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
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.