The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress.A description of the mechanisms of transcriptional and posttranscriptional regulat...

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.

Glutathione transferases--structure and catalytic activity.

The glutathione transferases, a family of multifunctional proteins, catalyze the glutathione conjugation reaction with electrophilic compounds biotransformed from xenobiotics, including carcinogens. In preneoplastic cells as well as neoplastic cells, specific molecular forms of glutathione transferase are known to be expressed and have been known to participate in the mechanisms of their resistance to drugs. In this article, following a brief description of recently identified molecular forms, we review new findings regarding the respective molecular forms involved in carcinogenesis and anticancer drug resistance, with particular emphasis on Pi class forms in preneoplastic tissues. The rat Pi class form, GST-P (GST 7-7), is strongly expressed not only in hepatic foci and hepatomas, but also in initiated cells that occur at the very early stages of chemical hepatocarcinogenesis, and is regarded as one of the most reliable markers for preneoplastic lesions in the rat liver. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-responsive element-like sequences have been identified in upstream regions of the GST-P gene, and oncogene products c-jun and c-fos are suggested to activate the gene. The Pi-class forms possess unique enzymatic properties, including broad substrate specificity, glutathione peroxidase activity toward lipid hydroperoxides, low sensitivity to organic anion inhibitors, and high sensitivity to active oxygen species. The possible functions of Pi class glutathione transferases in neoplastic tissues and drug-resistant cells are discussed.

Glutathione Transferases and Cancer

The glyoxalase system in health and disease.

(1990). The Role of Glutathione and Glutathione Transferases in Chemical Cardnogenesi. Critical Reviews in Biochemistry and Molecular Biology: Vol. 25, No. 1, pp. 47-70.

The role of glutathione and glutathione transferases in chemical carcinogenesis.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is known to be detoxified by a denitrosation reaction catalyzed by glutathione-dependent enzymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., 11:175-176, 1983). Using a modification of their procedure, we have measured the ability of different purified rat glutathione transferase isoenzymes to denitrosate BCNU. The catalytic efficiencies of the isoenzymes for the denitrosation reaction expressed as the ratio of Vmax to Km were as follows (isoenzyme, Vmax/Km): 1-2, 2.3; 3-3, 12.2; 3-4, 29.2; and 4-4, 26.1. Thus, the class mu isoenzymes containing subunit 4 are by far the best catalysts of the BCNU denitrosation reaction. The class pi transferase 7-7 and class alpha transferases 1-1 and 1-2 demonstrated very weak catalytic activity with BCNU. Determination of the glutathione transferase isoenzyme profiles of 9L rat brain tumor cells and the BCNU-resistant 9L-2 subline by immunoblotting revealed that although the resistant 9L-2 cells contain lower total glutathione transferase activity than 9L cells, they have elevated levels of the class mu transferases. Also, the class pi transferases were found to be down-regulated in 9L-2 as compared with 9L cells. Thus, the increased resistance of 9L-2 cells to BCNU may, in part, be explained by up-regulation of class mu transferase expression with consequent increased capacity for BCNU detoxication. Further support for this hypothesis comes from the fact that pretreatment of 9L-2 cells with the glutathione transferase inhibitors ethacrynic acid or triphenyltin chloride enhanced the cytotoxic effects of BCNU. These results suggest that the class mu transferases play a role in the resistance of brain tumor cells to BCNU.

Denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells.

Glutathione transferases are enzymes implied in the resistance of tumor cells to bifunctional alkylating cytostatic drugs. We have investigated the effect of the glutathione transferase inhibitor by ethacrynic acid on the cytotoxicity of melphalan to a human melanoma cell line (RPMI 8322) with a high level of glutathione transferase activity. Using 1-chloro-2,4-dinitrobenzene as substrate, ethacrynic acid was shown to inhibit the activity of purified human glutathione transferases, with 50% inhibition values of 1, 10, and 15 microM for transferase mu (class mu), transferase epsilon (class alpha) and transferase pi (class pi), respectively, all of which occur in RPMI 8322 cells. Ethacrynic acid at a concentration of 20 microM, which by itself was noncytotoxic, increased the cytotoxicity of melphalan to RPMI 8322 human melanoma cells approximately 2-fold. The induction of DNA interstrand cross-links by 40 microM melphalan was increased 1.4-fold by 30 microM ethacrynic acid. These results indicate that a potentiation of the cytotoxic effect of bifunctional alkylating agents can be achieved by inhibition of glutathione transferase and that the enhanced cytotoxicity may be caused at least in part by increased formation of drug-DNA adducts.

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Sensitization of Human Melanoma Cells to the Cytotoxic Effect of Melphalan by the Glutathione Transferase Inhibitor Ethacrynic Acid

The occurrence of glutathione transferase in human malignant melanoma cell lines and solid tumor material has been analyzed and compared with the enzyme composition in fibroblasts and naevus samples. All cells and tissues investigated contained essentially only the acidic class Pi glutathione transferase as demonstrated by SDS-PAGE and immunoblotting. The enzyme was purified from tumor material and characterized. Its intracellular concentration was significantly higher in all the melanoma cell preparations analyzed than in the non-malignant cells, supporting the view that the class Pi glutathione transferase may contribute to the drug resistance that is characteristic of malignant melanoma.

Expression of class Pi glutathione transferase in human malignant melanoma cells.

A large number of human tumor cell lines of various origins have been investigated with respect to expression of glutathione-linked enzymes in the cytosol fraction. The amounts of the different enzymes were estimated by use of activity measurements and by silver staining or immunoblot analysis after electrophoresis of cytosol fractions purified by affinity chromatography on S-hexylglutathione Sepharose. Class Pi glutathione transferase was the most abundant enzyme in most tumor cells; the cell lines HepG2 and Raji were exceptions in not expressing significant amounts of this enzyme. HepG2 cells derive from hepatocytes, which normally do not express the class Pi enzyme, whereas Raji cells originate from B-lymphocytes, which normally do express a class Pi glutathione transferase. The highest level of the class Pi transferase, in terms of protein reacting with antibodies as well as enzyme activity, was noted in the colon carcinoma cell line LS174T. Hu549Pat cells, EBV-transformed B-lymphocytes, also expressed high levels of a protein reacting with antibodies specific for class Pi glutathione transferases, but did not display any significant activity with ethacrynic acid, a substrate characteristic for this class. Class Alpha and class Mu glutathione transferases, in cell lines expressing these isoenzymes, were present in significantly lower concentrations than the class Pi enzyme. Most of the tumor cells contained a class Alpha transferase composed of 27.5 kd subunits, which has the physicochemical and immunological properties of the most basic glutathione transferase found in human skin. In several cell lines, a protein was detected with an apparent subunit Mr value of 30 kd that was tentatively identified as an additional class Alpha glutathione transferase not previously described. In addition, other glutathione-linked enzyme activities, namely glutathione peroxidase, glutathione reductase and glyoxalase I, were assayed with specific substrates in the cytosolic fraction of the tumor cells; glyoxalase I could also be estimated semiquantitatively by silver staining of SDS-PAGE cells after affinity chromatography. Like the glutathione transferases, these enzymes displayed distinctly different levels of expression in the various cell lines. Thus, virtually every cell line was found to have a unique pattern of glutathione-linked enzymes, suggesting that the resistance phenotypes of the cells differ accordingly.

Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Victor M. Castro

Papers

Denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells.

Sensitization of Human Melanoma Cells to the Cytotoxic Effect of Melphalan by the Glutathione Transferase Inhibitor Ethacrynic Acid

Expression of class Pi glutathione transferase in human malignant melanoma cells.

Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines