Markedly decreased expression of glutathione S-transferase π gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis

A. Yokomizo, K. Kohno, M. Wada, M. Ono, C. S. Morrow, K. H. Cowan, M. Kuwano

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Abstract

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, γ-glutamyl-cysteine synthetase (γ-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 π (GST-π) 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-π was decreased in BSO-resistant cells, and transient transfection of GST-π 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-π gene. By contrast, γ-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the γ-GCS gene was found to be amplified in the BSO-resistant cell lines. GST-π mRNA levels appeared to be inversely correlated with γ-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-π1 and KB/BSO3-π2, that overexpressed GST-π, from KB/BSO3, after introducing a GST-π expression plasmid. These two transfectants had similar levels in γ-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content as those of KB cells. These findings suggest that the cellular levels of GST-π and γ-GCS might be co-regulated in these novel BSO-resistant cells.

Original languageEnglish
Pages (from-to)19451-19457
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number33
DOIs
Publication statusPublished - Jan 1 1995

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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