Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents

Authors
Citation
M. Zhu et We. Fahl, Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents, ANALYT BIOC, 287(2), 2000, pp. 210-217
Citations number
24
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
287
Issue
2
Year of publication
2000
Pages
210 - 217
Database
ISI
SICI code
0003-2697(200012)287:2<210:DOAGFP>2.0.ZU;2-Q
Abstract
Here we develop a rapid, cell-based, functional assay to screen and identif y naturally occurring or synthetic chemicals with chemopreventive activity. We constructed a reporter gene that consists of the gene-encoding green fl uorescent protein (GFP) under the transcriptional control of the thymidine kinase (TK) promoter adjacent to which concatamerized EpRE regulatory eleme nts were inserted. Human hepatoma HepG2 cells were transfected with the EpR E/TK-GFP reporter plasmid, and clones with low GFP background expression an d high tBHQ-induced GFP expression were isolated. These GFP reporter cells were seeded into a 96-well microtiter plate, incubated for 24 h, and then t reated with test compounds for an additional 24 h. The GFP level and DNA co ntent (as an internal cell survival control) of cells in the 96-well plate were measured subsequently using a fluorescence plate reader. Known inducer s of phase II enzymes, such as tert-butylhydroquinone, beta -naphthoflavone , and sulforaphane, significantly increased the GFP level in the HepG2 repo rter cells. In an initial screening of a chemical library, we identified a synthetic compound whose inducing ability significantly exceeds (1.6-fold) that of the best currently known phase II enzyme inducers. The experimental results indicate that this cell system makes possible a new high throughpu t screening approach to identify novel chemopreventive molecules. (C) 2000 Academic Press.