Comparison of the protection of cells from antifolates by transduced human dihydrofolate reductase mutants.
Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.
Patel
M
M
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Sleep
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Lewis
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Spencer
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Mareya
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Sorrentino
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Blakley
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D000630:Aminopterin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D000818:Animals; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015870:Gene Expression; D014644:Genetic Variation; D006131:Growth Inhibitors / Q000037:antagonists & inhibitors; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000037:antagonists & inhibitors; D011817:Rabbits; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D016597:Trimetrexate / Q000037:antagonists & inhibitors
D005493:Folic Acid Antagonists; D006131:Growth Inhibitors; D011743:Pyrimidines; D013762:Tetrahydrofolate Dehydrogenase; C040210:edatrexate; D000630:Aminopterin; C024530:piritrexim; D016597:Trimetrexate; D013936:Thymidine; D008727:Methotrexate