Chromosomal localization of the murine RFC-1 gene encoding a folate transporter and its amplification in an antifolate resistant variant overproducing the transporter

Cancer Genet Cytogenet. 1998 Aug;105(1):29-38. doi: 10.1016/s0165-4608(98)00005-3.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / genetics*
  • Chromosome Aberrations / genetics
  • Chromosome Disorders
  • Chromosome Mapping
  • Chromosomes, Human, Pair 10 / genetics*
  • Drug Resistance, Neoplasm*
  • Folic Acid Antagonists / pharmacology*
  • Gene Amplification*
  • Gene Dosage
  • Humans
  • Karyotyping
  • Leukemia L1210 / genetics
  • Leukemia L1210 / metabolism
  • Membrane Proteins / antagonists & inhibitors
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / genetics*
  • Membrane Transport Proteins*
  • Mice
  • RNA, Messenger / analysis
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tumor Cells, Cultured

Substances

  • Carrier Proteins
  • Folic Acid Antagonists
  • Membrane Proteins
  • Membrane Transport Proteins
  • RNA, Messenger
  • SLC19A2 protein, human
  • Slc19a2 protein, mouse
  • Tetrahydrofolate Dehydrogenase