Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

gamma-Glutamyl hydrolase (also known as conjugase) is a ubiquitous enzyme that has the capacity to cleave folyl- and antifolylpolyglutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. H35 cells have lower cellular levels of gamma-glutamyl hydrolase than do hepatocytes but secrete a greater proportion of gamma-glutamyl hydrolase. More than 99% of the total enzyme from H35 cells accumulated in the medium after 48 h. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase, and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. Using the substrate 4-amino-10-methyl-pteroyldiglutamate (4-NH2-10-CH3-Pte-Glu2), the intracellular and secreted enzyme form(s) from H35 cells were found to have the following properties (a) Km values of 24.3 +/- 3.7 microM and 34.8 +/- 8.6 microM, respectively, and (b) maximal activity at pH 5 to 7 and apparent molecular weights of 120,000 by gel filtration. Both the cellular and secreted enzymes convert 4-NH2-10-CH3-PteGlu4 and pteroylpentaglutamate acid, to the corresponding monoglutamates with little or no appearance of intermediate chain length polyglutamates. This suggests that both act primarily as endopeptidases. Thus far, the cellular and secreted enzymes cannot be differentiated although the current studies do not establish this point unequivocally. Alterations in the cellular and secreted H35 cell gamma-glutamyl hydrolase levels in response to changes in culture conditions revealed that glutamine enhances activity while insulin diminishes it. Other transformed cells found to secrete this protein are Hep-G2 human hepatoma, JAR human choriocarcinoma, HeLa, and rat glioma. gamma-Glutamyl hydrolase could not be detected in medium conditioned by human MCF-7 breast cancer cells, and relatively low activities were found in the medium from CCRF-CEM or K562 leukemia cells. These studies directly establish for the first time the secretion of gamma-glutamyl hydrolase in vitro.
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PMID:Secretion of gamma-glutamyl hydrolase in vitro. 171 22

Methotrexate (MTX), a widely used antineoplastic agent, is metabolized to a non-active derivative, 7-OH-MTX, and to some active poly-gamma-glutamyl derivatives (MTX-Gn) which are retained within cells. Pharmacokinetic studies in humans indicate (i) a higher concentration of 7-OH-MTX than of MTX in plasma after a 24-h infusion and (ii) a time-dependent relationship for MTX and 7-OH-MTX kinetics in plasma and urine which might be explained by the variation of MTX metabolism. The intracellular metabolism of MTX and 7-OH-MTX has been investigated using a specific ion-paired method of high-performance liquid chromatography (HPLC) which permits the simultaneous determination of DAMPA, MTX, 7-OH-MTX and their respective polyglutamate derivatives. The formation of 7-OH-MTX polyglutamates and the possible effect of 7-OH-MTX on the transport and/or metabolism of the unchanged MTX in a human acute lymphoblastic leukaemia cell line (Molt 4) have been studied. After incubation of the cells to 1 microM (3H) 7-OH-MTX, four radiolabelled peaks, representing 75% of the intracellular 3H, were converted to 7-OH-MTX upon exposure to hog kidney conjugase indicating the formation of 7-OH-MTX polyglutamyl derivatives. The effects of 7-OH-MTX on MTX-PG formation were analysed after simultaneous incubation of the cells to 1 microM (3H) MTX and 10 microM 7-OH-MTX. The formation of the higher glutamyl derivatives, MTX-G3 and MTX-G4 was completely inhibited and the total intracellular accumulation of the MTX-PG's decreased by 35% compared to control. These data suggest that the 7-OH-MTX and the 7-OH-MTX-PG might modify the chemotherapeutic activity of this agent in vivo.
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PMID:In vitro approach to 7-hydroxymethotrexate interaction on methotrexate metabolism as tool of pharmacokinetic study. 659 Apr 70

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.
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PMID:Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM. 783 26

We have applied capillary electrophoresis to the separation of methotrexate (MTX)-polyglutamates, and gamma-glutamyl hydrolase (GGH) activities in tumor cells were measured by using this new analytical method. MTX-polyglutamates were sufficiently separated in 15min by capillary electrophoresis with silica fused capillary (phi 50 microns x 75cm), being electrophoresed at 25kV and 30 degrees C in a buffer which contained 20mM sodium tetraborate, 20mM SDS and adjusted pH to 9.5. MTX-polyglutamates eluted were detected at 300nm UV. Cellular extracts obtained from the sensitive and antifolate-resistant human leukemia cell lines, MOLT-3 and K562, were incubated with MTX-glu5 at 37 degrees C for 1, 2 and 4 hr, and the amounts of the degradation products (glu1-glu4) were measured for GGH activity by capillary electrophoresis. There was no significant difference in the production of the metabolites between MOLT-3 and K562 cells (867 +/- 109 vs 799 +/- 56 pmol products/min/1 x 10(7) cells), however, the MTX-resistant MOLT-3 cells with a diminished polyglutamation of folates (MOLT-3/MTX.P-17) and the ZD1694-resistant K562 cells with the impaired membrane transport for reduced folates/MTX/ZD1694 (K562/ZD1694.C) showed decreased activities of GGH (519 +/- 52 and 680 +/- 99 pmol products/min/1 x 10(7) cells, respectively), suggesting the down-regulation of the enzyme in these antifolate-resistant cells concomitant with the intracellular substrate depletion. This study indicates that capillary electrophoresis is a rapid, cost-efficacious method with a sufficient reproducibility in the measurement of GGH activity and must be more suitable for the analysis of clinical samples than HPLC method which requires a large volume of the material.
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PMID:[Separation of methotrexate-polyglutamates by capillary electrophoresis and its application to the measurement of gamma-glutamyl hydrolase activity in human leukemia cells in culture]. 869 40

Decreased methotrexate (MTX) long-chain polyglutamate formation is associated with MTX resistance whereas high levels of MTX polyglutamate accumulation are found in the blasts of leukemia patients who respond to therapy and have improved outcome. The steady-state level of long-chain MTX polyglutamates depends on the balance of activities of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamates to MTX in a gamma-carboxyl linkage, and gamma-glutamyl hydrolase (GGH) or conjugase, which sequentially removes the terminal glutamate residue of MTX polyglutamates. FPGS and GGH activities as well as the formation of total and long-chain MTX polyglutamates were measured after incubation with [3H]MTX in 15 blast samples from patients with acute leukemias (myeloid and lymphoid). The ratio between GGH and FPGS activities was better at predicting the amount of polyglutamate accumulated in the 24-h [3H]MTX assay compared to the determination of either activity alone. The linear regression curve relating the relative levels of long-chain polyglutamates/total polyglutamates with the ratio of GGH/FPGS showed an r value of 0.81 (P < 0.001). These data suggest that the evaluation of both these enzymes at diagnosis may be used as a predictor of MTX polyglutamylation and therefore for response to MTX therapy and outcome.
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PMID:gamma-Glutamyl hydrolase and folylpolyglutamate synthetase activities predict polyglutamylation of methotrexate in acute leukemias. 930 33

A previously identified cDNA encoding a human gamma-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had molecular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein contained asparagine-linked glycosylation. Sequence analysis of the expressed protein indicated that a 24-amino-acid signal peptide had been removed. A polyclonal antibody to the expressed enzyme was used in Western blot analysis of partially purified lysates of HL-60 promyeloid leukemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamates. Methotrexate-gamma-Glu is a poor substrate for the human enzyme relative to methotrexate gamma-Glu2-5. During hydrolysis of methotrexate-gamma-Glu4, all possible pterin-containing cleavage products (methotrexate and methotrexate-gamma-Glu1-3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate gamma-linkages of methotrexate polyglutamates. Glutamate was released as either glutamic acid or gamma-Glu2. Longer chain species of gamma-Glun>2 were not observed. Inhibition by iodoacetic acid suggested that both the expressed enzyme and the HL-60 enzyme may contain a catalytically essential cysteine. These results indicate that the identified cDNA encodes the intracellular gamma-glutamyl hydrolase found in a variety of human tumor cells and that the baculovirus-expressed enzyme is a suitable model for further structural and enzymatic studies.
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PMID:Characterization of human cellular gamma-glutamyl hydrolase. 961 6

Methotrexate (MTX) is an antifolate that is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL) and a number of other malignant and nonmalignant diseases. Within cells, MTX is metabolized to more active methotrexate polyglutamates (MTXPG), and these polyglutamates are subsequently cleaved in lysosomes by gamma-glutamyl hydrolase (GGH). GGH is reported to act as either an endopeptidase or an exopeptidase, exhibiting species differences in these functions. To better define the in vivo functions of GGH in human leukemia cells, we characterized GGH activity with different MTXPG substrates (MTX with three to five glutamates) in human T- and B-lineage leukemia cell lines, and in primary leukemia cells from newly diagnosed patients with ALL. Parameters estimated from fitting a series of hypothetical mathematical models to the data revealed that the experimental data were best fit by a model where GGH simultaneously cleaved multiple glutamyl residues, with highest activity at cleaving the outermost or two outermost residues from a polyglutamate chain. The model also revealed that GGH has a higher affinity for longer chain polyglutamates. Together, these findings provide new insights to the intracellular disposition of MTX in human ALL cells, and provides a mechanism-based model for characterizing differences among patients and genetic subtypes of ALL.
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PMID:Methotrexate intracellular disposition in acute lymphoblastic leukemia: a mathematical model of gamma-glutamyl hydrolase activity. 1211 48

Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymphoblastic leukemia (ALL). Interindividual differences in lymphoblast accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), may contribute to the effectiveness of treatment among ALL subtypes. To better understand these differences in MTXPG accumulation, we developed a model to characterize the cellular influx and efflux of MTX, formation of MTXPG by the addition of glutamyl residues catalyzed by FPGS (folylpolyglutamate synthetase), and cleavage of glutamyl residues from MTXPG by GGH (gamma-glutamyl hydrolase). The model was fitted to in vivo intracellular MTXPG concentrations measured serially in leukemic blasts from 20 newly diagnosed patients with ALL treated with 24-h intravenous infusions of MTX. The observed median concentrations of total MTXPG at 44 h was higher in B-lineage than in T-cell ALL (1706 vs 518 pmol/10(9) cells, P<0.025), consistent with the higher estimated Vmax for FPGS activity in B-lineage vs T-lineage blasts (414 vs 93 pmol/10(9) cells/h, P<0.008). Simulations based on the model-estimated parameters indicated greater accumulation of MTX, MTXPGs (MTXPG(2-7)) and total MTX (MTXPG(1-7)) with longer MTX infusions and with higher MTX doses, with the highest concentrations in hyperdiploid B-lineage, intermediate in non-hyperdiploid B-lineage, and lowest in T-cell ALL. These differences provide mechanistic and treatment insights for lineage and ploidy differences in MTXPG accumulation in human leukemia cells in vivo.
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PMID:A mathematical model of in vivo methotrexate accumulation in acute lymphoblastic leukemia. 1243 1

We found a significant inverse relationship between gamma-glutamyl hydrolase (GGH) activity and the accumulation of long-chain methotrexate polyglutamates (MTXPG4-7) in non-hyperdiploid B-lineage acute lymphoblastic leukaemia (ALL) cells after uniform treatment with high-dose methotrexate (HDMTX) (1 g/m i.v.). To identify genetic polymorphisms that alter the function of human GGH, we sequenced the GGH exons of genomic DNA from children with ALL, who had a 7.8-fold range of GGH activity in their ALL cells at diagnosis. A single nucleotide polymorphism (452C>T, T127I) was found among patients with low GGH activity, but not found in patients with high GGH activity. Computational modelling indicated that the T127I substitution alters the molecular surface conformation at the catalytic cleft-tail on GGH, which is predicted to alter binding affinity with long chain but not short-chain methotrexate polyglutamates. Enzyme kinetic analysis of heterologously expressed GGH revealed a significantly higher Km (2.7-fold) and lower catalytic efficiency (Vmax/Km reduced 67%) of the T127I variant compared to wild-type GGH using long-chain MTXPG5 as substrate, but not a significant change with short-chain MTXPG2. The 452C>T single nucleotide polymorphism (SNP) was also associated with lower GGH activity in hyperdiploid B-lineage and T lineage ALL cells. Caucasians [10.0%; 95% confidence interval (CI) 6.7-13.3%; n = 155] were found to have a significantly higher frequency of the Ile allele than African-Americans (4.4%; 95% CI 1.2-7.5%; n = 80) (P = 0.033). These studies demonstrate a substrate specific functional SNP (452C>T) in the human GGH gene that is associated with lower catalytic activity and higher accumulation of long-chain MTX-PG in leukaemia cells of patients treated with HDMTX.
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PMID:A substrate specific functional polymorphism of human gamma-glutamyl hydrolase alters catalytic activity and methotrexate polyglutamate accumulation in acute lymphoblastic leukaemia cells. 1528 38

The nature of mendelian inheritance assumes that all tissues in which a phenotype of interest is expressed have a uniform diploid karyotype, which is often not the case in cancer cells. Owing to nonrandom gains of chromosomes, trisomies are present in many cases of leukemia and other malignances. We used polymorphisms in the genes encoding thiopurine S-methyltransferase (TPMT), gamma-glutamyl hydrolase (GGH) and the reduced folate carrier (SLC19A1) to assess the nature of chromosomal acquisition and its influence on genotype-phenotype concordance in cancer cells. TPMT and GGH activities in somatic cells were concordant with germline genotypes, whereas activities in leukemia cells were determined by chromosomal number and whether the acquired chromosomes contained a wild-type or variant allele. Leukemia cells that had acquired an additional chromosome containing a wild-type TPMT or GGH allele had significantly lower accumulation of thioguanine nucleotides or methotrexate polyglutamates, respectively. Among these genes, there was a comparable number of acquired chromosomes with wild-type and variant alleles. Therefore, chromosomal gain can alter the concordance of germline genotype and cancer cell phenotypes, indicating that allele-specific quantitative genotyping may be required to define cancer pharmacogenomics unequivocally.
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PMID:Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes. 1604 4


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