Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.
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PMID:Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents. 153 4

Many in vitro tumor models have been examined to help understand the precise mechanisms responsible for drug resistance. The importance of these results in vivo remains uncertain. MatB 13762 is a rat mammary adenocarcinoma cell line that can be grown both in vitro and as a solid tumor in Fischer 344 rats, thus permitting the examination of tumor cell drug resistance under both conditions. Two cell lines have been selected in vitro for resistance to Adriamycin (AdrR) and melphalan (MlnR), respectively. Each subline has the following features: AdrR, increased mdr-1 messenger RNA, a high level of cross-resistance to vincristine and atypical low level resistance to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea, decreased cellular glutathione content, and increased expression of Yc and Yp glutathione S-transferase isozymes; MlnR, low level drug resistance to melphalan and cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, and vincristine; increased cellular concentration of glutathione; elevated glutathione S-transferase activity; and greatly increased messenger RNA specific to the Yc and Yp glutathione-S-transferase subunits. Most of the biochemical and molecular features described above are present but significantly less prominent in tumors grown in vivo. This model provides the opportunity to examine the magnitude of expression and the clinical significance of in vitro resistance in an in vivo model.
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PMID:In vivo and in vitro mechanisms of drug resistance in a rat mammary carcinoma model. 199 82

Induction of transient thermotolerance by heat or other cytotoxic stressors has been reported to confer a moderate degree of drug resistance to tumor cells in vitro. In this study, a genetically stable, heat-resistant mouse B16 melanoma variant (W-H75) was tested for its sensitivity to various cytotoxic and antiproliferative agents. The heat-resistant W-H75 cells displayed a moderate two- to threefold resistance to doxorubicin, VP-16, VM-26, colchicine, cis-dichlorodiammineplatinum(II), HgCl2, and CdCl2. Marginal resistance to 4'(9-acridinylamino)methanesulfon-m-anisidide vinblastine, 1,3-bis(2-chloroethyl)-1-nitro-sourea, and NaAsO2 was observed, while no difference in sensitivity to the anticancer drugs, actinomycin D and camptothecin, was observed. Although W-H75 cells were generally more resistant than the parental cells to most of the agents that were tested, they were collaterally sensitive to the antimetabolites methotrexate and 6-mercaptopurine. Resistance of the W-H75 cells to epipodophyllotoxins and anthracyclines was not due to differences in steady-state drug accumulation. For the epipodophyllotoxin VP-16, resistance may be related to a relative decrease in the number of drug-induced DNA strand breaks in W-H75 cells. However, no difference in DNA strand breakage was observed between W-H75 and parental cells which were treated with doxorubicin, suggesting that resistance to this drug occurred by a different mechanism. The possible involvement of glutathione and glutathione S-transferase in resistance was also investigated. The glutathione content in W-H75 cells was 35% higher than that in the parental line. However, glutathione S-transferase activity appeared to be identical in both cell lines. Two other heat-resistant B16 melanoma variants, B-H103 and R-H92, were also tested for sensitivity to doxorubicin and VP-16. In contrast to the W-H75 cells, these two heat-resistant variants were hypersensitive to doxorubicin. The B-H103 cells were also hypersensitive to VP-16. This study suggests that selection for cellular resistance to heat may result in cells that have an altered sensitivity to drugs.
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PMID:Drug sensitivity of heat-resistant mouse B16 melanoma variants. 223 92

Perfusion of the bovine eye with a buffer solution containing t-butyl hydroperoxide and the glutathione reductase inhibitor nitrofurantoin caused significant decreases in reduced glutathione level in ciliary body and iris. The result was interpreted to suggest that the organic hydroperoxide was decomposed by the glutathione peroxidase-reductase system. The glutathione reductase reaction requires NADPH. Since the level of NADPH is maintained by the hexose monophosphate shunt in many tissues, we investigated whether this is also the case with bovine uveal tissues. CO2 formation from [1-14C]glucose but not from [6-14C]glucose was markedly stimulated by t-butyl hydroperoxide and was inhibited by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, thus supporting the importance of the hexose monophosphate shunt for hydroperoxide decomposition through the glutathione peroxidase-reductase system. The peroxidase-reductase activity was found both in non-pigmented and pigmented ciliary epithelial cells in culture. Purification studies isolated two forms of glutathione reductase [GR I (140 kDa) with subunit Mr of 70 kDa and GR II (greater than 670 kDa) with subunit Mr of 45 kDa] and a novel glutathione peroxidase (112 kDa with subunit Mr of 29 kDa). The peroxidase is active both with H2O2 and organic hydroperoxides, does not contain selenium and shows no glutathione S-transferase activity.
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PMID:Glutathione-dependent detoxification of peroxide in bovine ciliary body. 237 73

Although both direct and glutathione S-transferase (GST)-catalyzed interactions between many electrophiles and GSH generally result in inactivation of the former, there are several reports of compounds whose electrophilic, alkylating, and cytotoxic activities are potentiated by GSH. This study investigates the effects of direct in vitro interaction between GSH and BCNU at physiological pH (7.2) and temperature (37 degrees C) and how this affects the cytotoxic and DNA cross-linking activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in target human malignant brain tumor cells. The kinetics and dose-response relationship of this interaction were determined by measuring residual GSH and residual BCNU-cytotoxicity in aGSH/BCNU mixture over a 45-min period and at varying BCNU concentrations. The results demonstrate that reaction of BCNU with four times its molar concentration of GSH for 45 min significantly inactivates BCNU, as expressed by a 32% decrease in induction of cellular DNA cross-linking, a 21% increase in DNA synthesis, and a 15% increase in clonogenic survival of human brain tumor cells compared to incubates of BCNU alone. Equine liver (EL)-GST increased the inactivation of BCNU only slightly (insignificant at p = 0.05). These results suggest that, in contrast to agents such as the alkyl-N-nitro-N'-nitrosoguanidines which become more potent alkylators after reacting with GSH, the 2-chloroethylnitrosoureas (CENUs) undergo inactivation by GSH. We propose that such interactions between GSH and the CENUs may constitute an important aspect of CENU metabolism and provide a potential means by which brain tumor cells can circumvent CENU toxicity and exhibit resistance to this class of agents.
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PMID:Decreased DNA interstrand cross-linking and cytotoxicity induced in human brain tumor cells by 1,3-bis(2-chloroethyl)-1-nitrosourea after in vitro reaction with glutathione. 255 96

Reduced glutathione (GSH) and activities of several glutathione-related enzymes were measured in two 9L rat brain tumor cell lines with differing sensitivities to both 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and nitrogen mustard. GSH, measured by a specific high-performance liquid chromatographic method, was found to be approximately twice as high in 9L cells sensitive to BCNU but resistant to nitrogen mustard. The nitrogen mustard resistant cell line was also found to have 2.5-fold more bulk glutathione transferase activity and approximately 3-fold more gamma-glutamyl transpeptidase activity. Glutathione reductase activity, protein thiol, and total protein content were similar in the two cell lines. Pretreatment of 9L cells with 50 microM buthionine sulfoximine for 24 h to deplete GSH only slightly potentiated BCNU cytotoxicity in a clonogenic assay whereas that of nitrogen mustard was markedly potentiated in both cell lines. Similarly, buthionine sulfoximine pretreatment had little effect on the induction of sister chromatid exchanges by BCNU, but significantly increased the number of sister chromatid exchanges induced by nitrogen mustard in both cell lines. Depleting GSH also had no significant effect on the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea to 9L cells. Pretreatment of 9L cells with 1 mM GSH significantly protected against nitrogen mustard cytotoxicity. Moreover, nitrogen mustard incubated with GSH and glutathione transferase was 4-fold less cytotoxic than nitrogen mustard incubated with GSH alone. Incubation of BCNU with GSH alone or with glutathione transferase had no effect on BCNU cytotoxicity. These results indicate that elevated GSH and glutathione transferase activity is one mechanism of cellular resistance to nitrogen mustard in the 9L cell line, but it does not correlate with resistance to BCNU or other clinically important nitrosoureas.
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PMID:Glutathione and related enzymes in rat brain tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea and nitrogen mustard. 288 34

