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PubMed1996

Biological activity and intracellular metabolism of ZD1694 in human leukemia cell lines with different resistance mechanisms to antifolate drugs.

The biological activity and cellular metabolism of ZD1694, a novel folate-based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT-3, and its antifolate-resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717). MOLT-3/CB3717(40), which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [3H]ZD1694-polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross-resistance (12-fold) of this subline to ZD1694. On the other hand, there was little or no cross-resistance to this drug in a subline (MOLT-3/TMQ800) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7-fold higher in the TMQ-resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT-3/TMQ800-MTX10000 cells, which were established by sequential exposure of the TMQ-resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross-resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.

Antimetabolites, Antineoplastic↗
PubMed1987

Biochemical factors in the selectivity of leucovorin rescue: selective inhibition of leucovorin reactivation of dihydrofolate reductase and leucovorin utilization in purine and pyrimidine biosynthesis by methotrexate and dihydrofolate polyglutamates.

Recent studies have clarified the critical role that polyglutamylation plays in methotrexate (MTX) action. Polyglutamate derivatives of MTX bind to dihydrofolate reductase (DHFR) with affinities comparable to the monoglutamate, but their retention in cells results in a sustained block in tetrahydrofolate (FH4) synthesis. One important element in the selectivity of MTX action is the preferential buildup and retention of these polyglutamyl forms in susceptible tumor cells as compared to host cells of the bone marrow or gastrointestinal mucosa. This selectivity in the accumulation of MTX polyglutamyl forms has now been further shown to play an important role in the selectivity of leucovorin rescue and may provide a unique new approach to nucleoside protection as well. This paper reviews the current understanding of the biochemical basis for leucovorin rescue and its selectivity. Important elements in leucovorin rescue are reactivation of DHFR with depression of cellular dihydrofolate (FH2) and provision of folate substrate to circumvent the block in FH4 synthesis. Selectivity of leucovorin rescue may be attributed to direct inhibition by MTX polyglutamyl forms, as well as FH2 polyglutamates that accumulate in their presence, at the levels of thymidylate synthase and transformylation during purine nucleotide biosynthesis. The presence of cellular MTX polyglutamates impairs reactivation of endogenous DHFR activity by leucovorin metabolites, and the resultant maintenance of high cellular levels of cellular FH2 and the polyglutamyl derivations of MTX impair the utilization of added FH4 in susceptible tumor cells. This paper also develops the concept of "early" nucleoside protection in antifolate therapy. In this approach, nucleosides are administered simultaneously with a pulse of MTX to provide early host protection from the cytotoxic effects of modest doses of MTX. Cessation of protection occurs at a time when extracellular and intracellular monoglutamate has fallen to low levels, and the polyglutamyl forms of the drug are present in susceptible tumors but not in host tissues of the gut and bone marrow. Data are presented to demonstrate that increased doses of MTX can be administered in normal and tumor-bearing animal systems as well as in humans by this technique.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Hydroxylation of 4-amino-antifolates by partially purified aldehyde oxidase from rabbit liver.

This paper explores the interaction between 4-amino-antifolates and aldehyde oxidase (aldehyde: O2 oxidoreductase, EC 1.2.3.1) that was purified 60- to 120-fold from rabbit liver with yields of 5-15%. The purification procedure consisted of one heat and two ammonium sulfate precipitations followed by chromatography on hydroxylapatite and then Sephacryl S-200. Analysis of initial rates of hydroxylation of methotrexate, aminopterin and dichloromethotrexate indicated an order of affinities of dichloromethotrexate (10 microM) greater than methotrexate (35 microM) greater than aminopterin (272 microM). There was no difference in the Vmax of methotrexate and dichloromethotrexate (248 and 231 nmoles/min/mg protein respectively); aminopterin (130 nmoles/min/mg protein) was less than that of the other two. The Vmax/Km ratios were 24.1, 7.20 and 0.48 for dichloromethotrexate, methotrexate and aminopterin respectively. This enzyme preparation also mediated the hydroxylation of methotrexate polyglutamyl derivatives with a decrease in the rates of hydroxylation, as the total number of glutamyl residues was increased to four, a consequence of a marked increase in Km values and/or decrease in Vmax; the ratios of the Vmax/Km for the di-, tri-, and tetraglutamates were 0.94, 0.31 and 0.21 respectively. This low activity of the polyglutamyl derivatives of methotrexate for aldehyde oxidase is consistent with the observations that the predominant forms of 4-amino-antifolate polyglutamates found in human liver after administration of methotrexate are the polyglutamyl derivatives of the parent compound. Finally, substrate inhibition for methotrexate and dichloromethotrexate was observed at concentrations in excess of 150 and 30 microM, respectively, about 5- and 3-fold higher than their respective Km values. Hence, while dichloromethotrexate had the lowest Km for aldehyde oxidase amongst the 4-amino-antifolates studied, the actual rates of hydroxylation depended upon the concentration employed because of substrate inhibition. Aminopterin was a very poor substrate for this enzyme at low and saturating concentrations. These properties of the hydroxylation of 4-amino-antifolates may be of importance in the design of clinical regimens with these agents--in particular, regimens that employ infusion of these drugs into the hepatic artery. However, the relevance of these observations to the hydroxylation of 4-amino-antifolates by human liver remains to be established.

