Folylpolyglutamate Synthetase Activity Research Articles

Potent Inhibition of Human Folylpolyglutamate Synthetase by Suramin

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 μM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 μM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuireet al., Adv. Exptl. Med. Biol.163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (Kii= 0.9 μM;Kis= 1.1 μM) and glutamic acid (Kii= 1.0 μM;Kis= 5.2 μM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (Kii= 3.4 μM;Kis= 0.35 μM), since the major effect is on itsKm. Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 μM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 μM; MTX, 48 μM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideoutet al.(Int. J. Cancer61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.

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.

Resistance to Tomudex (ZD1694): Multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 μM, resulted in stably resistant cell lines (MCF TDX and H630 TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCF TDX and a 52-fold increase in TS levels in H630 TDXcell lines. Despite continued exposure of these cells to 2.0 μM TDX, TS protein and TS mRNA expression decreased to parental levels in H630 TDX cells, whereas in MCF TDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCF TDXcell line. TDX uptake was 2-fold higher in resistant MCF TDX cells than in parental MCF-7 cells, whereas in H630 TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630 TDX cells was noted when compared with the H630 parental cells. In H630 TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630 TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCF TDX and H630 TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.

Critical factors for optimizing the 5-fluorouracil-folinic acid association in cancer chemotherapy

The 5-fluorouracil (FU)-folinic acid (FA) association has demonstrated clinical efficacy in colorectal cancer, both in adjuvant and metastatic situations. However, there is no clear consensus about the optimal FU-FA schedule and dose. In addition, it would be of interest to identify FU-FA-responsive tumors. Our purpose was to review preclinical and clinical data dealing with prediction of FU-FA sensitivity and optimization of FU-FA schedules. Preclinical studies have highlighted the importance of thymidylate synthase (TS), the cellular target of the FU-FA mechanism of action, for predicting FU sensitivity. It appears that the more sensitive cell lines express the lowest TS activity. Interestingly, the cell lines sensitive to FA supplementation are those more sensitive to FU. The role of TS in FU-FA responsiveness has been clearly demonstrated in patients with colorectal and gastric cancers. Preliminary in vitro and clinical data have shown that the folylpolyglutamate synthetase (FPGS), the enzyme responsible for folate polyglutamylation, is another promising tool for identifying FU-FA-responsive tumors. So far, results of clinical trials do not form a clear consensus regarding the need to administer high FA doses for improving FU-FA treatment. Experimental studies on human cancer cell lines have demonstrated the wide variability among cell lines, ranging from 0.05 to 200 microns, of 1 FA concentrations required for maximal FU potentiation. In addition, pharmacokinetic studies have reported a significant variability of active folates in plasma after administration of standard-dose FA. Altogether, these observations favour high-dose FA administration to achieve high folate concentrations in plasma and thus to counteract the variability of the 1 FA concentrations required. With respect to the choice of FU-FA schedule, it appears from experimental data that increasing the duration of exposure to FA enhances FU-FA cytotoxicity, probably through an increased formation of reduced folate polyglutamate forms. Considering the S-phase specificity of FU cytotoxicity as well as its rapid elimination from plasma, a schedule of prolonged exposure to both FU and FA should be considered preferable. Results of the new FU-FA administration schedules such as the one consisting of a 2-hour FA administration followed by a combination of FU bolus and FU infusion, or the chronomodulated FU-FA infusion, open up promising approaches for improving the therapeutic index of FU-FA chemotherapy. Finally, future clinical studies should investigate tumoral parameters pharmacologically linked to FU-FA sensitivity such as pre-treatment TS and FPGS activities. Such tumoral investigations along with FU and FA pharmacokinetic investigations should provide a better understanding of inter-patient variability in response to FU-FA therapy and an optimal management of this chemotherapy regimen.

The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD 50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Different Antifolate-resistant L1210 Cell Variants with either Increased or Decreased Folylpolyglutamate Synthetase Gene Expression at the Level of mRNA Transcription

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

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.

