Abstract
Extreme resistance of L5178Y (R4) mouse leukemia cells to methotrexate is associated with the expression of increased levels of dihydrofolate reductase activity represented by two forms of the enzyme (Forms 1 and 2). Form 2 has been shown to be markedly insensitive to methotrexate inhibition. Rabbit serum antibodies against highly purified preparations of the two forms of dihydrofolate reductase have differential affinities for the two forms. Anti-Form 1 immunoglobulin G exhibited approximately 65% higher affinity for Form 1 dihydrofolate reductase than for Form 2, demonstrating considerable antigenic diversity between the two forms. Some cross-reactivity between the two antigens is present, compatible with the fact that although they differed markedly in their inhibition by methotrexate, both forms retained their catalytic activity for the reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid. Anti-Form 2 immunoglobulin G exhibited a higher affinity for Form 2 dihydrofolate reductase than for Form 1 enzyme, although not to as great an extent.
The increased catalytic activity of dihydrofolate reductase in these cells as compared to that in the wild-type sensitive cells is shown to be due to the overproduction of the enzyme in the resistant cells as determined by radiolabeling of the cellular proteins followed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and fluorography of these proteins. A high proportion of the radioactivity was associated with a protein band corresponding to the molecular weight of dihydrofolate reductase. This overproduction is associated with the presence of abundant messenger RNA (mRNA) activity for the enzyme, as demonstrated by in vitro translation of mRNA (polyadenylic acid-containing) from the resistant and wild-type sensitive cells. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of 35S-labeled translation products followed by fluorography showed a major protein band corresponding to dihydrofolate reductase when mRNA from the resistant cells was used to program translation but not when mRNA from the wild-type sensitive cells was used. Immunoglobulin G preparations against both forms of dihydrofolate reductase, which showed considerable antigenic diversity, were able to immunoprecipitate a single band, corresponding to dihydrofolate reductase, from the translation products programmed by resistant cell mRNA. Chromosome analysis by Giemsa-trypsin banding revealed a prominent homogeneously staining region in one of the chromosomes of the resistant cells. The presence of abundant mRNA activity for dihydrofolate reductase and the chromosome with the homogeneously staining region is compatible with amplification of the dihydrofolate reductase gene resulting in the overproduction, in this case, of two antigenically distinct enzymes.