Aldehyde Oxidase↗
PubMed1999

The capsule of bacillus anthracis, a review

The capsule of Bacillus anthracis, composed of poly-D-glutamic acid, serves as one of the principal virulence factors during anthrax infection. By virtue of its negative charge, the capsule is purported to inhibit host defence through inhibition of phagocytosis of the vegetative cells by macrophages. In conjunction with lethal toxin and oedema toxin, whose target cells include macrophages and neutrophils, respectively, the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. Spores germinating in the presence of serum and elevated CO2 release capsule through openings on the spore surface in the form of blebs which may coalesce before sloughing of the exosporium and outgrowth of the fully encapsulated vegetative cell. It has not been established that spore encapsulation plays a role in the early events of anthrax infection. The capsule appears exterior to the S-layer of the vegetative cell and does not require the S-layer for its attachment to the cell surface. The three membrane-associated enzymes required for synthesis of the capsule are encoded by the 60-MDa pX02 plasmid. The cistrons are arranged in the order capB, capC and capA and encode for proteins of 44, 16 and 46 kDa, respectively. The synthesis of capsule and toxin is, in part, under bicarbonate regulation by interaction of transacting proteins of the atxA gene on the 100-MDa pX01 toxin-encoding plasmid and the acpA gene on the pX02 plasmid. Therefore, capsule synthesis is enhanced in the presence of the atxA gene on the pX01 plasmid. An additional protein (with a predicted size of 51 kDa) is encoded by the dep gene located downstream from the cap region and appears to be a depolymerase that catalyses the hydrolysis of poly-D-glutamic acid into lower molecular weight polyglutamates. Although the biological function of the Dep protein is unknown, it has been proposed that the low molecular weight polyglutamates produced by the action of the enzyme may act to inhibit host defence mechanisms.

Journal Article↗
PubMed1987

Kinetics of methotrexate and its metabolites in red blood cells.

It has been suggested that the intra-erythrocyte levels of methotrexate (MTX) and its polyglutamized derivatives could offer a method for evaluating intracellular MTX accumulation and its metabolism in children with acute lymphoblastic leukemia. We were interested in measuring the intra-erythrocyte levels of MTX in patients with solid tumors receiving high-dose MTX treatment. After a first incorporation stage occurring during infusion, MTX concentrations subsequently increased 9-12 days after the treatment as polyglutamized derivatives. Thirty days after the infusion, MTX and its polyglutamates were still measurable in erythrocytes. The percentage of polyglutamization varied on an individual basis, but two groups of patients could be separated according to their ability to form polyglutamates. We also noted the presence of 7-hydroxy-methotrexate (7-OH-MTX) appearing 48 hours after the beginning of the infusion which was still present in 17/20 samples 30 days after the treatment.

Adult↗
PubMed1982

Factors controlling the concentrations of methotrexate in cultured hepatic cells.

