Comparison of the Protection of Cells from Antifolates by Transduced Human Dihydrofolate Reductase Mutants

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Abstract
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 (≤7.5-fold increased resistance), piritrexim (≤16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (~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. Introduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into hematopoietic cells may alleviate the myelosuppression associated with these drugs. We used retroviral transduction to introduce into CCRF-CEM cells wild-type hDHFR and 20 variants to compare their ability to increase resistance to methotrexate, trimetrexate, piritrexim, and edatrexate. Two of the variants make cells ~200-fold resistant to trimetrexate, but the maximum increase in resistance to methotrexate is 7.5-fold, and resistance to edatrexate is negligible. In polyclonal cultures of transduced cells, high resistance to trimetrexate-induced cytotoxicity is associated with low affinity of the drug for the variant enzyme, retention of reasonable catalytic efficiency, and relative thermal stability, but not with elevated expression of the mutant hDHFR.

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