Characterization of cross-resistance to methotrexate in a human breast cancer cell line selected for resistance to melphalan

We have demonstrated previously decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan (MelR MCF-7). Cross-resistance studies of MelR MCF-7 cells revealed that this cell line was 6.7-fold cross-resistant to methotrexate, but only 2-fold resistant to trimetrexate. Methotrexate transport studies in MelR MCF-7 cells showed a 2-fold decrease in initial methotrexate uptake and a 2-fold decrease in the V max for methotrexate uptake in the resistant cells. Methotrexate resistance in MelR MCF-7 cells was also associated with a decrease in non-effluxable methotrexate following incubation with radiolabeled drug for 24 hr. Characterization of intracellular methotrexate after accumulation for 24 hr demonstrated decreased levels of free methotrexate in MelR MCF-7 cells. Analysis of methotrexate polyglutamate formation in MelR MCF-7 cells indicated that the decrease in non-effluxable, non-protein-bound methotrexate was associated with a 3-fold decrease in higher order methotrexate polyglutamate formation. No difference was noted in folylpolyglutamate synthetase activity between the resistant and parental cell lines. Therefore, the observed decrease in methotrexate polyglutamate formation in MelR MCF-7 cells appeared to result from decreased availability of substrate. There was no evidence of any alteration in the amount of the catalytic activity of dihydrofolate reductase in MelR MCF-7 cells compared with parental MCF-7 (WT MCF-7) cells; moreover, the binding affinity of dihydrofolate reductase for methotrexate and the percentage of protein-bound methotrexate were similar in both cell lines. In addition, the total amounts of thymidylate synthase protein and thymidylate synthase catalytic activity in MelR MCF-7 cells were unchanged. Thus, acquired methotrexate resistance in MCF-7 cells selected for resistance to melphalan appears to result from down-regulation of methotrexate uptake.

Rapid Decline in Folylpolyglutamate Synthetase Activity and Gene Expression during Maturation of HL-60 Cells: NATURE OF THE EFFECT, IMPACT ON FOLATE COMPOUND POLYGLUTAMATE POOLS, AND EVIDENCE FOR PROGRAMMED DOWN-REGULATION DURING MATURATION

These studies in HL-60 cells examined the regulation of folylpolyglutamate synthetase (FPGS) activity at the level of gene expression during terminal maturation. Following addition of 210 mM Me2SO to cultures of HL-60 cells at a concentration that induces maturation of 85-90% of the cells, FPGS activity, but not folylpolyglutamate hydrolase (FPGH) activity, was reduced 2-7-fold within 1-5 days. The initial decline in FPGS activity preceded any effect of Me2SO on rate of growth and the increase in appearance of nitro blue tetrazolium-positive cells, a marker of cellular maturation, and the decrease after 5 days of exposure to Me2SO was solely accounted for by a 7-fold decrease in value for Vmax. The same time and concentration dependence for Me2SO was shown for the decline in FPGS activity, increase in nitro blue tetrazolium-positive cells, and decline in the level of a 2.1-kilobase FPGS mRNA during exposure to this inducer. This decline in FPGS mRNA was reversible when Me2SO was removed from the culture medium but only until that time when an appreciable number of cells were committed to terminal maturation. Following growth of HL-60 cells with [3H]MTX, used as a model folate compound, a large reduction in its intracellular polyglutamate pools was shown during maturation which quantitatively reflected the decline in FPGS activity as well as folate transport inward (Sirotnak, F.M., Jacobson, D.M., and Yang, C-H. (1986) J. Biol. Chem. 261, 11150-11156). Other data showed that folate status or obviation of the folate requirement during growth of these cells strongly influenced the rapidity of the onset of maturation following exposure to inducer. Overall, these results show that FPGS activity in HL-60 cells is a marker for proliferative capacity and that the underlying basis for the decline in FPGS activity during maturation is altered cognate gene expression which is manifested as early reversible and late irreversible phases. They also suggest that the coordinate reduction observed in folate transport, FPGS activity, dihydrofolate reductase, and probably other folate related enzymes by limiting macromolecular biosynthesis may be early programmed events in the maturation process that influence the switch from proliferation to senescence in these cells.

Folylpolyglutamate synthesis in Neurospora crassa: Transformation of polyglutamate-deficient mutants

The methionine auxotrophy of Neurospora crassa met-6 and mac mutants is related to an inability to synthesize long-chained folylpolyglutamates. Both of these lesions affect folylpolyglutamate synthetase activity, but it is not clear whether these mutations occur in different genes or in functional domains of the same gene. To address this question, copies of the met-6 + gene have been introduced into both mutants using plasmid and cosmid vectors. Transformation to prototrophy was achieved in both mutants. The ability of these mutant and transformant strains to synthesize folylpolyglutamates was assessed by HPLC analysis of folate cleavage products. Mycelial extracts of the wild type revealed a folate pool dominated by folylhexaglutamates. These folates were also detected in the transformants but were lacking in both mutants. In the latter strains, the conjugated folates were mainly di- and triglutamates. When incubated for 24 hr with [ 14 C]p- aminobenzoate , transformant and wild type cultures synthesized long chain folates, ca 60–80% of these being hexaglutamyl derivatives. In contrast, the labelled folates of mac and met-6 were mainly mono- and diglutamyl derivatives, respectively. Polyglutamate synthesis was also studied in vitro by partial purification and characterization of mycelial folylpolyglutamate synthetase protein. Mycelial extracts of the wild type and transformant cultures utilized 5,10-methylenetetrahydrofolate monoglutamate and its diglutamate as substrates in this synthetase reaction. In contrast, extracts of met-6 and mac mycelia utilized only one of these folate substrates. Gel filtration of folylpolyglutamate synthetase protein indicated apparent M r values of ca 66 000 in all strains. It is suggested that polyglutamate synthesis in Neurospora is probably mediated, as in other eukaryotic species, by a single folylpolyglutamate synthetase protein.

Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora

The possible association of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) in Neurospora crassa (FGSC 853, wild type) has been examined using mycelial pxtracts prepared and fractionated in the presence of protease inhibitors. DHFS and FPGS were assayed by following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. Both of these activities were predominately cytosolic in mycelia that were harvested 24 hr after spore inoculation of defined minimal medium. Relatively small amounts of total mycelial DHFS activity were associated with mitochondrial fractions isolated by differential centrifugation. In contrast, ca 20% of the mycelial FPGS activity was mitochondrial. Treatment of the mitochondrial fractions with Triton X-100 suggested that these activities were not latent under the assay conditions employed. Separate peaks of DHFS and FPGS activity were observed when (NH 4) 2SO 4-fractionated protein was desalted and chromatographed on columns of either Mono Q HR, DEAE-cellulose, heparin agarose, Matrex Green A or Reactive Green 5. Gel filtration indicated average M r values of 52 and 66 × 10 3 for DHFS and FPGS protein, respectively. Dihydrofolate synthetase protein was purified over 1000-fold by a protocol that included chromatography on DEAE-cellulose, DEAE-Sephacel, heparin agarose and Matrex Green A. The isolated protein lacked ability to glutamyl conjugate 5,10-methylene tetrahydrofolate. SDS-polyacrylamide gel electrophoresis of the Matrex Green A peak fractions revealed a major protein band of average M r 52 × 10 3 whose concentration appeared to parallel DHFS activity. FPGS protein (average M r 66 × 10 3), which lacked ability to glutamyl conjugate dihydropteroate, was recovered by a similar protocol. The reaction catalysed by DHFS protein displayed ATP dependency, was stabilized by glycerol, and product formation was favoured under alkaline conditions. The major catalytic properties of Neurospora DHFS are compared with those of other species.

Differences in constitutive and post-methotrexate folylpolyglutamate synthetase activity in B-lineage and T-lineage leukemia

Folylpolyglutamate synthetase (FPGS) is responsible for the metabolism of natural folates and a broad range of folate antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after treatment with MTX as a single agent, before conventional remission induction therapy. The levels of enzyme activity in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher (P = .003) than those found in acute nonlymphoblastic leukemia (ANLL) blasts (median of 181 pmol/h/mg protein). Comparable lineage differences in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression, FPGS activity increased in ALL blasts after in vivo exposure to MTX. The median increase in FPGS activity was significantly higher (P = .003) in B-lineage ALL (188%) than in T-lineage ALL (37%). Likewise, the percentage of intracellular long chain MTX-PG (Glu3–6) was significantly higher (P = .02) in B- lineage ALL (92%) than in T-lineage ALL (65%), consistent with higher FPGS activity in B-lineage blasts. This finding could explain, at least in part, the superior outcome in children with B-lineage ALL treated with antimetabolite therapy.

