PubMed2000
AIM: To establish a sensitive and efficient reporter gene based screening model and use it to screen compounds for discovering new ligands of estrogen receptor alpha subtype. METHODS: A recombinant Epstein-Barr virus episomal vector (pMT/ERE-CAT) was constructed by inserting a synthetic sequence composed of five estrogen response elements upstream of promoter and a chloramphenicol acetyltransferase (CAT) gene downstream of promoter. pMT/ERE-CAT was transfected into HepG2 cells expressing estrogen receptor alpha subtype (ER +HepG2). Hygromycin (200 micrograms.mL-1) was added 48 h after transfection for selection. One stably transfected clone was isolated and used to screen compounds for activity of stimulating CAT gene expression using colorimetric CAT assay. RESULTS: In the ER +HepG2 cells, the expression of CAT gene was induced by estradiol. A dose-dependent expression of CAT gene with half-maximal induction at 0.07 nmol.L-1 was observed. The ER +HepG2 cell was used to screen compounds for activity of stimulating CAT gene expression. Resveratrol was found to produce a maximal level of induction (1.75 times of estradiol). In vitro radiation survival experiment showed that the radioprotection activity of resveratrol (D0 = 3.18 Gy) is stronger than that of estradiol (D0 = 2.59 Gy). CONCLUSION: Vector pMT/ERE-CAT was used to generate stably transfected ER +HepG2 cell lines. The cell lines can be used to screen compounds for estrogen activity by testing extracts of cells grown in microtiter wells directly using colorimetric CAT assay. This system should provide an efficient method for screening and analyzing the activity of large numbers of ligands of estrogen receptor.
Chloramphenicol O-Acetyltransferase↗