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:2.5.1.47 (cysteine synthase)
625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Maintenance of cellular homeostasis is a critical survival trait in tumors when exposed to anticancer drugs. Because conjugation and elimination of drugs and their metabolites is dependent upon sequential and coordinated pathways, acquired drug resistance through a gradual adaptive response would rarely be expected to be the consequence of changes in the expression of one gene product. We have used a number of drug-resistant human cell lines to characterize those genes that are implicated in maintaining a resistant phenotype. Human HT29 colon cancer cells chronically exposed to ethacrynic acid (EA) [a glutathione (GSH) and glutathione S-transferase (GST) modulator] have acquired resistance to the drug. Commensurate with resistance, EA is more effectively conjugated to GSH and effluxed from the resistant cells. Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at elevated levels. These include: gamma-glutamyl cysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis); GST pi (the enzyme catalyzing the conjugation reaction); multidrug resistance associated protein (MRP) (the membrane pump responsible for effluxing the conjugate from the cell interior). In addition, other gene products not directly linked with EA metabolism were induced, including dihydrodiol dehydrogenase (an alpha-ketoreductase) (30-fold), DT-diaphorase (threefold), and a transcriptional regulator SSP 3521 (threefold). HL60 cells resistant to a GSH paralog Ter199 also show increased expression of some of these gene products. Furthermore, an adriamycin-resistant human HL60 cell line also shows overexpression of GST pi, gamma-GCS, and MRP, but in addition has approximately 20-fold more DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This enzyme is an early stress response gene that can phosphorylate and activate downstream transcription factors. Such overexpression could impact on the transcriptional control of the other detoxification gene products. Both adriamycin and a typical drug-GSH conjugate (APA-SG) are inhibitors of DNA-PK. Because cellular levels of these conjugates would presumably be a good indicator of stress, it would seem reasonable to speculate that DNA-PK may act as a receiver and transmitter of signals that are crucial to the drug-resistant phenotype. Additionally, this enzyme may prove to be a potentially important target for drug design based upon the inhibitory activity of GSH conjugates.
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PMID:Importance of glutathione and associated enzymes in drug response. 940 35

Cellular detoxification, such as that mediated by the glutathione (GSH) system, is involved in the metabolism of various cytotoxic agents. Little is known, however, about the clinical relevance of cellular detoxification in chemoresistance. To elucidate the relevance of the GSH system to the resistance to chemotherapy observed in patients with ovarian cancer, we assayed the expression of mRNA encoded by the multidrug resistance-associated protein (MRP) and gamma-glutamyl cysteine synthetase (gamma-GCS) genes, as well as the level of GSH protein in 32 patients with epithelial ovarian cancer after chemotherapy. Tumors of 14 of the 32 patients responded to chemotherapy, whereas 18 did not. The levels of MRP and gamma-GCS transcripts in tumors from nonresponders were each about 2-fold higher than in responders. In contrast, the level of GSH did not differ between the two groups. We observed coordinated expression of gamma-GCS mRNA and GSH protein levels, and between gamma-GCS and MRP in nonresponders, but not in responders. Expression of MRP-encoded mRNA did not correlate to GSH level, however, in either group. These results suggest that gamma-GCS may up-regulate GSH and MRP expression in tumors unresponsive to chemotherapeutic agents, and that the GSH system may be involved in the mechanism of chemoresistance in ovarian cancer.
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PMID:Gamma-glutamyl cysteine synthetase up-regulates glutathione and multidrug resistance-associated protein in patients with chemoresistant epithelial ovarian cancer. 967 49

The relationship between l-cystine transport and intracellular glutathione (GSH) levels was investigated in cultured pancreatic AR42J acinar and betaTC3 islet cells exposed to diethylmaleate, an electrophilic agent known to activate cellular antioxidant responses. Cystine transport was mediated predominantly by the Na+-independent anionic amino acid transport system x-c, with influx inhibited potently by glutamate and homocysteate but unaffected by cationic or neutral amino acids. Saturable cystine transport was 10-fold higher in AR42J (531 pmol (mg protein)-1 min-1) than in betaTC3 (49 pmol (mg protein)-1 min-1) cells, and GSH levels were higher in AR42J cells. Treatment with 2-mercaptoethanol increased GSH levels in betaTC3 cells from 7.5 to 36 nmol (mg protein)-1, whilst the GSH content in AR42J cells (64 nmol (mg protein)-1) was not altered significantly. Incubation of AR42J or betaTC3 cells with homocysteate (2.5 mM, 0-48 h), a competitive inhibitor of cystine transport via system x-c, reduced intracellular GSH levels and resulted in a time-dependent (6-24 h) induction of system x-c transport activity. Treatment of AR42J cells with diethylmaleate (100 microM, 0-48 h) resulted in a time- (5-10 h) and protein synthesis-dependent induction of cystine transport, with intracellular GSH levels initially decreasing and then increasing 2-fold above control levels after 24 h. Diethylmaleate also depressed GSH levels in betaTC3 cells, but cystine transport was not elevated significantly. In both AR42J and betaTC3 cells, inhibition of gamma-glutamyl cysteine synthetase by buthionine sulphoximine (100 microM, 24 h) reduced GSH levels but had no effect on cystine transport. The present findings establish that induction of system x-c leads to changes in GSH levels in pancreatic AR42J acinar and betaTC3 islet cells, with changes in the intracellular redox state stimulating transporter expression. Induction of activity of system x-c, together with adaptive increases in GSH synthesis in response to oxidative stress, may contribute to cellular antioxidant defences in pancreatic disease.
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PMID:Induction of cystine transport via system x-c and maintenance of intracellular glutathione levels in pancreatic acinar and islet cell lines. 980 3

