Biochemistry and regulation of folate and methotrexate transport in Leishmania major

Promastigotes of the protozoan parasite Leishmania major exhibit high affinity uptake of folate (Kt = 0.7 microM) and methotrexate (MTX) (Kt = 1.8 microM) which is saturable and sensitive to metabolic poisons. Influx of folate and MTX is competitively inhibited by 5-formyltetrahydrofolate and p-aminobenzoic acid-glutamate, but not by 4-deoxy-4-amino-10-methylpteroate, biopterin, or pteroate. A single carrier is inferred for both folate and MTX transport, as the Ki of each inhibitor for both folate and MTX influx is the same, and the apparent affinities (Kt) of the substrates folate and MTX are identical to their respective Ki values for inhibition of MTX and folate uptake. Folate influx is specifically regulated according to cellular growth phase, as stationary phase cells exhibit 7% of the Vmax of log phase cells, while energy-dependent glucose uptake is only moderately reduced in stationary phase. Folate influx is also regulated by external folate levels, as cells grown in 5 microM folate exhibit 30% of the Vmax of cells grown in folate-depleted medium. Comparison of bacterial, mammalian, and Leishmania folate transport activities indicates considerable diversity in both biochemical and regulatory properties, and suggests the possibility that selective inhibition or manipulation of folate transport may be exploited in parasite chemotherapy.     

Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.     

Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification

The methotrexate (MTX) and folate transport properties of five MTX-resistant lines of Leishmania major have been examined. These resistant lines all show a decreased Vmax for MTX influx, with no change in apparent affinity (Kt). The Vmax of folate influx is also proportionately decreased without alteration in Kt, supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two regions of DNA, the R region (encoding dihydrofolate reductase-thymidylate synthase) and the H region (Beverley, S.M., Coderre, J.A., Santi, D.V., and Schimke, R.T. (1984) Cell 38, 431-439), were also observed. In a given line, the amplifications occurred singly, in combination, or not at all. No other regions of amplification were detected. The phenotype of reduced MTX transport was moderately stable in the highly resistant R1000 line, being retained for more than 200 generations in the absence of MTX in vitro and during one passage through an infected mouse; in contrast, R- and H-amplified DNA were less stable. The lack of correlation of R and H amplification with reduced MTX transport suggests that alterations in transport are not causally mediated by gene amplification in Leishmania, but are a separate mode of MTX resistance. The onset of decreased MTX transport was also examined; wild-type Leishmania developed a reduced Vmax of MTX influx within 24 h following exposure to 1 microM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the Vmax of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As the folate/MTX transporter is regulated by exogenous folate, these data suggest that the initial rapid decrease in MTX transport may be a cellular regulatory response, in contrast to that found within the R1000 line which resembles a more stable genetic mutation.     

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities

Intestinal absorption offolates has been characterized as a facilitative process with a low pHoptimum. Studies with intestinal epithelial cells have suggested thatthis activity is mediated by the reduced folate carrier (RFC1). In thispaper, we report on folate transport characteristics in an immortalizedrat IEC-6 cell line that was found to exhibit the predominant influxactivity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs andhigh folic acid uptake only at the low pH. For the two clones with thehighest level of transport activity, relative MTX influx at the two pHswas reversed. Moreover, the low pH MTX influx activity([MTX]_e = 0.5 µM) was markedly inhibited by 20 µM folic acid while influx at neutral pH was not. Furthermore, in thepresence and absence of glucose at low pH, MTX and folic acid influxactivity was inhibited by azide, while MTX influx at pH 7.4 wasstimulated by azide in the absence of glucose but was unchanged in thepresence of glucose and azide. This was contrasted with the results oftransfection of the same RFC1 construct into an L1210 murine leukemiacell line bearing a nonfunctional endogenous carrier. In this case, theactivity expressed was only at pH 7.4. These data indicate that RFC1can exhibit two distinct types of folate transport activities inintestinal cells that must depend on tissue-specific modulators.

     

Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine

A mildly acidic pH in the lumen of the small intestine markedly enhances the transport of folate. This study investigated the relationship between pH and the affinity between folic acid and the apical membrane transporter using brush border membrane vesicles from rat jejunum and differentiated monolayer cultures of the colon carcinoma cell line, CaCo-2. Uptake studies with BBMV were conducted at folic acid concentrations of 0.1 to 50 mumol/l, conditions which were suitable for analyzing uptake data based on the Michaelis-Menten equation modified to include a nonsaturable component. These analyses yielded apparent Km values of 0.6 and 12.3 microM at pH 5.5 and pH 7.4, respectively (P less than 0.05). Values for Vmax were lower at pH 5.5 than at pH 7.4 (0.8 vs. 1.6 pmol/mg protein per 10 s, P less than 0.05). The studies with CaCo-2 cells employed folic acid concentrations of 0.1 to 5 mumol/l. Under these conditions the apparent Km for folic uptake was lowest at pH 6.0, where the Km was 0.7 mumol/l. The apparent Km increased sharply as a neutral pH was approached; reaching a value of 13.9 mumol/l at pH 7.1. These data suggest that the prominent pH effect on intestinal folate transport is, in part, explained by an increased affinity of the folate substrate for its membrane transporter.     

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.     

A new type of high affinity folic acid transporter in the protozoan parasite Leishmania and deletion of its gene in methotrexate-resistant cells

The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here that Leishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistant Leishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (K(m) 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions.     

Growth phase regulation of the main folate transporter of Leishmania infantum and its role in methotrexate resistance

The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (K(m), 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally.     

Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3H-folic acid at pH 5.5 was (i) Na+-independent; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX); (iii) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS); (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin); (vi) trans-inhibited by MTX and folic acid; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3H-folic acid was (i) Na+-independent; (ii) inhibited by folic acid and MTX, but not by 5-MTHF; (iii) inhibited by SITS, but not by DIDS; (iv) not affected by FCCP; (v) inhibited by monensin (but not by cytochalasin D); (vi) trans-inhibited by folic acid (but not by MTX); and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.     

Entry of methotrexate into Streptococcus pneumoniae: a study on a wild-type strain and a methotrexate resistant mutant

Entry of methotrexate (MTX) into the folate prototrophic bacterium Streptococcus pneumoniae was poorly inhibited by folate or its natural derivative folinic acid, suggesting that if MTX is transported via a folate transporter, the affinity of that transporter for MTX is higher than for folate. In the range of concentrations tested, MTX uptake was non-concentrative and decreased in ATP-depleted bacteria. When the external concentration of MTX was increased from 1 X 10(-7) M to 1 X 10(-6) M, uptake became saturated and was insensitive to ionophores. However when external MTX concentrations were increased to 1 X 10(-5) M, uptake increased linearly, and was inhibited by the ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, suggesting that the process was energized by the protonmotive force (delta p) at this concentration. A model for MTX entry in S. pneumoniae is proposed with respect to these results. The high level of resistance to MTX of the nonsense mutant amiA9 cannot be entirely explained by a decrease in MTX uptake.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines

Dendritic nanostructures can play a key role in drug delivery, due to the high density and variety of surface functional groups that can facilitate and modulate the delivery process. We have investigated the effect of dendrimer end-functionality on the activity of polyamido amine (PAMAM) dendrimer-methotrexate (MTX) conjugates in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer-MTX conjugates were prepared using a dicyclohexylcarbodiimide (DCC) coupling reaction: one between a carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX (conjugate A) and another between an amine-terminated G3 dendrimer and the carboxylic acid group of the MTX (conjugate B). Our studies suggest that conjugate A showed an increased drug activity compared to an equimolar amount of free MTX toward both sensitive and resistant cell lines, whereas conjugate B did not show significant activity on any of the cell lines. Despite substantially impaired MTX transport by MTX-resistant CEM/MTX and RII cells, conjugate A showed sensitivity increases of approximately 8- and 24-fold (based on IC50 values), respectively, compared to free MTX. Co-incubation of the cells with adenosine and thymidine along with either conjugate A or MTX resulted in almost complete protection, suggesting that the conjugate achieves its effect on dihyrofolate reductase (DHFR) enzyme through the same mechanism as that of MTX. The differences in cytotoxicity of these amide-bonded conjugates may be indicative of differences in the intracellular drug release from the cationic dendrimer (conjugate B) versus the anionic dendrimer (conjugate A), perhaps due to the differences in lysosomal residence times dictated by the surface functionality. These findings demonstrate the feasibility of using dendrimers as drug delivery vehicles for achieving higher therapeutic effects in chemotherapy, especially in drug-resistant cells.     

Pteridine Requirement of the Hemoflagellate Leishmania tarentolae

SYNOPSIS. Leishmania tarentolae grown in a defined medium required both folic acid and an unconjugated pteridine. Continuous growth was obtained with folic acid at a minimal level of 0.34 ng/ml and biopterin at 1.7 ng/ml. Neopterin was about 1/100 as active as biopterin.     

A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.

Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.     

Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates

We have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major. These cells readily hydrolyzed MTX to N-10-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 microM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA. MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h. Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX. In contrast to MTX, folate is extensively polyglutamylated in L. major (Santi, D. V., Nolan, P., and Shane, B. (1987) Biochem. Biophys. Res. Commun. 146, 1089-1092). MAPA was found to be 190-fold less potent than MTX as an inhibitor of leishmanial growth and to bind less tightly than MTX to leishmanial dihydrofolate reductase-thymidylate synthase. We therefore examined the possibility that MTX resistance is mediated by increased MTX hydrolysis to MAPA in drug-resistant Leishmania. However, enzymatic assays show that the rate of MTX hydrolysis was unaltered in the MTX-resistant R1000-3 line and the primaquine-resistant PQ-R30 line (which is 24-fold cross-resistant to MTX). In addition to MAPA, several minor unidentified metabolites were observed in the LT252, R1000-5B, and PQR30 cells but no consistent differences in the amounts of these metabolites were evident among these lines. These data indicate that alterations in the rate of MTX hydrolysis in vitro, or in the characteristics of MTX metabolites formed in vivo, do not underly the MTX resistance displayed by the H region-amplified R1000-5B and PQ-R30 lines.     

Synthesis and activity of a folate peptide camptothecin prodrug

A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.     

Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.