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PubMed1989

Inhibition of glycinamide ribonucleotide formyltransferase and other folate enzymes by homofolate polyglutamates in human lymphoma and murine leukemia cell extracts.

In order to determine the biochemical basis for the cytotoxicity of homofolates, poly-gamma-glutamyl derivatives of homofolate (HPteGlu) and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT), thymidylate synthase, and serine hydroxymethyltransferase (SHMT) in extracts of Manca human lymphoma and L1210 murine leukemia cells. The most striking inhibitions are that of GARFT by (6R,S)-H4HPteGlu4-6 with IC50 values from 1.3 to 0.3 microM. Both diastereomers, (6R)-H4HPteGlu6 and (6S)-H4HPteGlu6, inhibit GARFT activity. In Manca cell extracts, the (6S) form is more potent than the (6R) form whereas in the murine system the reverse is true. The (6R,S)-H4HPteGlu polyglutamates are weak inhibitors of human AICARFT (IC50, 6-10 microM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu4-6 having IC50 values close to 2 microM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 microM for HPteGlu5-6 and greater than 20 microM for H4HPteGlu1-5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, greater than 20 microM). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 microM, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhibitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 microM, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 microM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.

Acyltransferases↗
PubMed1976

The fate of folate polyglutamates in meat during storage and processing.

The rate of hydrolysis of chicken liver folate polyglutamates, by endogenous liver conjugases, under various conditions of storage, heat, and tissue disruption, were investigated. The procedure used was to allow a radioactive tracer dose of the vitamin to equilibrate into the folate polyglutamyl pool. After various storage periods and treatments the polyglutamyl state of the folate present was examined by analytical techniques based on oxidative degradation of native folate polyglutamates to the corresponding p-aminobenzoylpolyglutamate followed by chromatographic separation on DEAE cellulose anion exchange resin. Identification of folate polyglutamates present was made by simultaneous elution of known p-aminobenzoylpolyglutamate markers. In an intact tissue sample only slight degradation was found after 48 hr at 4 C; complete degradation of folate polyglutamates taking 120 hr. Samples of homogenized tissue show complete degradation to folate monoglutamates and a small amount of diglutamate after 48 hr storage. Superimposed on the above is the consideration that if at any time prior to or during storage the liver is heated to greater than 100 C irreversible inactivation of the endogenous conjugases takes place and the folate polyglutamate pattern is stabilized. It was also demonstrated that during two different heating procedures no extra deconjugation occurred.

Animals↗
PubMed1989

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase.

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.

Animals↗
PubMed1988

[Methotrexate and its drug resistance].

Methotrexate (MTX), a folic acid antagonist, is among the most active known drugs for clinical cancer therapy. The mechanisms of resistance to MTX have been extensively studied, and several mechanisms have been reported so far. Those are decreased cell permeability, increased synthesis of dihydrofolate reductase (DHFR), which is the target enzyme of MTX, decreased polyglutamation of MTX, and decreased binding capacity of MTX to DHFR. Recent basic studies revealed that an increase of DHFR synthesis is the result of a proportional amplification of DHFR gene, and that polyglutamation of MTX plays an important role in its action. High-dose methotrexate (HDMTX) therapy has a good rationale for overcoming MTX resistance. However, many clinical trials of HDMTX during past decade have not demonstrated any apparent clinical efficacy except childhood ALL and ostosarcoma. Further research would be necessary for the improvement of the therapy using MTX.

Acute Disease↗
PubMed1975

Transport and metabolism of folates by bacteria.

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.

Azides↗
PubMed1982

Cell cycle patterns of thymidylate synthetase and 5,10-methylenetetrahydrofolate polyglutamates in cultured mouse hepatoma cells.

The regulation of thymidylate synthetase activity was investigated throughout the first cell cycle after release from an isoleucine block in synchronous cultures of mouse hepatoma (Hepa) cells. Activity in cell extracts increased with the onset of S phase and the increased activity was attributed to a parallel increase in enzyme concentration as determined by titration with tritiated fluorodeoxyuridylate. The polyglutamate chain length of reduced folate cofactors, which could also influence thymidylate synthetase activity, was unchanged.

