Biomedical subjects

I D Goldman

Publications and source records attributed to I D Goldman.

At least 37 records · Page 2Linked to original sources

PubMed1999

Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis.

Chemical mutagenesis with N-methyl-N-nitrosourea was employed to study the pattern of mutations in the reduced folate carrier (RFC1) that results in transport-related methotrexate resistance and to identify amino acid residues that are critical to carrier structure and/or function. Thirty-four methotrexate transport-defective L1210 leukemia cell lines were isolated with folic acid as the sole folate source under antifolate selective pressure. The RFC1 mRNA levels were comparable with, or not substantially decreased, in most of these cell lines relative to wild-type L1210 cells. The molecular basis for the transport defects was investigated by sequencing multiple RFC1 cDNA clones isolated from these mutants by reverse transcription-polymerase chain reaction, which encompassed the entire coding region. The mutations identified were further confirmed either by direct sequencing or, when applicable, by restriction analysis of total reverse transcription-polymerase chain reaction products. The majority of mutations (21) led to single amino acid substitutions that were in, or near, 9 of 12 predicted transmembrane domains, with the highest frequencies in the first, fifth, and eighth. There were no mutations in the sixth, ninth, and twelfth transmembrane domains. Glycine, serine, and arginine were the most frequently mutated residues. These data suggest that several transmembrane domains, rather than the amino- and carboxyl-termini, and the large intracellular loop between the sixth and seventh transmembrane domains play key roles as sites for RFC1 inactivation because of single point mutations. This panel of mutated cell lines offers an important resource for studies on RFC1 structure-function and for the evaluation of transport-related cross-resistance patterns with new-generation antifolate inhibitors of tetrahydrofolate cofactor-dependent enzymes.

Animals↗

PubMed1999

Recent advances in the understanding of the mechanism of membrane transport of folates and antifolates.

Transport of folates and antifolates in most cells is mediated by the reduced folate carrier (RFCI), an anion exchanging concentrative process, which is opposed by independent exit pump(s) that are directly coupled to energy metabolism. The balance of these processes governs the free intracellular folate level that is the substrate for folylpolyglutamate synthetase and determines the rate of metabolism of many folates and antifolates to polyglutamate derivatives. The RFCI has a high affinity for new-generation antifolates, but the relationship between transport and polyglutamylation for all these compounds has not, as yet, been defined. Analysis of mutations in RFCI indicate highly selective functional changes in binding affinity and/or carrier mobility, raising the possibilities that (1) enhanced transport and accumulation of natural folates can result in resistance to antifolate inhibitors of purine and pyrimidine synthesis by blocking their polyglutamylation to active derivatives, or by competition at the target enzyme site without substantial changes in antifolate transport, and (2) transport-related resistance to one antifolate might not produce comparable cross-resistance to another. Impaired export pump function, which results in enhanced accumulation of folates in cells and inhibition of antifolate polyglutamylation, represents another potential mechanism of resistance to new-generation antifolates. Folate receptor-mediated transport is another route of entry of folates and antifolates into cells, but its importance has not been well-defined. Finally, a low pH transport mechanism is present in a variety of mammalian cell lines. However, its relationship to RFCI and other low pH epithelial transporters and its ability to transport new-generation antifolates remain to be established.

Animals↗

PubMed1998

A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function.

In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in MTXrA-E45K cells was characterized by a 12- and 3.6-fold decrease in influx Vmax and Kt respectively, relative to L1210 cells. The folic acid influx Ki in L1210 cells was more than 50-fold greater than that of MTX based upon inhibition of 5-CHO-THF influx. In comparison, the mutated carrier had comparable affinities for folic acid and MTX in MTXrA-E45K cells due to a 7-fold decrease in the folic acid influx Ki and 7-fold increase in the MTX influx Ki. Transport via native RFC1 is inhibited by a variety of anions in L1210 cells associated with an increase in influx Kt. However, influx of 5-CHO-THF in MTXrA-E45K cells in a HEPES buffer (9 mM chloride) was decreased by 70% due to a 3-fold fall in the Vmax. In the complete absence of chloride (K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10% that in HBS buffer. 5-CHO-THF influx was restored by addition of chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP which were all inhibitory over a broad range of concentrations. The data suggest that substitution of a positive for a negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when cells are placed under selective pressure with antifolates that utilize RFC1 as the major route of entry into mammalian cells.

