Folate Accumulation Research Articles

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania.

Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.

Retrovirally Mediated Complementation of the glyBPhenotype: CLONING OF A HUMAN GENE ENCODING THE CARRIER FOR ENTRY OF FOLATES INTO MITOCHONDRIA

The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells. A 2.6-kilobase pair cDNA insert flanked by retroviral sequences had integrated into genomic DNA in rescued cells. An open reading frame in this cDNA encoded a 35-kDa protein homologous to several inner mitochondrial wall transporters for intermediate metabolites. The subcloned cDNA complemented the glycine auxotrophy of glyB cells and reinstated folate accumulation in the mitochondria of transfected cells. The human origin, chromosomal location, and intron-exon organization of the isolated mitochondrial folate transporter gene were deduced from the expressed sequence tag database and human genome project data.

Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells.

Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3'-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating approximately 65-90 kDa native reduced folate carrier and a approximately 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed.

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity.

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.

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals.

It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs such as methotrexate and overexpressed on various tumor cells. However, these receptors are highly restricted in normal differentiated tissues. Results of limited in vitro and in vivo animal studies suggest that folate receptors could be a potential target for tumor imaging. This study aimed to develop a 99mTc-labeled folic acid using ethylenedicysteine (EC) as a chelator and evaluate its labeling efficiency and potential use as a tumor seeking agent. Tissue distribution of 99mTc-EC-folate was determined in breast tumor-bearing rats at 20 min, 1, 2, and 4 h (n = 3/time interval, 370 KBq/rat, i.v.). Blocking study was employed to determine receptor-mediated process; 99mTc-EC-folate was co-administrated with 50 and 150 mumol/kg of cold folic acid to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 99mTc-EC (control). In animal studies, tumor/blood count density ratios at 20 min-4 h increased from 0.81 +/- 0.09 to 1.23 +/- 0.13 with 99mTc-EC-folate. Conversely, these values showed time-dependent decrease from 0.77 +/- 0.32 to 0.65 +/- 0.01 with 99mTc-EC in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with folic acid co-administrations. Planar images and autoradiograms confirmed that the tumors could be visualized clearly with 99mTc-EC-folate.

Imaging tumor folate receptors using 111In-DTPA-methotrexate.

It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs, such as methotrexate, and overexpressed on various tumor cells. This study was aimed to develop an 111In labelled DTPA-methotrexate (DTPA-MTX) to image tumor folate receptors in vivo. DTPA-MTX was synthesized by reacting ethylenediamine with MTX. The resulting amino analogue of MTX was reacted with DTPA dianhydride in basic aqueous solution followed by dialysis. Tissue distribution was determined in breast tumor-bearing rats at 0.5, 2, 24, and 48 h (n = 3/time interval). To determine receptor-mediated process 111In-DTPA-MTX was co-administrated with varying blocking doses of cold folate to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 111In-DTPA. In animal studies, tumor/blood count density ratios at 0.5-48 h gradually increased from 0.8 +/- 0.32 to 2.2 +/- 0.41 with 111In-DTPA-MTX. Conversely, these values showed time-dependent decrease from 1.19 +/- 0.69 to 0.56 +/- 0.10 with 111In-DTPA in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with high doses of folic acid co-administration. Planar images and autoradiograms confirmed that the tumors could be visualized acceptably with 111In-DTPA-MTX. The results indicate the feasibility of using 111In-DTPA-MTX to image tumors through a folate receptor-mediated process.

Effects of Antisense-based Folylpoly-γ-glutamate Synthetase Down-regulation on Reduced Folates and Cellular Proliferation in CCRF-CEM Cells

The effect of down-regulation of folylpoly-γ-glutamate synthetase (FPGS) activity on intracellular reduced folate accumulation and cellular proliferation was examined, using an inducible antisense expression system in the human T-lymphoblastic leukemia cell line CCRF-CEM. FPGS catalyzes the addition of γ-glutamyl residues to natural folates and classical antifolates, which results in their enhanced cellular retention and increased cytotoxicity. As such, this enzyme has become a focus as a potential anticancer drug target. However, direct evidence to support this concept has been elusive. Hence, a study was undertaken using an antisense-based expression system to down-regulate FPGS activity. This inducible expression system was used to demonstrate that lower FPGS activity can lead to substantially lower intracellular folate content, which coincides with suppression of thymidylate synthesis and inhibition of cellular proliferation.

Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Role of folate receptor and reduced folate carrier in the transport of 5-methyltetrahydrofolic acid in human ovarian carcinoma cells.

Folate receptor-alpha (FR-alpha) is generally over-expressed in non-mucinous human ovarian carcinomas. The meaning of FR-alpha over-expression and its role in the 5-methyltetrahydrofolic acid (N5-CH3-H4PteGlu) transport in such tumors is not clear, especially compared with the reduced folate carrier (RFC), the other known folate transporter. In this study, we analyzed molecular FR-alpha and RFC expression in 16 ovarian carcinoma tissues and in 5 ovarian carcinoma cell lines using competitive PCR. Co-expression of the 2 transporters was found both in vivo and in vitro. FR-alpha mRNA expression in the cell lines was in good agreement with the corresponding protein expression evaluated by cellular folic acid binding and immunofluorescence analysis, using a specific monoclonal antibody (MAb) (MOv18). Moreover, RFC mRNA expression levels were consistent with the selective cellular binding of N-hydroxysuccinimide of [3H]-methotrexate (NHS-MTX). The 5 ovarian carcinoma cell lines (IGROV-1, SW-626, SKOV-3, OVCAR-3 and OAW-42), grown at physiological N5-CH3-H4PteGlu concentrations (20 nM) and expressing FR-alpha and RFC levels superimposable to those observed in vivo, were used as in vitro cellular model to evaluate the different contribution of FR-alpha and RFC to the transport of N5-CH3-H4PteGlu. The cytoplasmic N5-CH3-[3H]H4PteGlu accumulation observed in each cell line was approximately linear over 4 hr of incubation, but there was no correlation between the rate of folate internalization and FR-alpha and RFC expression levels. Furthermore, the selective inhibition of FR-alpha and RFC functionality allowed us to distinguish their differential role on the overall N5-CH3-[3H]H4PteGlu intracellular delivery. Treatment with the N-hydroxysuccinimide of folic acid, which blocks FR-alpha activity, showed only a partial inhibition (about 20%) of folate internalization in all the cell lines. In contrast, the inhibition of RFC by NHS-MTX, under conditions that did not affect FR-alpha functionality, generally reduced folate accumulation by more than 70%. Only one cell line (IGROV-1) showed a comparable contribution of the 2 transport systems. Our findings suggest that in ovarian carcinomas, in spite of its over-expression, FR-alpha generally plays a minor role in N5-CH3-H4PteGlu transport compared with RFC.

Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells.

We previously described a 1,000-fold pyrimethamine-resistant Chinese hamster ovary cell line (PyrR100) which retains parental dihydrofolate reductase activity and methotrexate (MTX) sensitivity. This study characterizes the basis for the 14-fold decrease in folic acid and leucovorin concentrations required for clonogenic growth of PyrR100 cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, PyrR100 cells displayed relative to parental AA8 cells a: 1) 17- and 5-fold increase in the net transport of folic acid and MTX, respectively; 2) 23- and 5-fold decrease in the efflux rate constant for folic acid and MTX, respectively; and 3) 2-fold increase in folic acid influx with no significant change in MTX influx. The markedly increased net folic acid transport in PyrR100 cells could not be explained by cellular folic acid binding, mitochondrial sequestration, polyglutamylation, nor by a decreased membrane potential. The effect of energy deprivation on folic acid and MTX transport in both cell lines was quite different. Glucose and pyruvate deprivation nearly abolished the increase in net folic acid transport in PyrR100 cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with PyrR100 cells. Furthermore, while folic acid influx in PyrR100 and AA8 cells was markedly reduced with energy deprivation, MTX influx was not affected. Provision of glucose and pyruvate to energy-deprived cells resulted in a rapid onset of MTX efflux from parental AA8 cells but not from PyrR100 cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in PyrR100 cells is largely due to the complete loss of exit pump activity. Furthermore, the energy source that sustains the augmented levels of folic acid appears linked to the influx process and is distinct from the energy source that sustains MTX gradients under these conditions. We conclude that the loss of folic acid efflux is an efficient means of augmenting cellular uptake of folate cofactors and subsequent survival on picomolar folate concentrations. This constitutes the first demonstration of the loss of folic acid exporter activity in mammalian cells as a response to lipophilic antifolate selective pressure.

