Markedly decreased expression of glutathione S-transferase π gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis

Abstract

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, γ-glutamyl-cysteine synthetase (γ-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase π (GST-π) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST-π was decreased in BSO-resistant cells, and transient transfection of GST-π promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and - 80 base pairs of the 5' flanking region were at least partially responsible for the decreased expression of the GST-π gene. By contrast, γ-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the γ-GCS gene was found to be amplified in the BSO-resistant cell lines. GST-π mRNA levels appeared to be inversely correlated with γ-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-π1 and KB/BSO3-π2, that overexpressed GST-π, from KB/BSO3, after introducing a GST-π expression plasmid. These two transfectants had similar levels in γ-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content as those of KB cells. These findings suggest that the cellular levels of GST-π and γ-GCS might be co-regulated in these novel BSO-resistant cells.