A STRUCTURALLY ALTERED HUMAN REDUCED FOLATE CARRIER WITH INCREASED FOLIC-ACID TRANSPORT MEDIATES A NOVEL MECHANISM OF ANTIFOLATE RESISTANCE

CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays > 200-fold resistance to methotrexate (MTX) due to defective transport v ia the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, de rived by a gradual deprivation of folic acid from 2.3 mu M to 2 nM (LF ) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type K-m value for MTX transport but a 31-fold and 9-fold lower K-m values for folic acid and leucovorin, respectively, relative to wild type RF C; 2) a 10-fold RFC1 gene amplification along with a >20-fold increase d expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulati on of MTX transport by anions (i.e. chloride); and 4) a G --> A mutati on at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, r esulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the hu man RFC. Upon transfer of CEM/MTX-LF cells to folate replete medium (2 .3 mu M folic acid), the more efficient folic acid uptake in CEM/MTX-L F cells resulted in a 7- and 24-fold elevated total folate pool compar ed with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol /mg of protein, respectively). This markedly elevated intracellular fo late pool conferred a novel mechanism of resistance to polyglutamatabl e (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. t rimetrexate and pyrimethamine) by abolishing their polyglutamylation a nd circumventing target enzyme inhibition.