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Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with
A D Lewis1, I D Hickson, C N Robson
1Imperial Cancer Research Fund, University Department of Biochemistry, Edinburgh, Scotland, United Kingdom.
Abstract:
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.
Insights
Drug resistance in cancer cells involves glutathione-dependent enzymes. Researchers identified an elevated glutathione S-transferase (GST) protein in resistant cells, linked to gene amplification and altered enzyme activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Glutathione-dependent enzymes protect cells from cytotoxic chemicals.
- These enzymes are implicated in tumor resistance to chemotherapy drugs.
- Understanding drug resistance mechanisms is crucial for effective cancer treatment.
Purpose of the Study:
- To characterize the protein responsible for drug resistance in a Chinese hamster ovary (CHO) cell line.
- To investigate the molecular basis of acquired resistance to bifunctional nitrogen mustards.
- To explore the role of glutathione S-transferase (GST) and other enzymes in drug resistance.
Main Methods:
- Generated a drug-resistant CHO cell line.
- Isolated and purified the major phenotypic difference protein.
- Analyzed protein subunits, isoelectric point (pI), enzyme activity, mRNA levels, and gene amplification using Southern analysis.
Main Results:
- Identified an alpha class GST (YcYc subunits, pI ~8.0) elevated >40-fold in resistant cells.
- Observed increased GST activity towards specific substrates and elevated levels of other GST subunits and alpha class mRNA.
- Found 4- to 8-fold gene amplification for GST proteins and a 3.6-fold increase in gamma-glutamyl transpeptidase activity.
Conclusions:
- Gene amplification of GSTs is a key mechanism in resistance to alkylating agents.
- Increased gamma-glutamyl transpeptidase activity contributes to elevated cellular glutathione levels.
- Multiple enzymes in glutathione homeostasis are involved in acquired drug resistance.