The polyglutamate metabolites of methotrexate are as inhibitory to the target enzyme dihydrofolate reductase as is methotrexate. Because of their greater retention they have a longer half-life within the cells and thus a greater potential for cytotoxicity. These metabolites have been found in numerous cells and tissues and are extensively synthesized in cultured hepatic cells. Uptake of methotrexate by primary cultures of rat hepatocytes occurs by a pathway which is independent of the folate coenzymes but appears to be related in some way to cholic acid and organic anion uptake. The evidence for the commonality of these pathways is (a) an instability of both uptake systems in the absence of hormones in the culture medium, (b) nearly equal inhibition of uptake by PCMS and NEM, and (c) cross competition of cholic acid and methotrexate for entry into the cells. Cholic acid and BSP can also selectively inhibit methotrexate polyglutamate formation in hepatocytes. Methotrexate entry into H35 hepatoma cells is mediated by the transport system which is shared by folate coenzymes and is not inhibited by cholic acid, BSP or sulfhydryl reagents. At concentrations of cholic acid or BSP which inhibit methotrexate polyglutamate formation in hepatocytes there is little or no loss of polyglutamate formation in H35 cells, possibly because BSP and cholic acid are taken up less by H35 cells than by hepatocytes.

Animals↗
PubMed2000

Comparison of action of paclitaxel and poly(L-glutamic acid)-paclitaxel conjugate in human breast cancer cells.

The new anticancer agent poly(L-glutamic acid)-paclitaxel (PG-TXL) is a conjugate of paclitaxel and the water-soluble polyglutamate carrier. The observation that PG-TXL appears to possess antitumor activity superior to free paclitaxel in preclinical studies suggests that PG-TXL might possess favorable pharmacokinetic properties and/or have a mechanism of action different from that of paclitaxel. The purpose of this study was to compare the pharmacological action of PG-TXL and free paclitaxel in a panel of breast cancer cell lines with emphasis on their ability to induce apoptosis, their effects on cell cycle progression, and their cellular uptake. Morphological analysis and biochemical characterizations demonstrated that both compounds have similar abilities to induce apoptosis in cells expressing wild-type p53 (MCF-7) or mutant p53 (MDA-MB435 and MDA-MB453). Although MCF-7 cells were less sensitive to each compound than MDA-MB435 and MDA-MB453 cells, transfection experiments demonstrated that p53 did not appear to play a significant role in drug-induced cell death with either agent. Flow cytometry analysis further revealed that both free paclitaxel and PG-TXL induced a characteristic G2/M arrest in the cell cycle, consistent with the disturbance of microtubule polymerization as their mechanism of action. Western blot analysis showed that paclitaxel and PG-TXL downregulated HER2/neu expression in a similar fashion. HPLC analysis revealed that paclitaxel was released from the PG-TXL conjugate in vitro. The released paclitaxel, not the glutamic acid polymer, was subsequently transported into the cells. These results suggest that PG-TXL exerts its anticancer activity by continuous release of free paclitaxel, and that the favorable pharmacokinetics and drug distribution of the PG-TXL conjugate in vivo are likely the main factors contributing to its superior anticancer activity.

Antineoplastic Agents, Phytogenic↗
PubMed1975

Tissue-specific synthesis of methotrexate polyglutamates in the rat.

Purified tritium-labeled methotrexate was administered to rats and Sephadex G-15 gel chromatography was used to study the formation of poly-gamma-glutamyl metabolites of methotrexate in different tissues. These metabolities were recognized as tritium-labeled antifolate fractions and were identified by their response to serum pterolypolyglutamate hydrolases and by cochromatography with authentic standards. After doses equivalent to 15 to 20 mg for man, rat liver and kidney were found to contain both 4-amino 10-methylpterolyglutamyl-gamma-glutamic acid and 4-amino-10-methylpterolyglutamyl-gamma-glutamyl-gamma-glutamic acid. With one-third of the dose, only 4-amino 10-methylpteroylglutamyl-gamma-glutamic acid was found. Synthesis of methotrexate polyglutamates in liver and kidney was limited to the interval immediately following methotrexate administration and appeared to occur by sequential addition of single glutamyl residues to so-called "free" intracellular methotrexate. No synthesis of methotrexate polyglutamates was demonstrated in small intestine or thymus. After formation, 4-amino-10-methylpteroyglutamyl-gamma-glutamic acid disappeared from liver and kidney with a half-time of 6.5 days. The effect of these metabolites of methotrexate of cell metabolism is unknown.

Animals↗
PubMed1990

Folate analogues. 33. Synthesis of folate and antifolate poly-gamma-glutamates by [(9-fluorenylmethoxy)oxy]carbonyl chemistry and biological evaluation of certain methotrexate polyglutamate polylysine conjugates as inhibitors of the growth of H35 hepatoma cells.

Representative examples of folate and antifolate poly-gamma-glutamyl metabolites were synthesized via the [(9-fluorenylmethoxy)oxy]carbonyl (Fmoc) chemistry using the KH polyamide resin. Polyglutamate yields were consistently better in all cases compared to the previous Merrifield method, and the crude products were obtained in greater than 85% purity. The symmetrical anhydride (7) derived from alpha-tert-butyl N-Fmoc-L-glutamate (6) was used for the initial coupling of the first glutamate residue to the KH resin and also for subsequent chain elongation. The alpha-tert-butyl protective groups were not labile under the conditions used for the cleavage of the finished peptide from the resin. A series of poly-gamma-glutamyl metabolites of methotrexate (MTX) with a chain length ranging from two to five glutamyl residues were synthesized and coupled with poly(L-lysine) having an average molecular weight of 27,000 and 52,000. Each conjugate was tested for its ability to inhibit the growth of wild type (H35) and MTX transport resistant (H35R) strains of hepatoma cells in culture, the latter having a 100-fold reduced sensitivity to MTX. 4-Amino-4-deoxy-N10-methylpteroylglutamyl-gamma-glutamylpoly (L-lysine) conjugate [MTX(G2)-poly-L-Lys-52000] and MTX(G4)-poly-L-Lys-52000 were among the most active (I50 = 8.0 and 10 nM against H35 cells) MTX-polylysines synthesized to date, and they were somewhat more inhibitory to the transport resistant cells. MTX(G5)-poly-L-Lys-52000 was approximately 1000 times more effective than MTX(G5)-poly-D-Lys-52000 in inhibiting the growth of H35R hepatoma cells in culture, indicating that internal cleavage of the gamma-glutamate chain of the conjugate with subsequent release of MTX or shorter chain polyglutamates of MTX is unlikely to be an important determinant of MTX-polyglutamate polylysine cytotoxicity. The results indicate that MTX-polyglutamate poly(L-lysine) conjugates are taken up by the cells independently of MTX and probably via endocytosis.

Antimetabolites, Antineoplastic↗
PubMed2002

Polyglutamation of a novel antifolate, MX-68, is not necessary for its anti-arthritic effect.

N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid (MX-68), a derivative of methotrexate, was chemically designed to resist polyglutamation and to have a high affinity for dihydrofolate reductase, in an attempt to reduce the side effects of methotrexate. We confirmed that MX-68 did not undergo polyglutamation and investigated the pharmacological activities of MX-68 compared with methotrexate. (1) In vitro: MX-68 inhibited the activity of dihydrofolate reductase to the same degree as methotrexate-tetraglutamate. MX-68 treatment produced a similar anti-proliferative effect to that of methotrexate. However, the intracellular concentration of MX-68 was much lower than the sum of the levels of methotrexate and its polyglutamate, and the effects of MX-68 disappeared when it was removed from the culture medium. (2) In vivo: Oral administration of MX-68 suppressed the development of collagen-induced arthritis in mice and adjuvant-induced arthritis in rats, in a similar fashion to that of methotrexate. These results indicate that polyglutamation is not essential for the anti-arthritic effect of antifolates.

2-Aminoadipic Acid↗
PubMed2005

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles.

gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.

Arabidopsis↗
PubMed1994

Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase.

Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid. The products of the enzymatic reactions were purified by semipreparative C18 HPLC. The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y. Under standard reaction conditions, the E. coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position. Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates. The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors.

Escherichia coli↗
PubMed1985

gamma-Fluoromethotrexate: synthesis and biological activity of a potent inhibitor of dihydrofolate reductase with greatly diminished ability to form poly-gamma-glutamates.

