Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy.
A series of methotrexate (MTX)-resistant human sublines developed by step increases in selected MTX concentrations have been cloned and examined for dihydrofolate reductase (DHFR) content, relative DNA copy number, and sensitivity to MTX. These cloned sublines had increased DHFR levels which were dependent on the presence of MTX in the medium. The increased levels of DHFR in the absence of MTX were stable in all the clones examined for over a year. Antibody immunolocalization on Western blots showed good correlation of the intensity of the immunostained DHFR band with enzyme activities. Relative gene copy number in these sublines was low relative to the DHFR increases and was not dependent on the presence of MTX in the medium. The increase in gene copy number in these sublines did not correlate with either the levels of DHFR or the sensitivity to MTX.
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005493:Folic Acid Antagonists; D005796:Genes; D006801:Humans; D007939:Leukemia L1210 / Q000201:enzymology; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification
D005493:Folic Acid Antagonists; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate