MARKEDLY DECREASED EXPRESSION OF GLUTATHIONE-S-TRANSFERASE-PI GENE INHUMAN CANCER CELL-LINES RESISTANT TO BUTHIONINE SULFOXIMINE, AN INHIBITOR OF CELLULAR GLUTATHIONE SYNTHESIS

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversib ly inhibits an enzyme, gamma-glutamyl-cysteine synthetase (gamma-GCS), which is a critical step in glutathione biosynthesis. We isolated thr ee BSO-resist ant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold high er resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan an d nitrosourea. Cellular levels of glutathione S-transferase pi (GST-pi ) and its mRNA in BSO-resistant cell lines were less than 10% of the p arental cells, Nuclear run-on assay showed that the transcriptional ac tivity of GST-pi was decreased in BSO-resistant cells, and transient t ransfection of GST-pi promoter-chloramphenicol acetyltransferase const ructs revealed that the sequences between -130 and -80 base pairs of t he 5'-flanking region were at least partially responsible for the decr eased expression of the GST-pi gene. By contrast, gamma-GCS mRNA level s were 3-to 5-fold higher in resistant cell lines than in KB cells, an d the gamma-GCS gene was found to be amplified in the BSO-resistant ce ll lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established t he transfectants, KB/BSO3-pi 1 and KB/BSO3-pi 2, that overexpressed GS T-pi; from KB/BSO3, after introducing a GST-pi expression plasmid. The se two transfectants had similar levels in gamma-GCS mRNA, drug sensit ivity to alkylating agents, and glutathione content as those of KB cel ls. These findings suggest that the cellular levels of GST-pi and gamm a-GCS might be co-regulated in these novel BSO-resistant cells.