Amino Acid Substitution↗

PubMed1998

Concentrating capacity of the human reduced folate carrier (hRFC1) in human ZR-75 breast cancer cell lines.

Human RFC1 (hRFC1) transfected in transport-deficient methotrexate MTXR(R)ZR-75-1 human breast carcinoma cells (MTX(R)ZR-75/RFC) were used to investigate the impact of hRFC1 overexpression on influx and concentrative transport of methotrexate (MTX). Eight-fold overexpression of hRFC1, as determined by northern analysis, resulted in a 4-fold increase in MTX influx accompanied by a 2.4-fold increase in the steady-state level of free drug as compared with wild-type ZR-75-1 cells when the extracellular MTX level was 0.5 microM. When extracellular MTX was increased to 10 microM, the increase in influx equaled the increase in the transmembrane chemical gradient for MTX in the transfectant relative to wild-type cells. By 50 min, approximately 16-20 and 25% of the intracellular 3H represented MTX polyglutamates by HPLC analysis at [MTX]e = 0.5 and 10 microM in wild-type and transfected cells, respectively. Overexpression of hRFC1 enhanced sensitivity to MTX in MTX(R)ZR-75-1 cells by more than 250-fold. The data indicate that overexpression of hRFC1 in human cells results in comparable increases in influx and transmembrane gradients. This is different from what was reported when mouse RFC1 was transfected into murine leukemia cells, resulting in large, more symmetrical increases in the MTX bidirectional transport kinetics with a much smaller change in steady-state levels. The changes in the human cells transfected with hRFC1 however, were similar to what has been observed by other investigators when RFC1 expression is increased by low folate selective pressure.

Animals↗

PubMed1998

Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function.

Folate influx at low pH was characterized in MTXrA cells, an L1210 mouse leukemia cell line with a functional defect in the reduced folate carrier. Folic acid influx in MTXrA cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistinguishable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTXrA cells at pH 6.0 was 15-fold higher than at pH 7.5, in L1210 cells it was decreased by half. Influx of MTX, folic acid, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate in MTXrA cells was increased at pH < 7.0, but their pH optima and profile differed substantially. Influx of MTX and 5-methyltetrahydrofolate at pH 6.0 showed saturability, with a Kt of 2.65 and 0.56 microM, and a Vmax of 0.45 and 0.083 nmol/g dry wt/min, respectively. MTX influx mediated by the low pH transporter was insensitive to the anionic composition of the transport buffer and affected minimally (approximately 20%) by Na+ substitution. The anion transport inhibitors sulfobromophthalein, diisothiocyanatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene disulfonic acid were not effective inhibitors of the low pH route. MTX transport at low pH did not increase in MTXrA-R16 cells, an MTXrA derivative with 10-fold overexpression of the reduced folate carrier (RFC) due to transfection with RFC1 cDNA. Inhibition of reduced folate carrier activity with acetamidoisothiocyanatostilbene disulfonic acid resulted in identical MTX influx in L1210, MTXrA, and MTXrA-R16 cells at pH 5.5. Finally, low pH-mediated MTX influx was reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensin >> valinomycin). The data indicate that L1210 and MTXrA cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1998

A reduced folate carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate.