A methotrexate (MTX) analog containing fluorine at the gamma-carbon of the glutamate moiety, gamma-fluoromethotrexate (FMTX), has been synthesized and evaluated for its biochemical and pharmacological properties. FMTX inhibition of dihydrofolate reductase from several sources is nearly equivalent to that shown by MTX. Most important, FMTX is an exceedingly poor substrate for folylpoly (gamma-glutamate) synthetase, the enzyme that catalyzes the biosynthesis of the highly-retained, cytotoxic MTX polyglutamates. Uptake experiments in H35 hepatoma cells show that FMTX accumulates to approximately the same extent as MTX at steady state. The rapid efflux of both derivatives is also very similar. The major difference detected in cells between the two compounds is the meager glutamylation of FMTX, due to the electronegative properties of the fluorine adjacent to the potential amide-forming carboxyl group. Exposure of dividing cells to 50 microM MTX for 2 and 6 hr results in the formation of 55 and 130 nmol, respectively, of the polyglutamates (more than two glutamate residues)/g of cell protein. With FMTX these values were reduced by 98% and 93%, respectively. Growth inhibition studies show that MTX is only 12-fold more toxic than FMTX when the cells are exposed to each derivative continuously for 72 hr. When the exposure time is reduced, a greater disparity between the inhibitory effects is observed; with a 2-hr pulse, MTX is 2300-fold more effective than FMTX. These data correlate with the effects of pulses of FMTX and MIX on de novo thymidylate biosynthesis in intact cells. The results indicate that of the parameters examined, the vastly reduced toxicity of FMTX after its removal from the culture medium is best correlated with impaired glutamylation. The data strongly suggest that prolonged toxicity of MTX is a result of metabolic conversion to MTX polyglutamates and that these effects are far more dramatic in short-term than in long-term exposure to the antifolates.

Animals↗
PubMed2002

Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system.

Human MRP4 (ABCC4, MOAT-B) is a lipophilic anion transporter that is able to confer resistance to nucleotide analogues and methotrexate (MTX). We previously investigated the implications of the ability of MRP4 to confer resistance to nucleotide analogues and determined that the pump is competent in the MgATP-energized transport of cyclic nucleotides and estradiol 17beta-D-glucuronide. Here we examine the potential role of MRP4 in conferring resistance to MTX and related processes by determining the selectivity of the transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, it is shown that MRP4 is active in the transport of MTX as well as the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin). MTX, FA, and leucovorin are subject to high capacity [V(max(MTX)), 0.24 +/- 0.05 nmol/mg/min; V(max (FA)), 0.68 +/- 0.14 nmol/mg/min; V(max(leucovorin)), 1.95 +/- 0.18 nmol/mg/min], low affinity [K(m(MTX)), 0.22 +/- 0.01 mM; K(m(FA)), 0.17 +/- 0.02 mM; K(m (leucovorin)), 0.64 +/- 0.23 mM] transport by MRP4. In addition, as would be expected were MRP4 a component of the MTX efflux system, its capacity to transport this agent is abrogated by the addition of a single glutamyl residue. It is also shown that glutamylation similarly affects the ability of MRP2 to transport MTX. On the basis of these transport properties, it is concluded that the efflux system for MTX includes MRP2 and MRP4, in addition to MRP1 and MRP3, and that MRP4 represents a common efflux system for both MTX and certain nucleotide analogues.

Animals↗
PubMed1985

Characteristics of the formation and membrane transport of 7-hydroxymethotrexate in freshly isolated rabbit hepatocytes.

The cellular pharmacology of methotrexate was evaluated in freshly isolated rabbit hepatocytes in suspension with an analysis of drug metabolism by high-performance liquid chromatography. After exposure of hepatocytes at a cytocrit of 5% to 5 microM [3H]-methotrexate, intracellular 7-hydroxymethotrexate appears rapidly within the cell; within 15 sec, the level of 7-hydroxymethotrexate exceeds the level of intracellular methotrexate, although the latter has not achieved the dihydrofolate reductase binding capacity. Within 20 min, virtually all methotrexate is hydroxylated. There is minimal formation of methotrexate polyglutamyl derivatives even after exposure of cells to very high levels of methotrexate, and 7-hydroxymethotrexate polyglutamates do not accumulate in the cell at all after incubation with [3H]-7-hydroxymethotrexate. Because of the rapidity of the hydroxylation of methotrexate, transport of this agent could not be characterized. However, some aspects of the transport properties of 7-hydroxymethotrexate could be studied since the catabolite is neither bound nor metabolized in this system. Net 7-hydroxymethotrexate transport was reduced by the addition of 5-formyltetrahydrofolate. As observed for 4-aminoantifolate transport in other cell systems, net 7-hydroxymethotrexate transport was markedly stimulated by sodium azide, an inhibitor of energy metabolism. The data suggest that hydroxylation of methotrexate proceeds at a rate at least comparable to the rate of association of the drug with dihydrofolate reductase and that transport of methotrexate into rabbit hepatocytes is slow relative to the rate of catabolism to the 7-hydroxy derivative. Rabbit hepatocytes may be a useful model for exploring methotrexate catabolism at the cellular level and may provide insights into the interaction between methotrexate and/or other 4-aminoantifolates and the human liver.

