Markedly decreased expression of glutathione S-transferase pi gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis.
Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, gamma-glutamylcysteine synthetase (gamma-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 pi (GST-pi) 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-pi was decreased in BSO-resistant cells, and transient transfection of GST-pi promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5'-flanking region wer at least partially responsible for the decreased expression of the GST-pi gene. By contrast, gamma-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the gamma-GCS gene was found to be amplified in the BSO-resistant cells lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-pi1 and KB/ BSO2-pi2, that overexpressed GST-pi, from KB/BSO3, after introducing a GST-pi expression plasmid. These two transfectants had similar levels in gamma-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content at those of KB cells. These findings suggest that the cellular levels of GST-pi and gamma-GCS might be co-regulated in these novel BSO-resistant cells.
Yokomizo
A
A
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Kohno
K
K
Wada
M
M
Ono
M
M
Morrow
C S
CS
Cowan
K H
KH
Kuwano
M
M
D015139:Blotting, Southern; D019328:Buthionine Sulfoximine; D018076:DNA, Complementary; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005979:Glutathione Peroxidase; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005723:gamma-Glutamyltransferase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione