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

Here we develop a rapid, cell-based, functional assay to screen and identify naturally occurring or synthetic chemicals with chemopreventive activity. We constructed a reporter gene that consists of the gene-encoding green fluorescent protein (GFP) under the transcriptional control of the thymidine kinase (TK) promoter adjacent to which concatamerized EpRE regulatory elements were inserted. Human hepatoma HepG2 cells were transfected with the EpRE/TK-GFP reporter plasmid, and clones with low GFP background expression and 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 treated with test compounds for an additional 24 h. The GFP level and DNA content (as an internal cell survival control) of cells in the 96-well plate were measured subsequently using a fluorescence plate reader. Known inducers of phase II enzymes, such as tert-butylhydroquinone, beta-naphthoflavone, and sulforaphane, significantly increased the GFP level in the HepG2 reporter 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 throughput screening approach to identify novel chemopreventive molecules.