Development of an assay system for the detection and classification of methotrexate resistance in fresh human leukemic cells.
An assay system was developed for the detection and classification of methotrexate resistance in fresh human leukemic cells. Mechanisms of resistance to be identified were: overexpression of dihydrofolate reductase, decreased cellular uptake of methotrexate, decreased affinity of dihydrofolate reductase for methotrexate, decreased polyglutamylation of methotrexate, and low thymidylate synthase activity. The initial screening procedure utilizes 3H release after addition of [5-3H]-2'-deoxyuridine as a measure of intracellular activity of thymidylate synthase and of DNA synthesis; 3H release is assayed after 3-h incubations with methotrexate, trimetrexate, or gamma-fluoromethotrexate and after 4-h incubations with these agents followed by a 6-h incubation in drug free medium. The pattern of DNA synthesis inhibition and recovery under these two sets of conditions establishes the presence or absence of methotrexate resistance and allows a tentative classification of the resistance mechanism involved. In combination with determinations of dihydrofolate reductase activity, methotrexate titration studies, and the determination of intracellular drug accumulations in vitro, the system is readily able to classify CCRF-CEM human leukemia cell lines possessing well defined mechanisms of resistance. The findings in seven leukemia patients are also reported. Applying tentative reference values, four patients showed biochemical evidence of methotrexate resistance: two patients had only a transport defect, one patient had evidence of both a transport defect and low thymidylate synthase activity, and one patient appeared to have decreased methotrexate polyglutamylation. Application of the assay system in larger numbers of patients is feasible and is required to establish adequate reference values for the evaluation of biochemical-clinical correlates.
D000293:Adolescent; D000328:Adult; D000368:Aged; D002460:Cell Line; D004273:DNA, Neoplasm / Q000096:biosynthesis; D003857:Deoxyuridine / Q000378:metabolism; D004351:Drug Resistance; D005260:Female; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008297:Male; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis; D016597:Trimetrexate; D014316:Tritium
D004273:DNA, Neoplasm; D011799:Quinazolines; D014316:Tritium; D013762:Tetrahydrofolate Dehydrogenase; D016597:Trimetrexate; D003857:Deoxyuridine; D008727:Methotrexate