Structural Organization of the Reduced Folate Carrier Gene in Chinese Hamster Ovary Cells

The reduced folate carrier gene (rfc) encodes a putative protein that is involved in the intracellular accumulation of folates. In this report, we describe the organization of the rfc gene from Chinese hamster ovary cells. The hamster rfc gene contains 7 exons and 6 introns, which span 15.3 kilobases. It codes for two alternatively spliced messenger RNAs, one that contains all 7 exons and one that lacks exon 2 but contains the remaining 6 exons. The transcriptional start of the gene has been mapped to six sites approximately 200 base pairs upstream of the putative ATG initiation codon. The promoter region has no TATA box-like sequence but contains a consensus Sp1 binding site. This is the first report of the genomic structure of the reduced folate carrier gene from any species.

Purification and Properties of Human Cytosolic Folylpoly-γ-glutamate Synthetase and Organization, Localization, and Differential Splicing of Its Gene

Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity. Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates. Most pteroyldiglutamates were better substrates than monoglutamates. The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns. A single FPGS gene was located to chromosome region 9q34.1. Four exon 1 variants were identified, each of which was spliced to exon 2. The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584). Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5' to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites. Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools. The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

Structural and Functional Analysis of the Human KB Cell Folate Receptor Gene P4 Promoter: Cooperation of Three Clustered Sp1-Binding Sites with Initiator Region for Basal Promoter Activity

The human folate receptors (hFRs) are important in the cellular accumulation of folates and antifolates. We described the structure of the human KB cell FR (hFR-KB) gene and identified two discrete promoter regions (P1 and P4) upstream from exons 1 and 4, respectively (Elwood et al., 1993). To further understand the molecular basis of hFR expression, we have now analyzed the basal transcription of the P4 promoter localized upstream of a major transcription start site. The sequence upstream from exon 4 contains several potential transcriptional factor-binding sites and a consensus initiator region sequence at the transcription start site but does not contain canonical TATA or CAAT boxes. While deletion of a 5' flanking sequence from nt -1023 to nt -605 of P4 promoter region decreases the luciferase reporter gene expression in KB cells to 54-70% of control construct, the removal of the sequence between nt -292 and nt -46 markedly decreases the activity to 3%. DNase I footprints and competitive mobility shift and supershift mobility assays indicate that Sp1 or Sp1-related nuclear protein(s) bind to three clustered GC-rich regions within the sequence between nt -292 and nt -46 of the hFR-KB P4 promoter. Both in vitro and in vivo analyses of the expression of promoter constructs containing site-specific mutation(s) of these three Sp1-binding sites and initiator sequence demonstrate that each of three Sp1 sites and the initiator sequence are required for optimum promoter activity and that they interact cooperatively in this P4 promoter of the hFR-KB gene.

Mechanisms of resistance to N-[5-[ N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line

N-[5-[ N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694) is a folate-based thymidylate synthase (TS; EC 2.1.1.45) inhibitor. Metabolism to higher chain length polyglutamates is essential for its optimal cytotoxic effect. A ZD1694-resistant (300-fold) human ileocecal carcinoma cell line (HCT-8/DW2) was developed, and its mechanism of resistance was evaluated. TS activities in situ and TS protein levels in the HCT-8 parental line and HCT-8/DW2 were similar (168 ± 47 vs 137 ± 25 pmol/hr/10 6 cells and 2.05 ±0.28 vs 2.07 ± 0.19 pmol/mg protein, respectively). The ic 50 values of ZD1694 for TS inhibition in cell-free extracts were similar in both lines, but the ic 50 of ZD1694 for TS inhibition in situ in HCT-8/DW2 cells was 27- and 268-fold higher than that in HCT-8 cells at 0 and 24 hr, respectively, after a 2-hr drug exposure. Folylpolyglutamate synthetase (FPGS; EC 6.3.2.17) activity was significantly lower in resistant HCT-8/DW2 cells as compared with parental HCT-8 cells (88 ± 40 vs 1065 ± 438 pmol/hr/mg protein when ZD1694 was used as substrate). The combined endogenous pool of methylenetetrahydrofolate and tetrahydrofolate in HCT-8/DW2 cells was also decreased. In addition, HCT-8/DW2 cells accumulated lower levels of methotrexate (MTX) in a 2-hr period, although the initial velocity of MTX transport was similar to that in parental HCT-8 cells. The lower level of FPGS activity and the lower level of (anti)folate accumulation in HCT-8/DW2 correlated with drug resistance and with the higher ic 50 of ZD1694 for in situ TS inhibition. In addition, drug resistance was also correlated with the rapid recovery of in situ TS activity after drug treatment. In brief, in this highly ZD1694-resistant HCT-8 cell line, resistance is associated with decreased FPGS activity, which, in turn, affects the metabolism of ZD1694 and consequently the extent and duration of in situ TS inhibition by the drug.

