Liver Cancer
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1460-1464
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1460
Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals
Zhi-Ren Zhang, Shun-Qing Xu, Xi Sun, Yong-Jun Xu, Xiao-Kun Cai, Zhi-Wei Liu, Xiang-Lin Tan, Yi-Kai Zhou, Jun-Yue Zhang, Hong Yan
Zhi-Ren Zhang, Shun-Qing Xu, Xi Sun, Yong-Jun Xu, Xiao-Kun Cai, Zhi-Wei Liu, Xiang-Lin Tan, Yi-Kai Zhou, Jun-Yue Zhang, Hong Yan, Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 29877020 and 20107002
Correspondence to: Dr. Shun-Qing Xu, Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China. shunqing@public.wh.hb.cn
Telephone: +86-27-83693417 Fax: +86-27-83657705
Received: January 28, 2002
Revised: March 4, 2002
Accepted: May 11, 2002
Published online: July 15, 2003
Abstract

AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs.

METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL3-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG2, both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG2 cells stably transfected by the recombinant vector (HepG2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG2 cells (HepG2-wt).

RESULTS: The inducible luciferase expression of HepG2-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG2-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG2-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 × 10-12 to 5 × 10-9 mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG2-luc and HepG2-wt cells. The correlation between TCDD doses from 1.1 × 10-13 to 1 × 10-8 mol/L and luciferase activities was also found to be significant in HepG2-luc cells (r = 0.997, P < 0.001), but not in their HepG2-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG2-luc cells were both better than that of EROD in HepG2-wt cells, the former was at 1.1 × 10-13 mol/L and 3.5 × 10-12 mol/L, and the coefficients of variation (CV) of the latter was 15%-30% and 22%-38%, respectively.

CONCLUSION: The luciferase expression of HepG2-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.

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