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

Rongbao Zhao, Yehuda G. Assaraf, I. David Goldman

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Abstract

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 μM, 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 V(max) without a significant change in K(m). 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 MTXτA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXτA-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 RF1 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.

Original languageEnglish (US)
Pages (from-to)7873-7879
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number14
DOIs
StatePublished - Apr 3 1998

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Reduced Folate Carrier Protein
Leucovorin
Carrier mobility
Methotrexate
Conservation
Leukemia
Mutation
Folic Acid
Substrates
Growth
Cells
Cell Line
Mutagenesis
Asparagine
Serine
Inhibitory Concentration 50
Transfection
Tumors
Blood
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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",
abstract = "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 μM, 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 V(max) without a significant change in K(m). 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 MTXτA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXτA-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 RF1 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.",
author = "Rongbao Zhao and Assaraf, {Yehuda G.} and Goldman, {I. David}",
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T1 - 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

AU - Zhao, Rongbao

AU - Assaraf, Yehuda G.

AU - Goldman, I. David

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N2 - 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 μM, 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 V(max) without a significant change in K(m). 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 MTXτA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXτA-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 RF1 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.

AB - 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 μM, 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 V(max) without a significant change in K(m). 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 MTXτA cells, which lack functional endogenous carrier, resulted in a clonal derivative MTXτA-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 RF1 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.

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