With 5-formyltetrahydrofolate (5-CHO-THF) as the folate source a methotrexate (MTX) transport-deficient murine leukemia cell line, L1210-G1a, was isolated after chemical mutagenesis and MTX selection. This cell line was 10-fold resistant to MTX in comparison to parental L1210 cells, yet the EC50 for 5-CHO-THF was increased by a factor of only 2. The initial uptake of MTX, at a concentration of 1 microM, was decreased by a factor of 40, whereas influx of 5-CHO-THF dropped by a factor of only 8. This difference in initial uptake rates was attributed solely to changes in influx Vmax without a significant change in Km. Whereas the RFC1 mRNA level in L1210-G1a cells was indistinguishable from that of parental L1210 cells, a serine to asparagine substitution was identified at amino acid 46 within the first predicted transmembrane domain. This was a result of a homozygous mutation of G-->A in the genome. Transfection of the mutated RFC1 cDNA into MTXrA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXrA-S46N. The increase in influx of 5-CHO-THF and 5-CH3THF was 5 and 13 times greater than that for MTX in the transfectant, consistent with the influx ratio in the L1210-G1a line. The functional expression of the mutated RFC1 reduced the growth requirement for 5-CHO-THF by a factor of 30, compared with only a 3-fold decrease in the MTX IC50. This represents the first reported RFC1 mutation that confers resistance to MTX due to a markedly impaired influx with relative conservation of reduced folate transport. The kinetic changes are consistent with a substrate-dependent alteration in carrier mobility that favors reduced folates over MTX. These changes may account for the development of MTX resistance due to impaired drug transport in vivo, allowing tumor cells to meet their folate requirement with 5-CH3THF, the predominant blood folate.

Animals↗

PubMed1998

Increased activity of a novel low pH folate transporter associated with lipophilic antifolate resistance in chinese hamster ovary cells.

Previous studies described a Chinese hamster ovary cell line, PyrR100, resistant to lipid-soluble antifolates due to the loss of an energy-coupled folate exporter resulting in a marked increase in intracellular folate cofactor accumulation. There was, in addition, an unexplained increase in folic acid influx in PyrR100 cells which is shown in this paper to be mediated by a transporter with a low pH optimum. The pH profile for folic acid influx in parental Chinese hamster ovary AA8 cells indicated peak activity at pH 6; this was increased >3-fold in PyrR100 cells. In contrast, methotrexate (MTX) influx in AA8 cells showed two peaks of comparable activities at pH 6 and 7.5; in PyrR100 cells, the component at pH 6 was increased 2-fold. Folic acid was a potent inhibitor of [3H]MTX or [3H]folic acid influx (1 microM) via the low pH route with IC50 values of approximately 1 microM. Prostaglandin A1 was a potent inhibitor of [3H]MTX influx via the reduced folate carrier 1 at pH 7.5 with only a small inhibitory effect on the low pH route. The addition of 10 microM folic acid to PyrR100 cells resulted in a MTX influx pH profile identical to that of AA8 cells, consistent with suppression of the low pH route. In contrast, addition of 25 microM prostaglandin A1 to PyrR100 cells resulted in a MTX influx pH profile comparable to that of folic acid, consistent with the loss of the reduced folate carrier-mediated component. Inhibition ( approximately 70%) of [3H]folic acid influx by approximately 10 microM unlabeled folic acid at pH 7.5 indicated that the low pH transporter accounts for the majority of folic acid transport at physiological pH. This study demonstrates the functional importance of a low pH folate transporter that is increased when enhanced folic acid entry into cells is required as an adaptive response to antifolate selective pressure. This may represent a mechanism of resistance to new antifolate inhibitors of folate cofactor-dependent enzymes in which cytotoxic activity is limited by expanded cellular folate pools.

Animals↗

PubMed1997

Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells.

Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTXrA line, which lacks a functional endogenous carrier to obtain the MTXrA-R16 clonal derivative. Influx was increased approximately 9-fold in MTXrA-R16 cells without a change in Km. The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX]i, when [MTX]e was 1 microM. The concentrative advantage for RFC1 (the ratio of [MTX]i in MTXrA-R16 to L1210 cells) increased from 1.8 at 0.1 microM MTX to 3.8 at an [MTX]e level of 30 microM. Augmented transport in MTXrA-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a approximately 50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX]i and not the much larger change in influx at low [MTX]e levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.

Animals↗

PubMed1997

Organization of the murine reduced folate carrier gene and identification of variant splice forms.

RT-PCR analysis of the reduced folate carrier (RFC) from L1210 and murine erythroleukemia cells led to the identification of three clones which appeared to result from the use of alternative splice sites. The nucleotide sequence of each splice form predicts a protein that contains at least the first 7 transmembrane domains of the parental RFC protein followed by a novel hydrophilic carboxyl terminus of 33, 72, or 105 amino acid residues. Sequence analysis of cDNA clones isolated from murine liver and the results of 5'-RACE from L1210 cells indicated that RFC also utilizes alternate 5'-terminal exons. To understand how the alternatively spliced RFC transcripts and multiple 5'-termini were generated, the genomic organization of RFC was determined. The gene is comprised of at least 8 exons, the first two of which encode the alternative 5' termini. Based on sequence identity with cDNAs encoding RFC from hamster and rat, however, it appears that additional 5' exons may be present. Two of the RFC splice variants result from the use of a cryptic splice donor site within exon 4 and the third results from the use of a cryptic splice acceptor site within exon 5. In addition, the splice variant form that encodes the largest protein also utilizes an alternative exon located between exons 5 and 6. The apparent use of alternative transcriptional start sites and the identification of several RFC splice forms raises the possibility that unique RFC molecules may be generated that exhibit tissue- or cell line-specific distribution.

Alternative Splicing↗

PubMed1997

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.

Animals↗

PubMed1997

pH dependence of methotrexate transport by the reduced folate carrier and the folate receptor in L1210 leukemia cells. Further evidence for a third route mediated at low pH.

F2-MTX'A is an L1210 leukemia cell line with a functional detect in the reduced folate carrier and high level expression of folate receptor beta. The pH dependence of methotrexate (MTX) influx by folate receptor beta in F2-MTX'A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by folate receptor beta had a pH optimum of 6.5, whereas influx mediated by the reduced folate carrier showed a pH optimum of 7.5. Increased folate receptor beta-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTX'A cells proceeded by another mechanism. This transport route became active at pH < 7.5, operated optimally at pH 6.0 to 6.5, and, unlike folate receptor beta-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a Ki of 5.3 microM and a Vmax of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a Ki of 148 microM, and had similar relative affinities for folic acid, leucovorin, and 5 methyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTX'A cells, optimal at low pH and distinct from the reduced folate carrier or the folate receptor.

Animals↗

PubMed1996

Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Animals↗

PubMed1995

Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells.

This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-beta (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-beta and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 microM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-beta exhibited an influx kappa t one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO4(2-) was inhibitory. Anions enhanced MTX binding to folate receptor-beta with isethionate > SO4(2-) > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-beta-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

Animals↗

PubMed1995

Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line.

This laboratory previously described an L1210 leukemia cell line (MTXrA) selected for resistance to methotrexate by virtue of impaired transport due to a functional defect in the translocation process. We now report on the sequence analysis of cDNAs encoding the reduced folate carrier from this line and identify a single mutation that results in the substitution of a proline for an alanine in a highly conserved transmembrane region of the protein. Transfection of the parental reduced folate carrier into MTXrA cells resulted in a cell line which exhibited a complete restoration of methotrexate uptake and an enhanced sensitivity to methotrexate. Northern analysis and specific [3H]MTX cell surface binding indicated that expression of the reduced folate carrier was elevated approximately 5-fold in the transfectant compared to parental and MTXrA cells. The MTX influx properties of the transfectant cell line were identical to those of the well characterized reduced folate carrier from parental L1210 cells in terms of: 1) patterns of sensitivity to competing folates, 2) sensitivity to the organic anion sulfobromophthalein, 3) lack of energy dependence, and 4) capacity for trans-stimulation. We also provide new data which suggests that the nucleotide sequence 5' of the predicted ATG initiation codon may encode additional protein information in the form of a leader sequence. Finally, we demonstrate that the MTXrA line has both the mutant and the parental reduced folate carrier alleles but that expression appears to be restricted to the mutant allele. Thus, the methotrexate transport phenotype and resultant drug resistance in this cell line result from genetic/regulatory events at both alleles.

