TY - JOUR
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
PY - 1998/4/3
Y1 - 1998/4/3
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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U2 - 10.1074/jbc.273.14.7873
DO - 10.1074/jbc.273.14.7873
M3 - Article
C2 - 9525881
AN - SCOPUS:0032478826
SN - 0021-9258
VL - 273
SP - 7873
EP - 7879
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -