Phorbol ester exposure activates an AP-1-mediated increase in ERCC-1 messenger RNA expression in human ovarian tumor cells

Abstract.

ERCC-1 is an essential gene in the nucleotide excision repair pathway, and may be essential for life. However, the mechanism of transcriptional activation and regulation of ERCC-1 gene expression is unclear. We therefore investigated the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the ERCC-1 gene in A2780/CP70 human ovarian carcinoma cells. TPA induced a four- to sixfold increase in steady-state ERCC-1 messenger RNA (mRNA) levels that was time- and concentration-dependent. Nuclear run-on experiments demonstrated that the rate of transcription of ERCC-1 was approximately 2.8-fold higher in TPA-treated cells than in the controls. TPA stimulation of A2780/CP70 cells also resulted in a rapid but transient induction of c-jun and c-fos as determined by Northern and Western blot analyses, which peaked about 2 h before the peak in ERCC-1 expression. Electrophoretic mobility shift assays of nuclear extracts from TPA-treated cells revealed an increase in DNA-binding activity specific for the AP-1-like binding site in the 5′-flanking region of ERCC-1. c-Jun and c-Fos proteins were confirmed to be the components of the activated AP-1 complex by supershift analysis. The increase in AP-1 activity occurs immediately before the increase in ERCC-1 transcription. The increase in AP-1 DNA-binding activity and the increase in ERCC-1 mRNA expression were prevented by pretreatment with cycloheximide. These data suggest that AP-1 may contribute to the upregulation of ERCC-1 in response to TPA in human ovarian cancer cells.