Disposition of leucovorin and its metabolites in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of methotrexate-pretreated mice

Leucovorin (LV or 5-CHOFH4) has had long-standing clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH2FH4) plus tetrahydrofolate (FH4) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH4). The dihydrofolate (FH2) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH3FH4) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH4) in both intestinal epithelial and L1210 cells. The elevation of FH2 in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH2FH4 + FH4. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.

Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism.

The effect of folylpoly-gamma-glutamate synthetase (FPGS) levels on folate accumulation was investigated in Chinese hamster ovary cells expressing various levels of human and Escherichia coli FPGS activity. At low medium folate concentrations, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. Essentially all transported folate was metabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity. As medium folate concentration increased through the physiological to the pharmacological range, cellular folate accumulation became proportional to FPGS activity and the chain length of intracellular folates decreased. At high folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cell. Pteroyltriglutamates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosynthesis but were unable to support glycine and methionine synthesis. Transfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supporting glycine synthesis, and transfectants expressing higher levels of FPGS were able to grow in the absence of methionine. Growth in the absence of methionine required high (nonphysiological) intracellular folate levels and longer chain length polyglutamates.

Regulation of folate and one-carbon metabolism in mammalian cells. I. Folate metabolism in Chinese hamster ovary cells expressing Escherichia coli or human folylpoly-gamma-glutamate synthetase activity.

Chinese hamster ovary (CHO) cell transfectants expressing various levels of human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity and possessing different folylpolyglutamate chain length distributions have been developed as models for folate and antifolate metabolism. The synthesis of pteroyltriglutamate was sufficient for normal cellular retention of folate and also overcame the phenotypic requirement for purines and thymidine of AUXB1, a CHO cell mutant lacking FPGS activity and lacking folylpolyglutamates. Only low levels of FPGS are required to enable cellular metabolism of folates to forms that are retained by mammalian cells. The higher levels found in mammalian cells are required for the synthesis of the long chain polyglutamate derivatives characteristic of mammalian cells. At low medium folate concentrations, folate accumulation by transfectants expressing human FPGS was not responsive to FPGS levels as the limiting step in metabolism was beyond the triglutamate, the chain length required for retention. The rate-limiting step in folate metabolism in cells expressing the E. coli enzyme was the conversion of diglutamate to triglutamate, and, at low FPGS levels, the E. coli enzyme was about 50-fold less effective than the human FPGS in enabling cellular folate accumulation. These data suggest that cellular accumulation of any folate analog whose mono- or diglutamate derivative is a poor substrate for FPGS would be very responsive to the level of FPGS activity.

Regulation of folate and one-carbon metabolism in mammalian cells. III. Role of mitochondrial folylpoly-gamma-glutamate synthetase.

Wild-type Chinese hamster ovary (CHO) cells and CHO cell transfectants expressing human folylpoly-gamma-glutamate synthetase (FPGS) activity contain mitochondrial FPGS activity of higher specific activity than the cytosolic isozyme. Expression of mitochondrial FPGS activity is required for folate accumulation by mitochondria. The mitochondrial folate pool in CHO cells is not in equilibrium with the cytosolic pool and contains folylpolyglutamates of longer glutamate chain length than cytosolic folates. The inability of AUX-coli, a CHO cell expressing high levels of Escherichia coli FPGS activity and containing pteroyltriglutamate, to support glycine synthesis is due to a lack of mitochondrial FPGS activity. AUX-coli cells lack mitochondrial folate despite containing high levels of cytosolic folate.