Alleles↗

PubMed1995

Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells.

L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTXrA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXrA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXrA-TF1, which constitutively expresses high levels of FR-alpha. MTXrA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 microM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a approximately 20-fold lower influx Kb and Vmax for MTXrA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXrA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 microM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTXrA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXrA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXrA-TF1 cell lines; however, inhibition in MTXrA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.

Alkenes↗

PubMed1994

Increased expression and genomic organization of a folate-binding protein homologous to the human placental isoform in L1210 murine leukemia cell lines with a defective reduced folate carrier.

This laboratory previously described an L1210 leukemia cell line (MTXrA) selected for resistance to methotrexate by virtue of impaired transport. In this line, the reduced folate carrier had unchanged affinity for methotrexate, was present at the cell surface in usual quantity, but did not deliver drug into the cell, indicative of a functional defect in the translocation process. In this study, we further characterize this cell line along with a subline (F2-MTXrA) selected for growth in low levels of folic acid. This subline demonstrates continued high resistance to methotrexate and very low influx of [3H]methotrexate and 5-[3H]formyltetrahydrofolate, indicating the persistence of the defect in the reduced folate carrier. Both MTXrA and F2-MTXrA are shown to overexpress FBP2, the murine homolog of a folate-binding protein initially isolated from human placenta. Compared with parent L1210 cells, Northern analysis revealed FBP2 expression to be elevated 40-fold in the MTXrA line and 500-fold in F2-MTXrA. The large increase in FBP2 expression in the F2-MTXrA line correlates with a 10-fold increase in [3H]folic acid membrane surface binding and a 1000-fold decrease in the folic acid growth requirement compared with parental L1210 cells. Also, there are 20- and 500-fold decreases in the 5-formyltetrahydrofolate growth requirement compared with parent L1210 and MTXrA cells, respectively. Finally, the genomic organization of the FBP2 locus is presented. The results of Northern analyses using probes specific to FBP2 5'-untranslated sequences or to a splice junction within this region suggest that the up-regulated FBP2-specific message in F2-MTXrA utilizes 5'-noncoding sequences distinct from those used in the message encoded in L1210 cell lines with low level FBP2 expression. The MTXrA cells provide an example of a line selected for primary resistance to methotrexate that also exhibits concomitant increased expression of a folate-binding protein. Further overexpression of this folate-binding protein (which has homology to that initially identified in placenta) provides cells with the ability to meet cellular folate needs in a folate-deprived environment.

Animals↗

PubMed1994

Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells.

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

Amidohydrolases↗

PubMed1992

Insertion of an intracisternal A particle within the 5'-regulatory region of a gene encoding folate-binding protein in L1210 leukemia cells in response to low folate selection. Association with increased protein expression.

We have previously described a subline of L1210 murine leukemia cells (LL1) selected in a low level of 5-formyltetrahydrofolate which overexpresses a membrane-bound folate-binding protein (FBP1) and exhibits a rearrangement at the locus encoding this protein. Genomic clones containing the entire FBP1-encoding DNA from both L1210 and LL1 were isolated and characterized. Sequence analysis indicates that, with exception of the 5'-region, the FBP1-encoding locus in both cell lines is identical. The rearrangement in LL1 results from the insertion of an intracisternal A particle (IAP) in the head-to-head (antisense) orientation 72 base pairs (bp) upstream of the FBP1 ATG start codon. The IAP likely provides an alternative promoter for FBP1 expression which may produce a novel transcript with enhanced stability. Presence of the IAP appears to inactivate or relocate normal cis-acting regulatory sequences as expression of the FBP1 transcript in LL1 is not regulated by the folate status of the cell.

Amino Acid Sequence↗