Differences in Constitutive and Post-Methotrexate Folylpolyglutamate Synthetase Activity in B-Lineage and T-Lineage Leukemia

Folylpolyglutamate synthetase (FPGS) is responsible for the metabolism of natural folates and a broad range of folate antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after treatment with MTX as a single agent, before conventional remission induction therapy. The levels of enzyme activity in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher (P = .003) than those found in acute nonlymphoblastic leukemia (ANLL) blasts (median of 181 pmol/h/mg protein). Comparable lineage differences in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression, FPGS activity increased in ALL blasts after in vivo exposure to MTX. The median increase in FPGS activity was significantly higher (P = .003) in B-lineage ALL (188%) than in T-lineage ALL (37%). Likewise, the percentage of intracellular long chain MTX-PG (Glu3-6) was significantly higher (P = .02) in B-lineage ALL (92%) than in T-lineage ALL (65%), consistent with higher FPGS activity in B-lineage blasts. This finding could explain, at least in part, the superior outcome in children with B-lineage ALL treated with antimetabolite therapy.

Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazaacyclotetrahydrofolate, an anti-purine drug

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF), an anti-purine agent that requires conversion to polyglutamate forms to be a potent inhibitor of its target enzymes. The sensitivity of cells continuously exposed to DATHF was not influenced by FPGS activity. After short term exposure to DATHF, cells expressing higher levels of human FPGS were more sensitive to the drug while cells expressing low levels were resistant. Cells expressing E. coli FPGS solely in the cytosol were resistant to DATHF and accumulated only low levels of the drug. Expression of E. coli FPGS in the mitochondria of these cells restored drug sensitivity and cytosolic drug accumulation. DATHF is extensively metabolized in the mitochondria of mammalian cells and, despite an inability to enter the mitochondria, folylpolyglutamates and DATHF polyglutamates formed in the mitochondria are released into the cytosol without prior hydrolysis. DATHF was not solely an inhibitor of de novo purine biosynthesis but also inhibited glycine synthesis in the cell.

Single step LC method for determination of folypolyglutamate synthetase activity and separation of polyglutamates

An HPLC assay has been developed for measuring folylpolyglutamate synthetase (FPGS) activity. It is based on the incorporation of U(14C)-glutamic acid into a folate derivative. The method has the advantage over previous procedures of offering in a single step full separation of the unreacted U(14C)-glutamic acid from that incorporated in folylpolyglutamates, as well as the possibility of identifying the chain length of the polyglutamates formed. It has been applied to determine FPGS activity in murine leukaemic lymphoblasts L5178Y. Activity was proportional over a wide range both to the incubation time and the amount of protein. Maximum activity was observed with folinic acid and the antifolate aminopterin (AMT) as substrates.

Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus.

The folC gene from mutant strain SF4 was cloned into a pUC19 plasmid. Expression of the mutant gene from the lac promoter of the plasmid complemented the auxotrophy for methionine of the SF4 strain. The only difference in sequence between the mutant and wild-type genes was a G925A base change resulting in an A309T amino acid change. The mutant enzyme had a 30-fold higher Km for 10-formyltetrahydrofolate as well as a 60-fold higher Km for glutamate and a 200-fold higher Km for dihydropteroate of the dihydrofolate synthetase activity. Site-specific mutagenesis was used to substitute other amino acids at codon 309. Mutants with glycine, isoleucine, and valine substitutions at this position, when expressed from multicopy plasmids, complemented the SF4 strain. The glycine mutant had properties similar to the wild-type enzyme, whereas the isoleucine and valine mutants had properties similar to the threonine mutant, SF4. Mutant genes with arginine, glutamate, and leucine substitutions, which did not complement the SF4 strain, could complement a folC deletion strain, but produced smaller colonies on complex plates and did not grow on minimal medium. In the deletion strain, an increasing requirement for folate product supplements was observed as the folylpolyglutamate synthetase-dihydrofolate synthetase activities of the complementing mutants decreased.

Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.

Determinants of methotrexate (MTX) resistance in cell lines resistant to short, but not continuous, MTX exposure were investigated since such lines may have relevance to clinical resistance. CCRF-CEM R30dm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant subline of the CCRF-CEM human leukemia cell line), had growth characteristics similar to CCRF-CEM. R30dm was resistant to a 24-h exposure to levels as high as 300 microM MTX but was as sensitive as CCRF-CEM to continuous MTX exposure. MTX resistance of R30dm was stable for greater than 68 weeks in the absence of selective pressure. Initial velocities of MTX transport were comparable for R30dm and CCRF-CEM, as were dihydrofolate reductase specific activity and MTX binding. A 2-fold thymidylate synthase activity decrease for R30dm from that of CCRF-CEM was not a significant factor in R30dm MTX resistance. Decreased MTX poly(gamma-glutamate) synthesis resulted in lower levels of drug accumulation by R30dm. Decreased polyglutamylation was attributable to folylpolyglutamate synthetase (FPGS) activity in R30dm extracts which was 1, 2, and less than or equal to 10% of CCRF-CEM extracts with the substrates MTX, aminopterin, and naturally occurring folates, respectively. Comparison of cell lines with varying levels of resistance to short term MTX exposure indicated that the extent of MTX resistance was proportional to the reduction of FPGS activity. The evidence supported decreased FPGS activity as the mechanism of resistance to short MTX exposure in the cell lines investigated.

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5′ and the 3′ ends and by TAB linker insertion. A number of 5′-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site is at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4–16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6–19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the k cat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.

Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate

NaHCO 3 activated the folylpolyglutamate synthetase (FPGS) from rat liver and the human leukemia cell lines K562 and CCRF-CEM by 1.7- to 2.0-fold. Optimal activation was achieved by 10 m m NaHCO 3 in all cases; NaCl, sodium formate, sodium acetate, NaN 3, and Na 2SO 3 at 10 m m did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO 2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. K m and V max values for pteroyl substrates (aminopterin or methotrexate) and l-glutamate increased proportionally in the presence of NaHCO 3; there was thus no apparent change in the catalytic efficiency ( V max K m ) of the FPGS reaction with these substrates. However, NaHCO 3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent K m while increasing the V max of the reaction. NaHCO 3 also activated FPGS activity when folic acid, dihydrofolic acid and tetrahydrofolic acid were substrates. The relative distribution of products synthesized from methotrexate or tetrahydrofolate by FPGS was not altered by addition of NaHCO 3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO 3 (IC 50 = 0.4 μ m). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO 3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO 2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.

Impaired Milk Folate Secretion is not Corrected by Supplemental Folate during Iron Deficiency in Rats

Decreased milk folate secretion in iron-deficient rat dams contributes to the impairment of folate metabolism in nursing pups. The present study was designed to assess whether impaired milk folate secretion secondary to iron deficiency is due to a decrease in the supply of folate to the mammary gland or to an inability of the mammary gland to effectively use folate. Rats were fed diets containing 0.5, 2.0 or 7.0 mg folate/kg and 8 (-Fe) or 250 (+Fe) mg Fe/kg throughout gestation and until d 17 of lactation. Regardless of dietary Fe content, maternal plasma, red blood cell, liver and kidney folate concentrations correlated with dietary folate content (r = 0.75-0.85, p less than 0.0001). With the exception of plasma folate level, which was 46% lower for -Fe than +Fe dams fed 0.5 mg folate/kg, no other differences in indices of folate status were noted between +Fe and -Fe dams. Dietary folate content had a direct impact on milk folate content in +Fe dams but not in -Fe dams. Mammary tissue methionine synthase and folylpolyglutamate synthetase activities were not depressed in Fe deficiency; rather, mean activities were elevated among -Fe dams fed 0.5 mg folate/kg. In conclusion, the reduction in milk folate secretion during Fe deficiency is not due to a decrease in the amount of folate supplied to the mammary gland; rather, the defect causing this reduction is specific to the mammary gland.