Cellular balance of glutathione levels through the expression of γ-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison

• Published in: Biochimica et biophysica acta Vol. 1427; no. 3; pp. 367 - 377
• Main Authors: Tanaka, Toshiya, Uchiumi, Takeshi, Nomoto, Minoru, Kohno, Kimitoshi, Kondo, Takahito, Nishio, Kazuto, Saijo, Nagahiro, Kuwano, Michihiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 24.05.1999
• Subjects: Buthionine sulfoximine; Buthionine Sulfoximine - chemistry; Drug Resistance; Gene Expression Regulation; Glutamate-Cysteine Ligase - genetics; Glutathione; Glutathione - analysis; Glutathione - chemistry; Glutathione Peroxidase - genetics; Glutathione S-transferase; Glutathione Transferase - genetics; Hepatic cell (Human); Humans; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos - analysis; Proto-Oncogene Proteins c-jun - analysis; RNA, Messenger - analysis; Transcription; Transfection; Tumor Cells, Cultured; γ-Glutamylcysteine synthetase; GSH, reduced glutathione; Transcription; GST, glutathione S-transferase; γ-Glutamylcysteine synthetase; MEM, minimum essential medium; BSO, dl-buthionine-[ S, R]-sulfoximine; Buthionine sulfoximine; GPX, glutathione peroxidase; Hepatic cell (Human); GCSl, GCS light subunit; GCS, γ-glutamylcysteine synthetase; GSTP1, P i class GST; Glutathione S-transferase; GCSh, GCS heavy subunit; Glutathione
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(99)00016-1

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 P i class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, γ-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.