Cellular balance of glutathione levels through the expression of gamma-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison.
Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits glutathione biosynthesis and deranges reduced glutathione (GSH) metabolism in liver cells. We isolated two BSO-resistant lines, HLE/BSO2-1 and HLE/BSO2-2, from human hepatic HLE/WT cells. Cellular levels of the Pi class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCSh) mRNA levels were markedly decreased in HLE/BSO2-1 and HLE/BSO2-2 as compared with HLE/WT. The expression of a dominant-negative mutant of c-Jun inhibited the GCSh promoter activity in HLE/WT, but not in HLE/BSO2-1. Cellular levels of AP-1, however, were not decreased in either BSO-resistant cell line. Transfection of GCSh promoter of various lengths driven reporter constructs showed no sequence-specific increase in the promoter activities in HLE/BSO2-1. However, transfection of GSTP1 cDNA into HLE/BSO2-1 and HLE/BSO2-2 restored the levels of GCSh mRNA and the GCSh promoter activity to those of HLE/WT. Sequences between -315 and -241 bp of the 5' region contained an AP-1 site responsible for the enhanced GCSh promoter activity in GSTP1 transfectants of HLE/BSO2-1. In vivo footprint analysis showed a specific protection of the AP-1 site on GCSh promoter in GSTP1 transfected HLE/BSO2-1. GSH homeostasis thus appears to be maintained by an interaction between GSTP1 and GCS in human hepatic cells resistant to the GSH poison.
Tanaka
T
T
Department of Biochemistry, Kyushu University School of Medicine, Maidashi 3-1-1, Fukuoka 812-8582, Japan.
Uchiumi
T
T
Nomoto
M
M
Kohno
K
K
Kondo
T
T
Nishio
K
K
Saijo
N
N
Kuwano
M
M
D019328:Buthionine Sulfoximine / Q000737:chemistry; D004351:Drug Resistance; D005786:Gene Expression Regulation; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry; D005979:Glutathione Peroxidase / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D014407:Tumor Cells, Cultured
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D019328:Buthionine Sulfoximine; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione