Kiyomi Sato

Glutathione transferases and cancer

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 As Markers Of Preneoplasia And Neoplasia

Publisher Summary New markers, especially enzymes or isoenzymes, have proved useful for the detection of preneoplastic lesions in various organs of both animals and man. In addition, the description of preneoplastic marker enzymes has also played an important role in characterizing the metabolic patterns of preneoplastic cells, thus providing insights that have facilitated the analysis of carcinogenic processes. Recently, certain molecular forms of glutathione transferase (GT), one of the most important drug-metabolizing enzymes, have been reported as reliable markers for preneoplastic lesions and neoplastic tissue. This chapter reviews the known preneoplastic marker enzymes and molecular forms of GT in rat, human, and other species. Particular attention is focused on the unique properties of rat GT-P (7-7) and human GT-π, and what is known of their gene expression and possible functions. Furthermore, GT, including ligandin, have multipotential detoxifying functions and therefore, have been stimulated to study the changes in molecular forms and their roles in metabolism of carcinogens.

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.

Developmental and hormonal regulation of the major form of hepatic glutathione S-transferase in male mice

Among three forms of mouse hepatic glutathione S-transferase, the II form, which is immunologically related to rat 7-7 form, was the major form in adult male mice of all the five strains examined and the levels (about 5.0 mg/g of liver) were approximately ten-fold higher than those of females. This form markedly increased at puberty in male mice, whereas no change was observed in females. By castration, the levels in males decreased to those in females, while those in females increased to those in adult males by administration of testosterone. These results indicate that the expression of II form in mouse liver is regulated developmentally by testosterone, and this protein could be a useful marker for the male mouse.

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2α formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).

Modulation of Class Pi glutathione transferase activity by sulfhydryl group modification

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.

Drug resistance to cis-diamminedichloroplatinum(II) in Chinese hamster ovary cell lines transfected with glutathione S-transferase pi gene

Establishment of Chinese hamster ovary (CHO) cell lines expressing human glutathione S-transferase-pi (GST-pi) was performed after cotransfection of pSV2-neo and human GST-pi cDNA-carrying plasmid p beta actGPi-2. About 30 G418-resistant clones were tested for their expression of GST-pi by Northern blot analysis. Two clones, beta 2-3 and beta 2-5, expressed a significant amount of GST-pi mRNA; and one clone, beta 1-1, that did not was also used for further study. Western blot analysis with anti-GST-pi antibody showed significant increases of GST-pi in beta 2-3 and beta 2-5, but not in beta 1-1. Northern blot analysis with the human GST-pi cDNA probe showed that the increase in the expression of GST-pi-mRNA in beta 2-3 and beta 2-5 was respectively 2- and 4-fold higher than that in beta 1-1. Southern blotting analysis showed that beta 1-1, beta 2-3 and beta 2-5 contained about one copy of the human GST-pi cDNA sequence. beta 2-3 and beta 2-5 were resistant to 1.4- and 3.0-fold higher doses of CDDP than CHO, respectively, but beta 1-1 was not. Increased expression of GST-pi might be associated with CDDP-resistance in CHO cells.

Properties of the increased glutathione S-transferase a form in rat preneoplastic hepatic lesions induced by chemical carcinogens

Glutathione S-transferase A form (GST-A) is increased markedly in rat preneoplastic hepatic lesions such as hyperplastic nodules induced by diethylnitrosamine followed by administration of N-2-fluorenylacetamide. GST-A was also significantly increased in livers of rats after short-term administration of some drugs. The increased activity and protein content of GST-A were demonstrated by CM-Sephadex C-50 column chromatography as well as by two-dimensional polyacrylamide gel electrophoresis following immuno-affinity column chromatography using antibody against GST-A. Immunologically, GST-A crossreacted strongly with GST-C, weakly with GST-C2, but not with ligandin, GST-B, or GST-AA. It was confirmed by subunit recombination that GST-C is a heterodimer composed of the subunits of homodimers, GST-A and GST-C2.

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): Elucidation by oligonucleotide site-directed mutagenesis☆

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.

Characterization of glycogen phosphorylase isoenzymes present in cultured skeletal muscle from patients with McArdle's disease

Abstract Muscle biopsy specimens from patients with McArdles disease lack glycogen phosphorylase activity. Significant phosphorylase activity was detected in cultured muscle cells from these patients. The phosphorylase isoenzymes in the cells were identified electrophoretically and immunochemically. On polyacrylamide disc gel electrophoresis, two types of isoenzymes were separated in about equal amounts. Both differed the muscle type in migration, kinetic, and immunochemical properties. The first type corresponded to a fetal phosphorylase isoenzyme, and the second was a liver-like type which was completely absorbed with antibody against the rat liver isoenzyme. No adult skeletal muscle isoenzyme was detected.