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 capacity of K562.4CF cells (Matherly et al., Cancer Res. 51: 3420-3426, 1991) were studied with reduced folate carrier (RFC) cDNA, genomic, and antibody probes. Southern analysis showed that RFC gene copies were increased (approximately 4- to 5-fold) in K562.4CF over wild-type K562 cells. Fluorescence in situ hybridization using a genomic RFC probe confirmed the localization of the RFC gene to the q-arm of chromosome 21. 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) different chromosomal loci not seen in wild-type cells. Particularly intense RFC signals were mapped to homogeneously staining regions in chromosomes 2 and 15. Increased RFC gene copies were accompanied by a similar increase in the major 3.1 kb RFC transcript by northern blotting and an approximately 7-fold elevated level of the broadly migrating (80-95 kDa) RFC protein on a western blot probed with an RFC C-terminal peptide antibody. These results demonstrate that selection of cells with a growth-limiting concentration of reduced folates (0.4 nM of leucovorin) is sufficient to promote chromosomal aberrations, including gene amplification and translocations that result in increased RFC expression and folate transport.