Animals↗
PubMed1993

Acquisition of resistance to antifolates caused by enhanced gamma-glutamyl hydrolase activity.

A subline of H35 hepatoma cells has been developed which exhibits 80-fold resistance to 5,10-dideazatetrahydrofolate, an antifolate which inhibits glycinamideribonucleotide transformylase. The cells are cross-resistant to methotrexate, an inhibitor of dihydrofolate reductase and 10-propargyl-5,8-dideazafolate and its 2-desamino-2-methyl derivative, both inhibitors of thymidylate synthase. The resistant cells are characterized by an impaired activity of the reduced folate transport system which affects cellular import of methotrexate, 5,10-dideazatetrahydrofolate, and 2-desamino-2-methyl-10-propargyl-5,8-dideazafolate but not 10-propargyl-5,8-dideazafolate. In addition, the resistant cells exhibit a severalfold increased activity of gamma-glutamyl hydrolase, the enzyme which cleaves the intracellular polyglutamate derivatives of the antifolates. Evidence for the involvement of gamma-glutamyl hydrolase in resistance is derived from the observation that polyglutamate derivatives of 10-propargyl-5,8-dideazafolate in resistant cells are maintained at one-third the amount of that in parental cells in the presence of the same extracellular concentration. This is the first observation that an increase in gamma-glutamyl hydrolase contributes to acquired resistance to antifolates.

Animals↗
PubMed2001

Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport.

We have recently determined that human multidrug resistance protein (MRP) 3, which confers resistance to certain natural product agents and methotrexate (MTX), is competent in the MgATP-energized transport of MTX and the monoanionic bile constituent glycocholate as well as several glutathione and glucuronate conjugates. Of these capabilities, the facility of MRP3 in conferring resistance to and mediating the transport of MTX is of particular interest because it raises the possibility that this pump is a component of the previously described cellular efflux system for this antimetabolite. However, if this is to be the case, a critical property of cellular MTX efflux that must be addressed is its ability to mediate the export of MTX but not that of its intracellular polyglutamylated derivatives. Here we examine the role of MRP3 in these and related processes by determining the selectivity of this transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, we show that MRP3 is not only active in the transport of MTX but is also active in the transport the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin) and that polyglutamylation of MTX abolishes transport. Both FA and leucovorin are subject to high-capacity (V(max(FA)), 1.71 +/- 0.05 nmol/mg/min; V(max(leucovorin)), 3.63 +/- 1.20 nmol/mg/min), low-affinity (K(m(FA)), 1.96 +/- 0.13 mM; K(m(leucovorin)), 1.74 +/- 0.65 mM) transport by MRP3. Addition of a single glutamyl residue to MTX is sufficient to diminish transport by >95%. We also show that polyglutamylation similarly affects the capacity of MRP1 to transport MTX and that physiological folates are also subject to MgATP-stimulated transport by MRP1. On the basis of the capacity to transport MTX but not MTX-Glu(2), it is concluded that MRP3 and MRP1 represent components of the previously described cellular efflux system for MTX. The capacity of MRP3 to transport folates indicates that it may reduce intracellular levels of these compounds and thereby indirectly influence antifolate cytotoxicity, and it also implies that this pump may play a role in the response to chemotherapeutic regimens in which leucovorin is a component.

3T3 Cells↗
PubMed1997

Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase.

The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations. rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximately 50-fold tighter than previously reported, with Kd and Ki values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor. The binding of the hexaglutamate of DDATHF to rmGARFT had Kd and Ki values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono- or hexaglutamate substrate did not yield different values for the Ki for the hexaglutamate form of DDATHF, in contradiction with previous reports. Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low Km values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.

Acyltransferases↗