Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Accumulating evidence suggests a critical role of intracellular glutathione in tumor cell resistance to alkylating agents. The present study provides evidence for the direct interaction between cis-diamminedichloroplatinum(II) (cisplatin) and glutathione (GSH) both in a cell-free system, as well as in L1210 murine leukemia cells. We have isolated the reaction product and identified it by a combination of high performance liquid chromatography and atomic absorption spectroscopy. Stoichiometric analysis showed a 2:1 molar ratio of GSH/cisplatin for the reaction. The molecular mass assessed by mass spectroscopy was 809 Da, corresponding to a GS-platinum chelate complex, bis-(glutathionato)-platinum. The GS-platinum complex was detected in L1210 leukemia cells incubated with 20 microM cisplatin. The intracellular content of the GS-platinum complex reached a maximal level after 12 h, corresponding to about 60% of the intracellular platinum content. Thus, formation of the GS-platinum complex is considered a significant part of the cellular metabolism of cisplatin. The GS-platinum was found to inhibit cell-free protein synthesis in a rabbit reticulocyte lysate system using both chloramphenicol acetyltransferase mRNA and poly(A) mRNA from HL-60 human promyelocytic leukemia cells (IC50 = 190 microM the GS-platinum complex). Elimination of the GS-platinum complex from tumor cells may represent an important mechanism which reduces the intracellular accumulation of the platinum complex. Using plasma membrane vesicles prepared from L1210 cells, the transport of the GS-platinum complex across the plasma membrane was found to be an ATP-dependent process (apparent Km values: 49 microM, ATP; 110 microM, GS-platinum complex). The ATP-dependent transport of the GS-platinum complex was inhibited by vanadate (IC50 = 35 microM) as well as by S-(2,4-dinitrophenyl)-glutathione, leukotriene C4, and GSSG, but not by doxorubicin, daunorubicin, or verapamil. The ATP-dependent glutathione S-conjugate export pump, "GS-X pump" (Ishikawa, T. (1992) Trends Biochem. Sci. 17, 463-468), is suggested to play a role in the elimination of the GS-platinum complex from tumor cells.
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PMID:Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance. 837 70

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.
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PMID:Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells. 998 57

Species differences in the ability to cope with pollutant-mediated oxidative stress can provide insight into the mechanisms behind both the mode of toxicity of a specific chemical as well as the different ways in which an organism may deal with such stressors. In this study, the effects of exposure to model prooxidants on parameters of oxidative stress were investigated in liver cells from both fish (PLHC-1) and rat (H4IIE). The goals of this study were to compare the oxidative stress response of these cell lines and to assess the relative utility of several different measures of oxidative stress as signals preceding cytotoxicity. Cellular response to two model prooxidants, copper and Fenton reagents (ferrous sulfate plus hydrogen peroxide), was assessed by measuring cytotoxicity, lipid peroxidation, total glutathione (GSHT), and percent glutathione disulfide (%GSSG). Additionally, transcriptional activation of an antioxidant response element (ARE) reporter gene was measured using the chloramphenicol acetyltransferase (CAT) assay in response to these chemicals. In general, the fish cells were more sensitive than rat cells to prooxidants, and the assays for lipid peroxidation and ARE reporter gene activation were more sensitive for measuring oxidative stress than GSH or %GSSG. Fish cells were significantly (P < 0.0001) more sensitive to copper sulfate and Fenton reagent induced oxidative stress, as measured through lipid peroxidation and ARE reporter gene transcriptional activation. Copper sulfate and Fenton reagents caused a two-fold increase in %GSSG in both cell lines. Basal levels of GSHT were higher in the HII4E cells than the PLHC-1 cells, and Fenton reagents significantly reduced GSHT in fish cells but showed no effect on the rat cells. Significant differences were also observed in the cytotoxicity of the test chemicals to both cell lines, with the fish cells demonstrating a higher level of cell death. Lipid peroxidation and ARE transcriptional activation appeared to better reflect subsequent cytotoxicity than a change in GSHT or %GSSG. These results suggest that HII4E (rat) cells are more protected from oxidative stress than PLHC-1 (fish) cells. Additional studies are addressing oxidative stress-mediated signal transduction pathways that may play a role in the differential responses of these cells lines.
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PMID:Differential susceptibility of fish and rat liver cells to oxidative stress and cytotoxicity upon exposure to prooxidants. 1522 51

The Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate-cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE-Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gsta1/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, beta-NF (beta-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5'-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (-1391 to -1381) and ARE-1 (-95 to -85). Deletion analyses demonstrated that the proximal region (-185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2-protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.
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PMID:Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. 1642 33

Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously indentified cis-elements in the CHS promoter.
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PMID:Stress Responses in Alfalfa (Medicago sativa L.): VI. Differential Responsiveness of Chalcone Synthase Induction to Fungal Elicitor or Glutathione in Electroporated Protoplasts. 1666 19