Glutathione (GSH) levels are supposed to determine the vulnerability of many cells towards a wide array of insults. We investigated the effects of chronic inhibition of GSH synthesis and acute depletion of GSH on cerebellar granule neurons in vitro and determined cytoplasmic and mitochondrial GSH with relation to mitochondrial function and generation of reactive oxygen intermediates (ROI). l-buthionine sulfoximine (BSO), which irreversibly blocks gamma-glutamyl-cysteine synthase, led to a time- and concentration-dependent loss of cytoplasmic GSH, while mitochondrial GSH was relatively preserved. No increased generation of ROI was detected over 48 h and the mitochondrial membrane potential was largely maintained. Neuronal degeneration occurred when mitochondrial GSH levels had fallen below 50% of control after 24-36 h. In contrast, direct conjugation of mitochondrial and cytoplasmic GSH with etacrynic acid (EA), resulted in immediate loss of mitochondrial GSH, a large increase of ROI within 2 h, subsequent collapse of the mitochondrial membrane potential and complete cell death within 4-8 h. Electron microscopy studies revealed an as yet unknown change of the chromatin structure to a homogeneous granular pattern after BSO, while EA resulted in typical necrotic changes. No typical features of apoptosis, i.e., no chromatin condensation or DNA fragmentation were detected after GSH depletion after BSO or EA treatment.
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PMID:Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function. 1021 96

A wide spectrum of human lung diseases is characterized by the presence of granulomas. Although understanding of the pathways leading to their development remains incomplete, data from in vitro studies suggest that neutrophils, monocytes, and their secreted products (eg, hydrogen peroxide, H2O2) influence the pathogenesis of pulmonary granulomatous disease through the regulation of local chemokine and cytokine production. Using a well-characterized rat model of glucan-induced pulmonary granulomatous vasculitis, we sought to determine the role of intracellular glutathione (GSH) redox status in the expression of monocyte chemoattractant protein-1 (MCP-1). Previous studies have revealed that vascular wall MCP-1 expression is obligatory for granuloma development and that both neutrophils and hydrogen peroxide are required for MCP-1 induction. Because in vitro expression of MCP-1 is in part mediated by the redox-sensitive transcription factors nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), we studied their activation as a function of varying intracellular GSH redox status in the pathogenesis of glucan-induced pulmonary granulomatosis. Infusion of particulate yeast cell wall glucan into rats resulted in a rapid decrease in intracellular GSH concentrations which was accompanied by the activation of NF-kappaB and AP-1. The pattern of AP-1 and NF-kappaB activation in turn correlated temporally with the expression of MCP-1. Administration of L-buthionine-S, R-sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase, resulted in a significant reduction in intracellular GSH pools. GSH depletion resulted in a more than 100% increase in pulmonary MCP-1 concentrations and increased cytosolic to nuclear translocation of NF-kappaB while having no effect on AP-1 levels. These observations suggest that in the pathogenesis of pulmonary granulomatous disease, intracellular glutathione redox status modulates the expression of MCP-1 through redox-sensitive transcription factors.
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PMID:Intracellular glutathione redox status modulates MCP-1 expression in pulmonary granulomatous vasculitis. 1041 24

1-Cyano-3,4-epithiobutane (CEB), a naturally occurring nitrile derived from cruciferous plants, causes nephrotoxicity and increased renal glutathione (GSH) concentration in male F-344 rats. This CEB-induced nephrotoxicity is dependent on GSH conjugation and bioactivation. The objectives of the present study were to investigate the effect of CEB on several xenobiotic-metabolizing enzymes and to evaluate the effect of modulators of GSH transport and metabolism on CEB-induced nephrotoxicity and GSH concentration. Animals received 125 mg kg-1 CEB alone or following pretreatment with one of three selective inhibitors of GSH metabolism: acivicin, probenecid or aminooxyacetic acid. There were no significant alterations in epoxide hydrolase (EH), P-450, ethoxyresorufin O-deethylase (EROD) or pentoxyresorufin O-depentylase (PROD) enzyme activity, but renal glutamyl cysteine synthetase (GCS) activity was decreased at 12 and 24 h, as was renal glutathione S-transferase 4 h after CEB administration. Renal ECOD activity was also diminished at 24 h and at 12 and 24 h in liver. Aminooxyacetic acid (AOAA) abrogated the nephrotoxicity, the renal GSH-enhancing effect, and decreased GCS of CEB alone. These findings provide further evidence for the importance of GSH conjugation as a significant pathway in CEB metabolism and the role of a reactive thiol in nephrotoxicity and altered renal GSH.
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PMID:Protection from 1-cyano-3,4-epithiobutane nephrotoxicity by aminooxyacetic acid and effect on xenobiotic-metabolizing enzymes in male Fischer 344 rats. 1043 37

