Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization

被引:69
作者
Novotna, Arleta [1 ]
Petr, Pavek [2 ]
Dvorak, Zdenek [1 ]
机构
[1] Palacky Univ, Fac Sci, Dept Cell Biol & Genet, Olomouc 78371, Czech Republic
[2] Charles Univ Prague, Fac Pharm Hradec Kralove, Dept Pharmacol & Toxicol, Hradec Kralove 50005, Czech Republic
关键词
AH RECEPTOR; CALUX-BIOASSAY; INDUCTION; CYP1A1; EXPRESSION; 3-METHYLCHOLANTHRENE; PLASMIDS; AGONIST; SYSTEM;
D O I
10.1021/es2029334
中图分类号
X [环境科学、安全科学];
学科分类号
08 ; 0830 ;
摘要
We constructed stably transfected gene reporter cell line AZ-AHR, allowing measurement of aryl hydrocarbon receptor (AhR) transcriptional activity. Human hepatoma HepG2 cells were transfected with a construct containing several AhR binding sites upstream of luciferase reporter gene. We prepared 12 clones and we characterized the best five in responsiveness to TCDD. Dose response analyses were performed for various AhR ligands, including TCDD, 3-methylcholanthrene, indirubin, resveratrol, omeprazole, and SP600125. The EC(50) values were similar in all tested clones. Induction of luciferase was time-dependent, and treatment for 6 h with 5 nM TCDD was sufficient to evaluate AhR transcriptional activity in 96-well plate format (8-24 fold induction). Response to AhR ligands of cryopreserved cells after thawing was not significantly different from that of fresh cells. Cell line remained fully responsive to AhR ligands over 15 passages and 30 days in culture without significant alterations. Overall, we have developed novel human luciferase reporter cell line AZ-AHR for monitoring AhR transcriptional activity. The sensitivity of the assay allows high throughput format (96-well plate) and evaluation of luciferase activity as soon as after 6 h of incubation, which has potential implication for studies of cytotoxic compounds.
引用
收藏
页码:10133 / 10139
页数:7
相关论文
共 18 条
[1]  
Allen SW, 2001, DRUG METAB DISPOS, V29, P1074
[2]  
Bovee TFH, 1998, FOOD ADDIT CONTAM A, V15, P863, DOI 10.1080/02652039809374723
[3]   A high-throughput cell-based reporter gene system for measurement of CYP1A1 induction [J].
Cui, X ;
Palamanda, J ;
Norton, L ;
Thomas, A ;
Lau, YY ;
White, RE ;
Cheng, KC .
JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS, 2002, 47 (03) :143-151
[4]   JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes [J].
Dvorak, Zdenek ;
Vrzal, Radim ;
Henklova, Paula ;
Jancova, Petra ;
Anzenbacherova, Eva ;
Maurel, Patrick ;
Svecova, Lucie ;
Pavek, Petr ;
Ehrmann, Jiri ;
Havlik, Roman ;
Bednar, Petr ;
Lemr, Karel ;
Ulrichova, Jitka .
BIOCHEMICAL PHARMACOLOGY, 2008, 75 (02) :580-588
[5]   Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide [J].
Gerbal-Chaloin, S ;
Pichard-Garcia, L ;
Fabre, JM ;
Sa-Cunha, A ;
Poellinger, L ;
Maurel, P ;
Daujat-Chavanieu, M .
CELLULAR SIGNALLING, 2006, 18 (05) :740-750
[6]   Third-Generation Ah Receptor-Responsive Luciferase Reporter Plasmids: Amplification of Dioxin-Responsive Elements Dramatically Increases CALUX Bioassay Sensitivity and Responsiveness [J].
He, Guochun ;
Tsutsumi, Tomoaki ;
Zhao, Bin ;
Baston, David S. ;
Zhao, Jing ;
Heath-Pagliuso, Sharon ;
Denison, Michael S. .
TOXICOLOGICAL SCIENCES, 2011, 123 (02) :511-522
[7]   Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway [J].
Kwak, Mi-Kyoung ;
Kensler, Thomas W. .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2006, 345 (04) :1350-1357
[8]  
LUSSKA A, 1993, J BIOL CHEM, V268, P6575
[9]   Chemical-activated luciferase gene expression (CALUX): A novel in vitro bioassay for Ah receptor active compounds in sediments and pore water [J].
Murk, AJ ;
Legler, J ;
Denison, MS ;
Giesy, JP ;
vandeGuchte, C ;
Brouwer, A .
FUNDAMENTAL AND APPLIED TOXICOLOGY, 1996, 33 (01) :149-160
[10]   An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor [J].
Opitz, Christiane A. ;
Litzenburger, Ulrike M. ;
Sahm, Felix ;
Ott, Martina ;
Tritschler, Isabel ;
Trump, Saskia ;
Schumacher, Theresa ;
Jestaedt, Leonie ;
Schrenk, Dieter ;
Weller, Michael ;
Jugold, Manfred ;
Guillemin, Gilles J. ;
Miller, Christine L. ;
Lutz, Christian ;
Radlwimmer, Bernhard ;
Lehmann, Irina ;
von Deimling, Andreas ;
Wick, Wolfgang ;
Platten, Michael .
NATURE, 2011, 478 (7368) :197-203