Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, -glutamylcysteine synthetase (-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 (GST-) 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- was decreased in BSO-resistant cells, and transient transfection of GST- 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- gene. By contrast, -GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the -GCS gene was found to be amplified in the BSO-resistant cell lines. GST- mRNA levels appeared to be inversely correlated with -GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-1 and KB/BSO3-2, that overexpressed GST-, from KB/BSO3, after introducing a GST- expression plasmid. These two transfectants had similar levels in -GCS mRNA, drug sensitivity to alkylating agents, and glutathione content as those of KB cells. These findings suggest that the cellular levels of GST- and -GCS might be co-regulated in these novel BSO-resistant cells.

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