Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

Henderson, Gary B.; Grzelakowska-Sztabert, Barbara; Zevely, Edward M.; Huennekens, F.M.

doi:10.1016/0003-9861(80)90416-6

Cited by 53 publications

(27 citation statements)

References 17 publications

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“…9 (3, 5-7, 12, 15, 24-29). However, studies on folate uptake systems in several other intact mammalian cells including L-1210 cells (33)(34)(35), PHA stimulated lymphocytes (36), and human bone marrow cells (37) and human erythrocytes (10) suggest that transport was equally efficient and maximal for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and amethopterin, while that for PteGlu was poor.…”

Section: Resultsmentioning

confidence: 99%

Identification of high affinity folate binding proteins in human erythrocyte membranes.

Antony¹,

Kincade²,

Verma³

et al. 1987

J. Clin. Invest.

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Mature human erythrocyte membranes contained specific, high affinity (Kd 33 X 10-11 M) folate binding moieties. Folate binding was pH, time-and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with M, 2 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins) or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging.

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Section: Resultsmentioning

confidence: 99%

Identification of high affinity folate binding proteins in human erythrocyte membranes.

Antony¹,

Kincade²,

Verma³

et al. 1987

J. Clin. Invest.

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“…Net intracellular accumulation of (22,23) mediated internalization of this analogue, while simple diffusion through the plasma membrane at this temperature and concentration range is virtually nil. Under these conditions, the data documented (Table II) (Table II).…”

Section: Resultsmentioning

confidence: 99%

“…The measurement of specific binding of [ 3 H]MTX to tumor cells was carried out in HMO buffer (20 mM Hepes with 225 mM sucrose adjusted to pH 7.5 with MgO) using a modification of a previously described procedure (23). Following incubation for 5 min at 0°C with varying concentrations of [ 3 H]MTX with and without 1 mM DL-L5-formyl-tetrahydrofolate, the cells were centrifuged at 12,000 ϫ g for 1 min through a mixture (9:1) of silicone:mineral oil.…”

Section: Methodsmentioning

confidence: 99%

A Single Amino Acid Difference within the Folate Transporter Encoded by the Murine RFC-1 Gene Selectively Alters its Interaction with Folate Analogues

Roy

Tolner

Chiao

et al. 1998

Journal of Biological Chemistry

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The apparent K m , but not V max , for influx of methotrexate (MTX) mediated through the plasma membrane of S180 cells by the one-carbon, reduced folate transporter as well as the K D for binding to the transporter were 4-fold higher than in L1210 cells correlating with the greater intrinsic resistance of the former to this folate analogue. In contrast, no difference was observed between each cell type with regard to efflux of [ 3 H]MTX mediated by this same transporter in ATP-depleted cells. The difference in influx K m in the case of this 10-methyl substituted N1O analogue of folic acid was not seen with more effective permeants, such as the unsubstituted N1O aminopterin or C1O analogues. Thus, values for influx K m for aminopterin, which were 1-1.2 M in each cell type, increased as a result of substitution at N1O (MTX) 3-fold in L1210 cells but 12-fold in S180 cells. Nucleotide sequencing of reverse transcriptase-polymerase chain reaction-generated cDNA and of polymerase chain reaction-generated genomic DNA identified a single nucleotide difference between each cell type at ؉890 within exon 3 of the RFC-1 gene. This was in the form of a G (L1210 cells) to A (S180 cells) transition. Codon 297, the site of this transition, encodes either Ser or Asn in L1210 or S180 cells, respectively, which is located between the seventh and eight membrane-spanning helices. This amino acid difference had no effect on the electrophoretic mobility or amount of the transporter in each cell type that was shown by Western blotting with anti-RFC-1 peptide antibodies to migrate as 46 kDa in each case. Proof that this nucleotide difference alone accounted for the alteration in influx between each cell type was obtained by S180 RFC-1 cDNA versus L1210 RFC-1 cDNA transfection of an L1210 cell variant with undetectable MTX influx and RFC-1 gene expression. In this case, the higher K m for MTX influx associated with S180 cells was duplicated only in the S180 RFC-1 transfectants. These results appear to document the first example of a nucleotide alteration within the RFC-1 gene, which influences the interaction of MTX with the encoded plasma membrane transporter. An analysis of topology, in addition to other considerations, suggests that the site of the amino acid difference found in the transporter from L1210 and S180 cells occurs within or near the binding site on the external plasma membrane surface.Mammalian cells have an absolute requirement for exogenous folates (1, 2) for growth and macromolecular biosynthesis. This is fulfilled by the internalization of folate compounds into these cells by either carrier-mediated or receptor-mediated mechanisms (3-6) depending upon their level of expression. In the case of tumor cells, mediated internalization of folate compounds occurs primarily by the one-carbon, reduced folate transporter (3-5). The same carrier-mediated system is involved in the internalization of cytotoxic folate analogues. This and the low level of expression of this system in many tumor cells resistant to these analogues (7-...

