A study on the molecular diagnosis of antifolate resistance in leukemia cells : Establishment of anti-folate-resistant cell line and elucidation of molecular mechanisms of resistance

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Antifolate resistant cell lines were established to elucidate molecular mechanisms of resistance. Effects of reduced or oxidized folates on the development of methotrexate (MTX) or N^<10>-propargyl-5,8-dideazafolic (CB3717) acid resistance were examined. An approximately 60-fold MTX-resistant CCRF-CEM developed in pteroylglutamic acid (PGA) was resistant to MTX by virtue of decreased membrance transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX resistance as noted by increased dihydrofolate reductase (DHFR) due to gene amplification. An approximately 140-fold and subsequently 1500-fold MTX-resistant subline developed in leucovorin were resistant to MTX by virtue of increased DHFR due to gene amplification. Approximately 200-fold CB3717-resistant MOLT-3 sublines developed in PGA or leucovorin had decreased membrance transport. Only CB3717-resistant subline developed in PGA had increased thymidylate synthase due to gene amplification. Membrane transport defective anti-folate-resistant sublines showed collateral sensitivity to trimeterxate, which was reduced in the presence of leucovorin, suggesting collateral sensitivity to TMQ was caused by intracellular folate defficiency.