Gene Amplification and Increased Expression of the Reduced Folate Carrier in Transport Elevated K562 Cells

• Published in: Biochemical pharmacology Vol. 55; no. 7; pp. 1135 - 1138
• Main Authors: Wong, So C, Zhang, Long, Proefke, Susan A, Hukku, Bharati, Matherly, Larry H
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.04.1998; Elsevier Science
• Subjects: Antineoplastic agents; Biological and medical sciences; Blotting, Western; Carrier Proteins - biosynthesis; Carrier Proteins - genetics; Cell Line; Cell physiology; Chromosome Aberrations - genetics; Chromosomes, Human, 21-22 and Y; folates; Folic Acid - metabolism; Fundamental and applied biological sciences. Psychology; gene amplification; Gene Amplification - genetics; General aspects; homogeneously staining region; Humans; In Situ Hybridization; Medical sciences; Membrane and intracellular transports; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Membrane Transport Proteins; methotrexate; Molecular and cellular biology; Pharmacology. Drug treatments; Reduced Folate Carrier Protein; Translocation, Genetic; transport; folates; methotrexate; homogeneously staining region; transport; gene amplification; Antineoplastic agent; Antifolate; Translocation; Gene amplification; Antimetabolic; Biological transport; Established cell line; Calcium folinate; Gene expression; Methotrexate; In vitro; Tumor cell
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(97)00639-4

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 (≈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 ≈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.