Chromosomal localization of the murine RFC-1 gene encoding a folate transporter and its amplification in an antifolate resistant variant overproducing the transporter.
A variant of the L1210 cell (L1210/R83) selected in the presence of the lipophilic antifolate, metoprine, and a concentration of the natural diastereoisomer of 5-formyltetrahydrofolate, lL5CHO-folateH4, suboptimum for growth exhibited a 35-fold increase compared to parental L1210 cells in one-carbon, reduced folate transport. This was evidenced by the increase in Vmax for [3H]MTX (methotrexate) influx and a commensurate increase in the amount of the 46 kilodalton (kDa) transport protein and reduced folate carrier (RFC-1) mRNA. The variant is resistant to lipophilic antifolates, but shows collateral sensitivity to classical folate analogues. Karyotype analysis of L1210/R83 cells revealed the presence of several new chromosome abnormalities. One of these was a large, submetacentric marker chromosome comprising a normal #10 and a longer, abnormally banded arm of uncertain origin which exhibited an interstitial, palely staining, HSR-like segment. The results of Southern and Northern blotting showed that the RFC-1 gene copy number and RNA transcript level were markedly increased (30-35 fold) in L1210/R83 cells. Fluorescence in situ hybridization (FISH) analysis revealed that the HSR-like segment in these cells was the site of amplified RFC-1 genes. Independent revertant subclones, obtained following growth in the absence of selection pressure, showed four- to 12-fold decreases in [3H]MTX influx Vmax and in amount of NHS (N-hydroxysuccinimide)-[3H]MTX affinity labeled one-carbon, reduced folate transporter compared to L1210/R83 cells. RFC-1 gene copy number also decreased, and the mean length of the HSR in these revertants declined 1.6- to 5-fold. Based upon genomic nucleotide sequencing, the RFC-1 gene in the normal mouse genome was localized to chromosome 10 in close association with the alpha 1 (Col18a1) collagen gene at 10B3(locus 41cM). The close association of these genes was confirmed by other data showing that the alpha 1 collagen gene was co-amplified in L1210/R83 cells. These results document the amplification at the site of a putative HSR in an L1210 cell variant of the RFC-1 gene regulating expression of the one-carbon, reduced folate transporter.
Program in Cell Biology and Genetics, Graduate School of Medical Sciences, Cornell University, New York, New York, USA.
K
K
Roy
J H
JH
Chiao
B A
BA
Spengler
B
B
Tolner
C H
CH
Yang
J L
JL
Biedler
F M
FM
Sirotnak
D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D002869:Chromosome Aberrations / Q000235:genetics; D025063:Chromosome Disorders; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10 / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005784:Gene Amplification; D018628:Gene Dosage; D006801:Humans; D007621:Karyotyping; D007939:Leukemia L1210 / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D026901:Membrane Transport Proteins; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C120043:SLC19A2 protein, human; C492292:Slc19a2 protein, mouse; D013762:Tetrahydrofolate Dehydrogenase