Studies of the overproduction of dihydrofolate reductase by variant hamster cells in culture

Abstract

Recent studies in this laboratory have concerned the regulation of dihydrofolate reductase in cultured hamster cells. Variant sublines highly resistant to Methotrexate due to markedly increased reductase activity were isolated. The reductase, purified by affinity chromatography, exists in hamster cells in two basic forms, which have different physical and kinetic properties but the same fingerprint patterns, and which cannot be interconverted. In the variant cells the main form has the same fingerprint pattern, and the minor form also is probably not altered. Turnover studies, first with antiserum to the enzyme and subsequently with affinity chromatography, indicate that the increase in reductase activity is entirely due to an increased rate of enzyme synthesis rather than decreased degradation. Polysomal RNA from the resistant cells, combined with a cell-free protein-synthesizing system, stimulates the in vitro production of reductase at an increased rate which accounts for half or more of the increased rate of enzyme production in the resistant cells. Possible mechanisms causing overproduction of the reductase are discussed, in addition to our current related studies.