Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with resistance to nitrogen mustards

Glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals and have been implicated in the intrinsic and acquired resistance of tumors to cytotoxic drugs. We have generated a Chinese hamster ovary line resistant to bifunctional nitrogen mustards and in this report have characterized and isolated the protein that represents the major observable phenotypic difference between the drug-sensitive and drug-resistant cell lines. This purified protein is shown to be an alpha class glutathione S-transferase comprising YcYc subunits and possessing a pI value of approximately 8.0. The intracellular level of the Yc subunit is elevated greater than 40-fold in the drug-resistant cell line, which could account for the increase in glutathione S-transferase (RX:glutathione R-transferase; EC 2.5.1.18) activity toward both 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Other glutathione S-transferase subunits within this gene family are also elevated. These changes are accompanied by a significant elevation in alpha class mRNA levels. Southern analysis indicates that the genes coding for these proteins are amplified 4- to 8-fold in the drug-resistant cell line. In addition, gamma-glutamyl transpeptidase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase; EC 2.3.2.2] activity is increased 3.6-fold in the drug-resistant Chinese hamster ovary cell line, which may explain the increase in cellular glutathione level. In this case no gene amplification was seen. These data indicate that gene amplification may be important in drug resistance toward alkylating agents and also that other enzymes in glutathione homeostasis are involved.