Amplification and Organization of Dihydrofolate Reductase Genes in a Human Leukemic Cell Line, K-562, Resistant to Methotrexate

A subline of human leukemia cells (K-562), highly resistant to methotrexate, was developed by stepwise selection in the presence of increasing concentrations of this drug. The ED₅₀ of these resistant cells was 1 mM compared to 10 nM for the parental line. Comparison of certain folate-requiring enzymes from crude extracts of the parent and resistant cells showed a 240-fold elevation of dihydrofolate reductase activity in the resistant cells with no significant increase in the levels of the other enzymes. Purified dihydrofolate reductase from the resistant cells had the same physical and kinetic properties as the enzyme from the sensitive cells. Southern blot analysis showed a marked increase in the number of dihydrofolate reductase genes in the resistant line. The genomic organization of the human dihydrofolate reductase gene was determined by hybridization with specific cDNA sequences from a human cDNA to DNA fragments from K-562 cells generated by restriction endonucleases. The human dihydrofolate reductase gene contained at least four intervening sequences and was approximately 30 kb in size. Northern blot studies demonstrated an increase of dihydrofolate reductase mRNA species; the predominant message was 3.8 kb. Karyotype analysis revealed three elongated marker chromosomes, derived from chromosomes 5, 6, and 19 which contained homogeneous staining regions, which were not present in the parent cell line.