MUTATIONS IN THE GENE FOR HUMAN DIHYDROFOLATE-REDUCTASE - AN UNLIKELYCAUSE OF CLINICAL RELAPSE IN PEDIATRIC LEUKEMIA AFTER THERAPY WITH METHOTREXATE

Resistance to methotrexate (MTX) in some sublines of mammalian cells i s reported to be due to one of the following amino acid substitutions in dihydrofolate reductase (DHFR) that lower inhibition by MTX: Gly(15 ) to Trp, Leu(22) to Arg or Phe, or Phe(31) to Trp or Ser. We have pro duced variants of human DHFR (hDHFR) with these substitutions by direc ted mutagenesis. Recombinant hDHFR variants expressed in Escherichia c oli have greatly decreased inhibition by MTX, but decreased catalytic efficiency, and in one case decreased stability. When a retroviral vec tor encoding wild-type (wt) hDHFR or one of these variants was introdu ced into murine fibroblasts or bone marrow progenitors, modest protect ion from MTX was conferred, even by wt. Relapsed pediatric patients wi th acute lymphoblastic leukemia who have received multiple courses of high-dose MTX seem most likely to develop such MTX resistance. cDNA wa s reverse transcribed from blast mRNA from 17 of these patients. Howev er, upon amplification and sequencing of DHFR cDNA, no resistance muta tion was found. The explanation for this probably lies in the need for considerable gene amplification to offset lowered catalytic efficienc y, and the need for two-base changes for most substitutions, both of w hich are probably infrequent events.