5-METHYLTETRAHYDROFOLATE TRANSPORT BY HEPATOMA-CELLS AND METHOTREXATE-RESISTANT SUBLINES IN CULTURE

  • 1 January 1981
    • journal article
    • research article
    • Vol. 41  (5) , 1757-1762
Abstract
The properties of the folate transport system in H35 hepatoma cells [rat] were studied by measuring the transport of (+)-5-methyltetrahydrofolate. Using initial rates of uptake, it was demonstrated that the uptake is saturable, carrier mediated, and shared by methotrexate [an antitumor agent]. The accumulation of (+)-5-methyltetrahydrofolate is concentrative, demonstrating the presence of an active transport process. The initial uptake and steady-state level of (+)-5-methyltetrahydrofolate were markedly reduced in the resistant sublines as was the case with methotrexate. Triazinate (2-{chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1(2H)-ylphenoxyl]} -N,N-dimethyl-m-toluamide .cntdot. ethanesulfonic acid) an inhibitor of dihydrofolate reductase which enters the cells by a pathway independent of the folate coenzyme, was equally toxic to H35 cells and to an H35 subline resistant to 0.3 .mu.M methotrexate. Resistant sublines that are insensitive to methotrexate up to 1 .mu.M display a transport defect but have normal levels of dihydrofolate reductase. Sublines resistant to higher levels of methotrexate showed not only defective transport but also commensurate increases in dihydrofolate reductase. Attempts to demonstrate carrier-dependent transport of (+)-5-methyltetrahydrofolate or methotrexate in resistant sublines were negative, suggesting the lack of a functional carrier. These properties were readily demonstrated in H35 cells and included temperature dependence, competition for uptake with analogs and transstimulation.