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)

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.
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PMID:Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization. 816 96

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.
Leukemia 1993 Dec
PMID:Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines. 825 99

The de novo purine synthesis inhibitor 5,10-dideazatetrahydrofolate (DDATHF) has previously been shown to inhibit the growth of mouse L1210 and human CCRF-CEM leukemia cells. The present study demonstrates that both the 6R and 6S diastereomers of DDATHF are also cytotoxic to mammalian cells in a stereospecific manner. The cytotoxic potency of (6R)-DDATHF (also known as Lometrexol) towards different cell lines varied by approximately 14-fold and that of (6S)-DDATHF by as much as 156-fold. Compared to (6R)-DDATHF, (6S)-DDATHF was 6.0- and 7.2-fold more cytotoxic to human WiDr colon adenocarcinoma and Chinese hamster ovary (CHO) cells, respectively, and only 1.5- and 2.0-fold more cytotoxic to human T24 bladder carcinoma and mouse L1210 leukemia cells, respectively. However, compared to (6S)-DDATHF, (6R)-DDATHF was 8.7- and 6.9-fold more cytotoxic to C3H/10T1/2 clone 8 and clone 16 mouse fibroblasts, respectively. Weak inhibition of aminoimidazolecarboximide ribonucleotide formyltransferase (AICARFT, EC 2.1.2.3) appeared to have little role in the cytotoxicity of DDATHF diastereomers to WiDr cells during a 24-h exposure. Although glycinamide ribonucleotide formyltransferase (GARFT, EC 2.1.21) is the main biochemical target of DDATHF, DDATHF stereoisomers' cytotoxic potency showed no clear negative correlation with cellular GARFT levels. However, cellular folylpolyglutamate synthetase (FPGS, EC 6.3.2.17) levels correlated with cytotoxic potency in a positive manner. Surprisingly, two enzyme-dose/DDATHF LD90-response curves were observed for FPGS corresponding to differences in (6R) and (6S)-DDATHF cytotoxic potency among the six cell lines studied.
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PMID:The stereospecific cytotoxic potency of (6R) and (6S)-5,10- dideazatetrahydrofolate correlates with cellular folylpolyglutamate synthetase levels. 858 57

A clinically relevant mechanism of acquired methotrexate (MTX) resistance is decreased MTX polyglutamate (MTXGn) synthesis (McCloskey et al. J. Biol. Chem. 266:6181, 1991) secondary to deficient folylpolyglutamate synthetase (FPGS) activity. Earlier studies showed that this mechanism resulted after intermittent MTX treatment, but did not define its evolution in populations or its clonal frequency of occurrence and heterogeneity. We thus studied evolution of resistance in CCRF-CEM human leukemia cell populations and clones after repeated treatment with 30 microM MTX for 24 h. In populations, MTX resistance was detectable after 1 treatment and increased in degree as total cycles increased to 7. Defective MTXGn synthesis in populations was the only resistance mechanism detected, and FPGS activity in extracts decreased with treatment cycle. After 1 treatment, defective MTXGn synthesis was the major (27/48 clones) form of resistance; 18 clones were sensitive, while 1 clone with a DHFR-related change, no clones with decreased MTX uptake, and 2 complex phenotypes were observed. Sporadic clones with DHFR-mediated resistance appeared up to cycle 4, but defective MTXGn synthesis remained the major resistance mechanism. The degree of clonal resistance tended to increase with treatment cycle, but 1 clone in cycle 2 was similar to the clones from cycle 7. No change in FPGS gene copy number or restriction pattern, or FPGS mRNA level or size (2.3 Kb) was detected in populations. Decreased FPGS activity must result from decreased translation, increased protein turnover, or a point mutation affecting catalysis.
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PMID:Evolution of drug resistance in CCRF-CEM human leukemia cells selected by intermittent methotrexate exposure. 886 66

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 microM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 microM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuire et al., Adv. Exptl. Med. Biol. 163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (K(ii) = 0.9 microM; K(is) = 1.1 microM) and glutamic acid (K(ii) = 1.0 microM; K(is) = 5.2 microM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (K(ii) = 3.4 microM; K(is) = 0.35 microM), since the major effect is on its K(m). Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 microM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 microM; MTX, 48 microM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideout et al. (Int. J. Cancer 61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.
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PMID:Potent inhibition of human folylpolyglutamate synthetase by suramin. 891 44

Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) (p < 0.01) and was characterized by a saturable (Emax) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 microM), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (Emax in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4-6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC50) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.
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PMID:Differences in folylpolyglutamate synthetase and dihydrofolate reductase expression in human B-lineage versus T-lineage leukemic lymphoblasts: mechanisms for lineage differences in methotrexate polyglutamylation and cytotoxicity. 922 25

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.
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PMID:Disparate affinities of antifolates for folylpolyglutamate synthetase from human leukemia cells. 924 58

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 single human gene encodes both mitochondrial and cytosolic isoforms of the enzyme. The major mRNA species in human cells encodes the mitochondrial isoform but alternate translation initiation at a downstream in-frame ATG also generates the cytosolic isoform. Cytosolic FPGS may also be generated by use of alternate transcription initiation start sites 3' to the start ATG of the mitochondrial FPGS. Three additional human FPGS mRNAs differing in exon 1 have been identified. One of these is a major species in HEP-G2 cells and other tissue culture cells, and can encode a protein lacking the first 8 amino acids of cytosolic FPGS. A protein of the predicted size is observed in coupled transcription/translation systems. However, expression of this protein in E. coli does not generate an active enzyme. Mutagenesis studies indicate that Tyr-3 of the missing N terminal residues is required for enzyme activity. The major cellular folate pools are in the cytosol and mitochondria and FPGS activity is normally distributed in both compartments. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, and cells lacking this isozyme are auxotrophic for glycine. Overexpression of cytosolic FPGS does not complement the lack of mitochondrial activity. Cells expressing FPGS activity solely in the mitochondria are glycine prototrophs, but also possess cytosolic folylpolyglutamates and are prototrophic for thymidine and purines, products of cytosolic one carbon metabolism. Although cytosolic folylpolyglutamates cannot enter the mitochondrion, mitochondrial folylpolyglutamates are released intact into the cytosolic compartment. Cellular accumulation of some antifolates and their cytotoxic efficacy is highly responsive to the level of FPGS activity. Polyglutamylation of methotrexate (MTX) has little affect on its affinity for dihydrofolate reductase, its target enzyme, but does affect the cellular accumulation of the drug. The sensitivity of model cells, expressing a range of FPGS activities similar to that observed in leukemia blasts, to MTX varied over four orders of magnitude. MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX. The cytotoxic efficacy of other folate antagonists that are transported into the mitochondria was enhanced by mitochondrial FPGS activity, even when their loci of inhibition was a cytosolic enzyme. Mitochondrial metabolism of these drugs increased cytosolic drug levels. Compartmentalization of antifolate metabolism has to be considered in evaluating mechanisms for increased drug cytotoxicity and for the development of acquired resistance to these agents.
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PMID:Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity. 1047 Mar 77

We investigated the expression of the folylpolyglutamate synthetase (FPGS) gene at the mRNA level in MOLT-3 and K562 human leukemia cell lines sensitive, or made resistant, to methotrexate (MTX) and/or trimetrexate (TMQ), or raltitrexed (ZD1694). Northern blot analysis demonstrated approximately 3-fold higher FPGS mRNA expression in K562 cells than that in MOLT-3 cells, being consistent with graded polyglutamation capacities of these cell lines. A slight increase in the expression of the FPGS gene was observed in the TMQ-resistant MOLT-3 cells (MOLT-3/TMQ800); moreover, sequential development of MTX resistance in the TMQ-resistant cells (MOLT-3/TMQ800-MTX10,000) resulted in a further enhancement of FPGS mRNA expression despite of decreased polyglutamation capacity in this subline. Another MTX-resistant subline with impaired reduced folate carrier (MOLT-3/MTX10,000) also showed overexpression of FPGS mRNA. Conversely, both raltitrexed-resistant sublines (MOLT-3/ZD1694 x C and K562/ZD1694 x C) displayed a moderately decreased expression of FPGS mRNA. These findings did not correspond to the virtual absence of ZD1694 polyglutamates inside the former cells nor to possibly intact polyglutamation capacity in the latter cells. These results indicate that FPGS mRNA expression may predict cellular ability to produce polyglutamate metabolites of antifolate drugs in the sensitive cells, but does not necessarily reflect FPGS function at the enzyme level in the antifolate-resistant tumor cells.
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PMID:Variable expression of the folylpolyglutamate synthetase gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant, to various antifolate drugs. 1050 18


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