COMPARISON OF THE PROTECTION OF CELLS FROM ANTIFOLATES BY TRANSDUCED HUMAN DIHYDROFOLATE-REDUCTASE MUTANTS

Retroviral transduction of antifolate-resistant variants of human dihy drofolate reductase (hDHFR) into cells can increase their resistance t o the cytotoxic effects of these drugs, We evaluated the ability of wi ld-type hDHFR and 20 mutant enzymes (13 with single-amino acid substit utions, 7 with two substitutions) to prevent growth inhibition in anti folate-treated CCRF-CEM cells, The wild-type enzyme and all of the var iants significantly protected transduced cells from trimetrexate (TMTX )-induced growth inhibition, However, only half of the variants confer red more protection than does the wild-type enzyme, For the variants t ested, the observed protective effect was higher for TMTX than for met hotrexate (less than or equal to 7.5-fold increased resistance), pirtr exim (less than or equal to 16-fold), and edatrexate (negligible), Tra nsduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (similar to 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 fr om the level of expression: Efficient protection resulted from low aff inity of the variant for antifolates, reasonable catalytic activity, a nd good thermal stability, Clones isolated from a polyclonal populatio n of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression lev els.