Animals↗
PubMed1975

Folic acid conjugase from Plasma. I. Partial purification and properties.

Human plasma conjugase was partially purified by ammonium sulphate and DEAE-cellulose column fractionation. The pH optimum was found to be 4.5. The enzyme followed normal Michaelis-Menten Kinetics. The Km value was found to be 1.4 x 10(-7)M for folylheptaglutatmate assuming this compound to be the predominant form of folate in yeast extract. The enzyme required sulfhydryl compounds for full activity. Differential microbiological assay of the conjugase reaction product using L. casei and S. faecalis as the test organisms revealed that during the initial stage of the reaction considerable portion of the reaction product was at polyglutamyl stage. On prolonged incubation polyglutamates decreased due to further hydrolysis of the gamma-glutamyl residues. The plasma conjugase activity of different species of animals showed wide variations. Goat, dog and rabbit plasma had neglibible activity while monkey, guinea pig and chick plasma had moderate activity. Bovine plasma had low activity while rat plasma had high activity. The plasma conjugase activity of normal Indian adults ranged from 400 to 900 units per ml, and there was no significant sex difference. The values obtained are considerably higher than those reported in literature.

Animals↗
PubMed2003

Rational design, synthesis, evaluation, and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid.

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

Binding Sites↗
PubMed1978

Folate-induced remission in aplastic anemia with familial defect of cellular folate uptake.

Severe aplastic anemia developed in a young man with an extensive family history of leukemia, pancytopenia, and neutropenia. Megaloblastic changes became evident, and treatment with high doses of folic acid resulted in striking clinical improvement. However, red-cell folate levels remained persistently low despite high serum folate levels. A defect in cellular folate uptake was suspected, and, indeed, uptake of 5-14CH3-H4-folate by stimulated lymphocytes and by bone-marrow cells from the patient was significantly reduced (P less than 0.05 as compared to normal cells. Further characterization of folate metabolism showed that intestinal absorption of the vitamin, membrane transport of 5-14CH3-H4-folate by mature red cells, folate utilization in the conversion of deoxyuridylate to thymidylate and polyglutamate formation were all normal. At least five other family members manifest decreased uptake of 5-14CH3-H4-folate by stimulated lymphocytes. These studies suggest that a genetically induced abnormality of folate uptake contributed to this patient's severe, but reversible, aplasia.

Adult↗
PubMed1980

Folate assay: a comparison of radioassay and microbiological methods.

Folate mono- and polyglutamate standards were assayed by a microbiological method and by four commercially available radioassay methods to evaluate the usefulness of radioassay techniques for the quantitation of naturally occurring folates. Folate monoglutamates exhibited different responses in the radioassay procedures, depending on the one-carbon constituent and oxidation state. Folate polyglutamates exhibited an increased response that varied depending on the folate concentration. The varied responses of folate mono- and polyglutamates in the radioassay procedures make this technique unsuitable for the determination of the mixture of folate derivatives that are normally encountered in biological extracts.

Biological Assay↗
PubMed1997

Immunosuppressive properties of MX-68, a novel unpolyglutamatable antifolate.

MX-68 is a novel unpolyglutamatable antifolate. We here reported the in vitro and in vivo immunosuppressive properties of MX-68 compared with a polyglutamatable antifolate, methotrexate (MTX). MX-68 showed potent suppressive effects on mitogen-induced mouse splenic lymphocyte proliferation as well as immunoglobulin production from LPS-stimulated mouse splenic B cells. In in vivo studies, MX-68 significantly suppressed antigen-specific antibody production of both T cell-dependent antigen and T cell-independent antigen. Moreover, MX-68 inhibited a sheep red blood cell (SRBC)-induced delayed-type hypersensitivity (DTH) reaction by administration starting from the day of antigen immunization, but did not suppress the effector phase of the DTH reaction. MTX showed suppressive activities similar to MX-68 in all experiments. Interestingly, although MX-68 demonstrated somewhat stronger suppressive effects than MTX in vivo, the results from in vitro studies were reversed. These results suggest that polyglutamation is not always required to suppress immune responses and that MX-68 is a slightly stronger immunosuppressive drug than MTX in mice.

2-Aminoadipic Acid↗
PubMed1989

Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenograft.

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of less than 3 microM. The interaction of polyglutamates of [6R]CH2-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 microM) were greater than 200-fold and [6R]CH2-H4PteGlu2 (18.2 microM) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 microM) at stabilizing ternary complexes for a T1/2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 microM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 x 10(-8) M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 x 10(-9) M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 x 10(6) M-1 min-1 for 10 or 20 microM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 microM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n greater than or equal to 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Adenocarcinoma↗
PubMed1989

Sequence-dependent enhancement of HCT-8 cell kill by trimetrexate and fluoropyrimidines: implications for the mechanism of this interaction.

The new folate antagonist trimetrexate is an inhibitor of dihydrofolate reductase, but unlike methotrexate (MTX) it is not polyglutamylated. We have compared the cell killing effects of MTX and trimetrexate/5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) combinations on HCT-8 cells in vitro, in an attempt to explore indirectly the role of polyglutamylation of the antifol in determining the known sequence-dependent synergism between MTX and FUra. The comparisons were made in a number of equitoxic concentrations and times of exposure. Trimetrexate given for 4, 24 or 48 h followed by FUra, for 4, 24 or 196 h, produced synergistic HTC-8 cell kill, whereas antagonism was observed when FUra preceded or was given simultaneously with trimetrexate. The degree of interaction was essentially identical to those obtained when MTX was combined with FUra. The interactions between MTX/FdUrd and trimetrexate/FdUrd were also similar: synergistic cell kill resulted from the sequences trimetrexate or MTX followed by FdUrd, while additive effects were produced by trimetrexate or MTX + FdUrd combinations or FdUrd followed by MTX or trimetrexate. Because the same interactions observed with MTX/FUra or FdUrd combinations were also obtained when trimetrexate was combined with the fluoropyrimidines, it is unlikely that polyglutamylation of the antifols plays a significant role in determining the different sequence-dependent effects of these antimetabolites. However, these studies do not rule out the possibility that increased levels of dihydrofolate polyglutamates increase fluoropyrimidine cytotoxicity.

Adenocarcinoma↗
PubMed2006

Molecular basis for decreased folylpoly-gamma-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line.

A CCRF-CEM mutant, CEM-p, has been shown to exhibit resistance to methotrexate due to decreased methotrexate polyglutamate accumulation. To ascertain the mechanism(s) responsible for this phenotype, we analyzed FPGS and SLC19A1 mRNA expression, examined FPGS promoter activity, and determined nucleotide sequence of the FPGS promoter and full length cDNA from CCRF-CEM and CEM-p cells. We identified in FPGS from CEM-p cells three amino acid substitutions that altered the ATP binding P-loop, glutamate/folate binding, and a conserved domain located at the carboxyl-terminal. Our data demonstrated for the first time the importance of the highly conserved domain (VTGSLHLVGGV) located at the carboxyl-terminal for FPGS activity.

Amino Acid Sequence↗
PubMed1983

Pteroylpolyglutamates in liver of phenobarbitone-treated rats.

The effects of "acute" phenobarbitone treatment on some aspects of folate metabolism in rat liver, that is total folate activity, protein bound and free folate, and various-length pteroylpolyglutamate amounts, were studied. The administration of two doses of the anticonvulsant drug (8 mg/100 g of body weight), caused a severe depletion in folate pool, free folate and in the percentage of longer-chain polyglutamates, while the amount of protein bound folates was unchanged. Such modifications are more evident in the animals killed at 24 h after the last drug injection rather than in those killed 12 h later. The possible mechanisms by which phenobarbitone might interfere with the synthesis of pteroylpolyglutamates, the actual coenzymic forms of folic acid, are discussed.

Animals↗
PubMed1998

Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy.

Reduced folate carrier (RFC) transcripts in human leukemias were measured by a competitive PCR assay. Total RNAs were reverse transcribed and amplified in the presence of competitive templates for RFC and beta-actin. RFC transcripts were normalized to transcripts for beta-actin. In a series of K562 sublines, a approximately 30-fold range of RFC transcripts measured by PCR assay closely agreed with results of Northern analysis and varied in proportion to RFC protein on Western blots and [3H]methotrexate transport. RFC transcripts varied over a 88-fold range in 49 specimens from 48 children with acute lymphoblastic leukemia (ALL). Median RFC transcripts were similar for 15 T-cell and 33 B-precursor ALL samples (RFC/beta-actin = 6.13 x 10(-3) and 7.92 x 10(-3), respectively) and for 41 diagnostic (7.20 x 10(-3)) and 8 relapse (5.58 x 10(-3)) samples. Whereas PCR measurements of RFC transcripts approximated changes in methotrexate transport in B-precursor ALL blasts (n = 10), for T-ALL blasts (n = 12) there was no apparent relationship between these parameters. For hyperdiploid B-precursor blasts (n = 11) with greater than 52 chromosomes and three to five copies of chromosome 21, the median RFC transcript level was approximately 3-fold higher than that for diploid B-precursor blasts. RFC transcripts were also elevated for two of three B-precursor specimens with acquired trisomy 21. Our results suggest that RFC gene expression is far more predictive of methotrexate uptake capacity in B-precursor than T-ALL and that increased copies of chromosome 21 in B-precursor ALL blasts are generally associated with increased RFC transcripts. Hence, the good prognosis for children with hyperdiploid B-precursor ALL treated with antimetabolite-based chemotherapy and the high levels of methotrexate and methotrexate polyglutamates accumulated may, in part, reflect elevated RFC gene expression and capacities for methotrexate transport.

Adolescent↗
PubMed1992

l-folinic acid versus d,l-folinic acid in rescue of high-dose methotrexate therapy in children.

BACKGROUND: At this time, folinic acid (FA) is commercially available as the racemic mixture d,l-FA, whose biological activity is supported by natural l-FA. The administration of d,l-FA results in the accumulation of d-FA in plasma relative to the active l-FA form; in vitro studies have shown that d-FA can compete with the polyglutamation of methotrexate (MTX). PURPOSE: Our purpose was to compare, on a pharmacokinetic, biological, and clinical basis, the racemic mixture d,l-FA with the pure l-FA in rescue of high-dose MTX therapy (5 g/m2) in children with acute lymphocytic leukemia (ALL). METHODS: Eighteen children with ALL were entered in this trial, which was planned with a crossover design. Four cycles of MTX were administered to each patient, and rescue was achieved orally every 6 hours at a dose of 12 mg/m2 for d,l-FA and 6 mg/m2 for pure l-FA. The d,l-FA and l-FA rescues were alternated from one cycle to the next. d-FA, l-FA, and the active metabolite 5-methyltetrahydrofolate (5-MTHF) were measured in plasma using a stereospecific high-performance liquid chromatography assay. RESULTS: Considering total active folate levels (l-FA + 5-MTHF), mean residual concentrations were similar for rescue by d,l-FA and l-FA, after two and six intakes, respectively: 92 and 186 nM for d,l-FA rescue versus 100 and 184 nM for l-FA rescue. Intra-individual comparison of total active folates (l-FA + 5-MTHF) did not show any significant difference between d,l-FA rescue and l-FA rescue. After administration of d,l-FA, the accumulation of d-FA in plasma was confirmed. For both types of FA rescue, MTX terminal half-lives were identical (average value, 13.9 hours). Considering each type of toxic effect (hematologic, hepatic, renal, and digestive), there was no significant difference in the proportion of toxic cycles following l-FA rescue or d,l-FA rescue. CONCLUSION: The administration of the pure l-FA, compared with the administration of the racemic mixture, results in comparable blood profiles of active folates and MTX, leads to equivalent treatment tolerance, and avoids the plasma accumulation of d-FA.

Adolescent↗