Elevated intracellular levels of metallothionein have been associated with resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms responsible for this resistance, we used a pair of CHO cell lines consisting of normal K1-2 cells and their derivative K1-2MT, which overexpresses the human metallothionein II-A gene (Lohrer et al., 1989). K1-2MT cells were found to be resistant to cadmium chloride and the alkylating agents N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), but resistance did not extend to the alkylating agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea, nor to adriamycin, an inhibitor of DNA synthesis. The DNA damage caused by MNNG, was only marginally less in resistant cells compared with the parental cell line, thus excluding drug scavenging as a possible mechanism for resistance. Also, glutathione S-transferases (GSTs) were present at equal levels in both cell lines (acidic and basic type GST) or slightly reduced in drug resistant K1-2MT cells (neutral type GST), thereby ruling out metabolic inactivation of the alkylating agents. However, the drug resistant phenotype was accompanied by a more efficient block of DNA synthesis after MNNG treatment and by a 3-h delay in the G2 phase of the cell cycle. Using two-dimensional gel electrophoresis of total protein extracts, we identified a 24-kDa protein (MIP1), which is only present in the resistant K1-2MT cells, and a 23.5-kDa protein (MIP2) which is 2-3 times over-synthesized in K1-2MT cells. MNNG treatment reduced the level of both proteins MIP1 and MIP2. These results suggest that the proteins MIP1 and possibly MIP2 may be responsible for the alkylating agent resistant phenotype and are probably modulated by the human metallothionein II-A protein.
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PMID:Increased DNA-repair capacity and the modulation of 2 proteins in and metallothionein overexpressing Chinese hamster cell line. 751 Mar 64

MCF-7 human breast cancer cells possess high levels of O6-alkylguanine-DNA alkyltransferase and moderate levels of glutathione, and are more resistant to chloroethylnitrosoureas (CNUs) than cells with low levels of either molecule. The role of each as a component of CNU resistance was assessed using O6-benzylguanine (O6-bG) or O6-methylguanine (O6-mG) to deplete the alkyltransferase and L-buthionine sulfoxamine (L-BSO) to deplete glutathione. O6-bG and O6-mG potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, resulting in a dose modification factor of 5.4 and 2.3, respectively, which reflected the more potent inhibitory effect of O6-bG. L-BSO alone had little effect on the survival of MCF-7 cells following BCNU exposure, but when combined with O6-mG, BCNU cytotoxicity was additive, yielding a dose modification factor of 3.2. O6-bG or O6-mG and L-BSO acted independently, as neither class of inhibitor affected the other's mechanism of CNU resistance. Furthermore, MCF-7 cells overexpressing GST mu were not more resistant to BCNU than the parent cell line in either the presence or absence of O6-bG or L-BSO. These results indicate that on a relative basis in MCF-7 cells, the alkyltransferase is the cell's first line of defense against CNUs. This suggests that therapeutic trials based on O6-bG-induced biochemical modulation of CNU resistance may increase the efficacy of these chemotherapeutic agents against human malignant cells and that L-BSO may have little additive effect when used with these agents.
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PMID:Combined depletion of O6-alkylguanine-DNA alkyltransferase and glutathione to modulate nitrosourea resistance in breast cancer. 806 41

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.
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PMID:Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells. 814 7

Sixteen healthy donors were investigated for the presence or absence of glutathione transferase (GST) M1-1 in lymphocytes by immunodetection with polyclonal antibodies against human GST M1-1. Nine out of 16 individuals (56%) were categorized as GST M1-1 positive. Phytohaemagglutinin stimulated lymphocytes from GST M1-1 positive and negative donors were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and compared regarding inhibition of [3H]thymidine incorporation as a measure of cytotoxicity. No significant differences in the effect of BCNU were observed between the two groups, indicating that GST M1-1 is not an important resistance factor for BCNU.
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PMID:Similar toxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea on lymphocytes from human subjects differing in the expression of glutathione transferase M1-1. 820 94


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