Glutathione homeostasis in human hepatic cells. Overexpression of γ-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 cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO. In the absence of BSO, cellular levels of GSH were 3.0-fold higher, whereas levels of Pi class glutathione thiol transferase, GSTP1, were 2-fold lower, in the subclones than in the wild 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 between -315 and -241 base pairs of the 5@? region, which contain an AP1 site, 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-Jun was found to inhibit the AP1-dependent GCSh promoter activity in HLE/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.