The peptidomimetic drug gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine diethyl ester (TER199) is an analog of glutathione designed to be an isozyme-specific inhibitor of GSTP1-1 protein1-1. This compound (and the de-esterified moiety) is shown to be an effective inhibitor of multidrug resistance-associated protein1 (MRP1)-mediated drug resistance. Kinetic analyses revealed that gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine reversibly inhibits the transport of 2,4-dinitrophenyl-S-glutathione with a K(i) of 752 microM. TER199 reversed the accumulation deficit of daunorubicin in MRP1-transfected NIH3T3 fibroblasts and maintained intracellular levels for >2 h after daunorubicin removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1-transfected NIH3T3 cells for vincristine, doxorubicin, etoposide, and mitoxantrone. HL-60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of multidrug resistance-associated protein, MRP1, and gamma-glutamyl cysteine synthetase heavy and light subunits (the rate-limiting enzyme in GSH synthesis). In spite of increased gamma-glutamyl cysteine synthetase, their glutathione content was reduced approximately 35% from that of parental HL-60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 overexpressing phenotype, with resistance to Vinca alkaloids, epipodophyllotoxins, and anthracyclines; additional cross-resistance to paclitaxel (Taxol), mitoxantrone, and 5-fluorouracil was observed.
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PMID:Glutathione peptidomimetic drug modulator of multidrug resistance-associated protein. 1056 60

Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimer's disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.
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PMID:Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease. 1056 22

Previous studies indicate that the ability of cells to up-regulate levels of intracellular glutathione (GSH) synthesis may determine their sensitivity to MeHg exposure. The purpose of the current study is two-fold. First, we determined whether the vulnerability of the developing central nervous system (CNS) to MeHg lies in its intracellular GSH content. The intracellular GSH content and the activity of gamma-glutamyl cysteine synthetase (GCS) were determined with and without MeHg exposure in primary cultures of rat embryonic CNS cells. In addition, the effect of GSH modulation on MeHg-induced cytotoxicity was determined. Second, we characterized the mechanism of GCS regulation, initially by studying the GCS heavy chain subunit (GCS-HC). Primary embryonic limb bud cells were used as a reference cell type for comparing the response of CNS cells. The results indicate that constitutive intracellular GSH content, GCS activity, and GCS-HC mRNA and protein levels of CNS cells were approximately ten-, two-, five-, and ten-fold higher, respectively, than those in limb bud cells. A dose-dependent increase in GSH levels and GCS activity was observed in CNS and limb bud cells following 1 and 2 microM MeHg exposure for 20 hr. Further characterization of GCS up-regulation in CNS cells showed that the increase in GCS activity following MeHg exposure, unlike limb bud cells, was not accompanied by an elevation of GCS-HC mRNA and protein levels. Pretreatment with N-acetylcysteine led to a significant increase in intracellular GSH, while L-buthionine-(S,R)-sulfoximine (BSO) resulted in decreased GSH levels, however neither pretreatment had a significant impact on MeHg-induced cytotoxicity in either cell type. Our results suggest that although oxidative stress may mediate aspects of MeHg toxicity, disruption of GSH homeostasis alone is not responsible for the sensitivity of embryonic CNS cells to MeHg.
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PMID:The role of intracellular glutathione in methylmercury-induced toxicity in embryonic neuronal cells. 1059 15

Glutathione metabolism during infection has been poorly documented. Glutathione concentrations and synthesis rates were studied in infected rats (2 d after infection) and in pair-fed controls. Glutathione synthesis rates were determined in liver, spleen, lung, small and large intestine, skeletal muscle, heart and blood by a 4-h or 6-h (15)N cysteine infusion. The activities of four hepatic enzymes involved in glutathione metabolism were also determined. Glutathione synthesis rates were significantly greater in liver (+465%), spleen (+388%), large intestine (+109%), lung (+100%), muscle (+91%) and heart (+80%) of infected rats compared with pair-fed controls. Glutathione concentrations were also greater in these tissues but were unaffected in small intestine and lower in blood. In keeping with the stimulation of liver glutathione synthesis, the activities of liver gamma-glutamyl-cysteine synthetase and glutathione reductase were significantly greater in liver of infected rats than of pair-fed rats. From the present study, we estimate that glutathione synthesis accounts for at least 40% of the enhanced cysteine utilization during infection. This increased utilization may be the primary cause of an enhanced cysteine requirement in infection.
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PMID:Glutathione turnover is increased during the acute phase of sepsis in rats. 1080 25


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