Sensitivity to 5,10-dideazatetrahydrofolate is fully conserved in a murineleukemia cell line highly resistant to methotrexate due to impaired transport mediated by the reduced folate carrier

A murine leukemia cell line was identified that is highly resistant to meth otrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dide azatetrahydrofolate (DDATHF), A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in dr ug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell l ine markedly increased DDATHF influx (32x) but only modestly increased infl ux of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively). The g rowth inhibition or growth requirements for these folates fell by factors o f 18, 2, and 4, respectively, in the transfectant. Preservation of DDATHF i nflux in cells with V104M RFC1 resulted in even greater preservation (60%) of the exchangeable drug level, Another major element in the preservation o f DDATHF activity was the impact of the mutated carrier on cellular folate pools. For folic acid, folate pools mere essentially unchanged but DDATHF p olyglutamate levels decreased in lines that express the V104M carrier. Howe ver, with 5-formyltetrahydrofolate as the growth source, there was a marked decrease in folate pools in the lines carrying the mutated carrier, and DD ATHF polyglutamate levels were unchanged, Hence, DDATHF activity was preser ved in cells with V104M RFC1 due to (a) relative conservation of DDATHF tra nsport, and (b) depletion of cellular THF cofactors with diminishing folate cofactor competition at folylpolyglutamate synthetase and possibly glycina mide ribonucleotide formyltransferase, Hence, resistance to one antifolate, in this case MTX, because of a loss of RFC1 transport activity need not ex clude the subsequent utility of another antifolate that uses the same carri er.