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“…Of particular interest to this and other laboratories has been the resistance associated with impaired transport of the drug via the reduced folate carrier system. In murine L1210 leukemia cells, this carrier system mediates the rapid transport of reduced folates and the 4-amino analogs of folic acid and its kinetic and thermodynamic properties have been characterized in detail by several laboratories (3)(4)(5). Clones encoding a transmembrane carrier protein have recently been isolated from murine, hamster, and human cDNA libraries (6 -9) and shown to restore folate transport in cell lines defective in carrier-mediated transport.…”

Section: Resistance To Methotrexate (Mtx)mentioning

confidence: 99%

Characterization of a Mutation in the Reduced Folate Carrier in a Transport Defective L1210 Murine Leukemia Cell Line

Brigle

Spinella

Sierra

et al. 1995

Journal of Biological Chemistry

106

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This laboratory previously described an L1210 leukemia cell line (MTX r A) 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 MTX r A cells resulted in a cell line which exhibited a complete restoration of methotrexate uptake and an enhanced sensitivity to methotrexate. Northern analysis and specific [3 H]MTX cell surface binding indicated that expression of the reduced folate carrier was elevated ϳ5-fold in the transfectant compared to parental and MTX r A 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 MTX r A 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. Resistance to methotrexate (MTX)1 is a major limiting factor in the clinical utility of this agent and can occur by a variety of mechanisms (1, 2). Of particular interest to this and other laboratories has been the resistance associated with impaired transport of the drug via the reduced folate carrier system. In murine L1210 leukemia cells, this carrier system mediates the rapid transport of reduced folates and the 4-amino analogs of folic acid and its kinetic and thermodynamic properties have been characterized in detail by several laboratories (3-5). Clones encoding a transmembrane carrier protein have recently been isolated from murine, hamster, and human cDNA libraries (6 -9) and shown to restore folate transport in cell lines defective in carrier-mediated transport. Hydropathy plots of this ϳ60-kDa protein, designated RFC1 in the murine system, predict 12 ␣-helical transmembrane regions and suggested that it was likely a member of a superfamily of membrane spanning transporters. Based upon the relative molecular mass of membrane components which are labeled when murine cells are treated with various MTX-affinity analogs (M r ϭ 43-48), the predicted M r of the cloned RFC1 protein is different from that predicted for the reduced folate carrier (10 -12). This suggests that, in the murine system, there is either significant post-trans...

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Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

Cited by 53 publications

References 17 publications

Identification of high affinity folate binding proteins in human erythrocyte membranes.

Identification of high affinity folate binding proteins in human erythrocyte membranes.

A Single Amino Acid Difference within the Folate Transporter Encoded by the Murine RFC-1 Gene Selectively Alters its Interaction with Folate Analogues

Characterization of a Mutation in the Reduced Folate Carrier in a Transport Defective L1210 Murine Leukemia Cell Line

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