GLUTATHIONE HOMEOSTASIS IN HUMAN HEPATIC CELLS - OVEREXPRESSION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE GENE IN CELL-LINES RESISTANT TO BUTHIONINE SULFOXIMINE, AN INHIBITOR OF GLUTATHIONE SYNTHESIS

The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cel lular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepati c HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversi ble inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 a nd HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, t han the wild type to BSO. In the absence of BSO, cellular levels of GS H were 3.0-fold higher, whereas levels of Pi class glutathione thiol t ransferase, GSTP1, were a-fold lower, in the subclones than in the wil d type cells. GCS heavy subunit (GCSh) mRNA level were 2.5-fold higher in HLE/BSO1-1 and HLE/BSO1-2 as compared with HLE/WT. Sequences betwe en -315 and -241 base pairs of the 5' region, which contain an AP1 sit e, were shown to be responsible for the enhanced expression of GCSh in HLE/BSO1-1 cells. The expression of a dominant-negative mutant of c-J un was found to inhibit the AP1-dependent GCSh promoter activity in HL E/WT and HLE/BSO1-1. Both protein level of c-Jun and binding activity of AP-1 were increased in both HLE/BSO1-1 and HLE/BSO1-2 cells. The up -regulation of GCSh gene appeared to be due to enhanced GCSh promoter acting through AP-1 activation in BSO-resistant hepatic cells. (C) 199 8 Academic Press.