GENE AMPLIFICATION AND INCREASED EXPRESSION OF THE REDUCED FOLATE CARRIER IN TRANSPORT ELEVATED K562 CELLS

The molecular bases for the 6-fold elevated methotrexate transport cap acity of K562.4CF cells (Matherly et al., Cancer Res. 51: 3420-3426, 1 991) were studied with reduced folate carrier (RFC) cDNA, genomic, and antibody probes. Southern analysis showed that RFC gene copies were i ncreased (approximate to 4- to 5-fold) in K562.4CF over wild-type K562 cells. Fluorescence in situ hybridization using a genomic RFC probe c onfirmed the localization of the RFC gene to the q-arm of chromosome 2 1. In K562.4CF cells, the frequent loss of a normal copy of chromosome 21 (61% of metaphases) was accompanied by RFC gene amplification and translocations of amplified RFC gene fragments to several (2 to 6) dif ferent chromosomal loci not seen in wild type cells. Particularly inte nse RFC signals were mapped to homogeneously staining regions in chrom osomes 2 and 15. Increased RFC gene copies were accompanied by a simil ar increase in the major 3.1 kb RFC transcript by northern blotting an d an approximate to 7-fold elevated level of the broadly migrating (80 -95 kDa) RFC protein on a western blot probed with an RFC C-terminal p eptide antibody. These results demonstrate that selection of cells wit h a growth-limiting concentration of reduced folates (0.4 nM of leucov orin) is sufficient to promote chromosomal aberrations, including gene amplification and translocations that result in increased RFC express ion and folate transport. (C) 1998 Elsevier Science Inc.