INHIBITION BY 5-FLUOROURACIL OF ERCC1 AND GAMMA-GLUTAMYLCYSTEINE SYNTHETASE MESSENGER-RNA EXPRESSION IN A CISPLATIN-RESISTANT HST-1 HUMAN SQUAMOUS CARCINOMA CELL-LINE

Pretreatment of 5-fluorouracil (5-FU), but not posttreatment, has been shown to augment the cytotoxicity of cisplatin (CDDP) or even circumv ent CDDP resistance by inhibiting repair of platinum-DNA interstrand c rosslinks as well as by reducing the cellular glutathione (GSH) conten ts in CDDP-resistant HST-1/CP0.2 human squamous carcinoma cells. Becau se exogenous thymidine, which compensates for 5-FU-mediated inhibition of de novo DNA synthesis via salvage pathway, did not affect this sch edule-dependent synergism, the modulatory effect of 5-FU on CDDP resis tance would be attributed to the 5-FU-induced RNA damage. We therefore examined the effect of 5-FU on the steady-state levels of messenger R NA (mRNA) of a human excision repair gene ERCCl and gamma-glutamylcyst eine synthetase (gamma-GCS) gene coding for a rate-limiting enzyme for GSH synthesis. The HST-1/CP0.2 cells were found to have significantly more mRNA expression of these respective genes than do parental HST-1 cells. In these cells, 5-FU pretreatment progressively inhibited mRNA expression of both ERCCl and gamma-GCS after removal of 5-FU, without affecting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A ma ximal mRNA suppression was observed at 48 h posttreatment. Such 5-FU-i nduced suppression of mRNA transcripts of these genes seems to be cons istent with its inhibitory activity on DNA repair capacity and cellula r GSH contents. In contrast, 5-FU did not reduce the level of glutathi one-S-transferase-pi (GST-pi) or DNA topoisomerase I mRNA. Although no t convinced, our data suggest that 5-FU, when incorporated into RNA, m ay inhibit both GSH synthesis and repair of platinum-DNA adducts by do wnregulating the ERCCl and gamma-GCS genes, thereby enhancing antitumo r activity of CDDP and reversing resistance to CDDP in HST-1/CP0.2 cel ls.