vitro and in vivo.


METHODS
(1) Recombinant expression vectors of HSV-TK driven by hTERT promoter and SV40 promoter (pGL3-hTp-TK and pGL3-SV40-TK) were transfected into telomerase-positive human lung adenocarcinoma cell A549 and telomerase-negative human embryonic lung fibroblast cell MRC-5. The mRNA expression of TK gene was detected with RT-PCR method; (2) With the treatment of GCV, the proliferation of above transfected cells was investigated by MTT assay; Influence of GCV on apoptosis and cell cycle of these cells was evaluated with flow cytometry; (3) After the subcutaneously transplantation of A549 cells into nude mice, intra-tumor injection of plasmid-liposome as well as intra-peritoneal injection of GCV were performed to stUdy anti-tumor effects of pGL3-hTp-TK/GCV and pGL3-SV40-TK/GCV in vivo.


RESULTS
(1) Enzyme digestion and PCR suggested that recombinant plasmids of pGL3-hTp-TK and pGL3-SV40-TK were successfully constructed; TK mRNA expression was detected in both A549 and MRC-5 cells transfected with pGL3-SV40-TK, also in A549 transfected with pGL3-hTp-TK, but not in MRC-5 transfected with pGL3-hTp-TK; (2) GCV showed significant inhibition effect on proliferation of A549 and MRC-5 transfected with pGL3-SV40-TK in vitro, also on that of A549 transfected with pGL3-hTp-TK, but not of MRC-5 transfected with pGL3-hTp-TK; With the treatment of GCV, apoptosis index (AI) of A549 cells transfected with pGL3-SV40-TK and pGL3-hTp-TK (21.58% and 23.19% respectively) increased significantly, compared with that of A549 transfected with pGL3-hTp and blank control; GCV enhanced the effects on AI in MRC-5 transfected with pGL3-SV40-TK (9.35%), but not with pGL3-hTp-TK (0.88%); (3) Inhibition ratio of pGL3-SV40-TK/GCV and pGL3-hTp-TK/GCV to transplanted tumor of A549 in nude mice (46.7% and 40.5% respectively) were significantly higher than that of negative control groups (9.7%, 14.3%, 7.0% and 8.0% respectively).


CONCLUSIONS
TK gene driven by hTERT promoter could express selectively in lung cancer cell. Lung cancer cell could be specifically inhibited by HSV-TK/GCV driven by hTERT promoter in vitro and in vivo.
Wang
Yan-ping
YP
The Laboratory of Molecular Diagnosis of Cancer, West China Hospital, Sichuan University, Chengdu 610041, China.
Tang
Xiao-jun
XJ
Zhou
Qing-hua
QH
Che
Guo-wei
GW
Chen
Xiao-he
XH
Zhu
Da-xing
DX
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology / Q000628:therapy; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D016503:Drug Delivery Systems; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D051379:Mice; D008819:Mice, Nude; D011401:Promoter Regions, Genetic / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D013937:Thymidine Kinase; C509186:TERT protein, human; D019098:Telomerase; D015774:Ganciclovir