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 t he lipophilic antifolate, metoprine, and a concentration of the natura l diastereoisomer of 5-formyltetrahydrofolate, lLCHO-folateH(4), subop timum for growth exhibited a 35-fold increase compared to parental L12 10 cells in one-carbon, reduced folate transport. This was evidenced b y the increase in V-max for [H-3]MTX (methotrexate) influx and a comme nsurate increase in the amount of the 46 kilodalton (kDa) transport pr otein and reduced folate carrier (RFC-1) mRNA. The variant is resistan t to lipophilic antifolates, but shows collateral sensitivity to class ical 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 a n interstitial, palely staining, HSR-like segment. The results of Sout hern 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 th at 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 i n [H-3]MTX influx V-max and in amount of NHS (N-hydroxysuccinimide)-[H -3]MTX affinity labeled one-carbon, reduced folate transporter compare d to L1210/R83 cells. RFC-1 gene copy number also decreased, and the m ean length of the HSR in these revertants declined 1.6- to 5-fold. Bas ed upon genomic nude otide sequencing, the RFC-1 gene in the normal mo use genome was localized to chromosome 10 in close association with th e alpha 1 (Col18a1) collagen gene at 10B3(locus 41cM). The close assoc iation of these genes was confirmed by of her data showing that the al pha 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. (C) Elsevier Science Inc., 1998.