COMPARISON OF METHOTREXATE POLYGLUTAMYLATION IN L1210 LEUKEMIA-CELLS WHEN INFLUX IS MEDIATED BY THE REDUCED FOLATE CARRIER OR THE FOLATE RECEPTOR - LACK OF EVIDENCE FOR INFLUX ROUTE-SPECIFIC EFFECTS

We previously described a methotrexate-resistant L1210 cell line (MTX( r)A) that lacks a functional reduced folate carrier and does not appre ciably express the folate receptor. In the present study, we utilized MTX(r)A cell lines stably transfected with cDNAs encoding either the f olate receptor or the reduced folate carrier to investigate the influe nce of the route of folate influx on the rate and extent of methotrexa te polyglutamylation. At an extracellular methotrexate concentration o f 0.1 mu M, influx in the folate receptor transfectant (MTX(r)A-TF1) a nd in the reduced folate carrier transfectant (MTX(r)A-R1) was equal a nd methotrexate polyglutamates accumulated at an identical rate, but t he onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (similar to 3 hr). The onset of polyglutamate format ion was immediate and identical among the lines in cells pretreated wi th the lipophilic dihydrofolate reductase inhibitor trimetrexate to bl ock methotrexate binding to dihydrofolate reductase. The spectra of in dividual methotrexate polyglutamates that accumulated were similar, wi th the tetraglutamate present as the predominant form. A 100-fold high er methotrexate concentration was required to detect methotrexate upta ke and polyglutamylation in the transport defective parent MTX(r)A lin e, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the red uced folate carrier achieve nearly identical rates of polyglutamylatio n despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglut amate synthetase is unlikely to play a role in tetrahydrofolate metabo lism. This study supports the notion that it is the intracellular conc entration of methotrexate achieved within the cell that drives polyglu tamylation irrespective of its route of entry.