DETERMINANTS OF THE DISPARATE ANTITUMOR ACTIVITIES OF (6R)-5,10-DIDEAZA-5, 6,7,8-TETRAHYDROFOLATE AND METHOTREXATE TOWARD HUMAN LYMPHOBLASTIC-LEUKEMIA CELLS, CHARACTERIZED BY SEVERELY IMPAIRED ANTIFOLATE MEMBRANE-TRANSPORT

We previously reported (Matherly et al., J Biol Chem 267: 23253-23260, 1992) that impaired methotrexate transport in a drug-resistant CCRF-C EM variant (CEM/MTX) involved the synthesis of a structurally altered isoform of the ''classical'' carrier for methotrexate and related deri vatives. Although CEM/MTX cells were highly resistant (162- to 300-fol d) to assorted antifolate substrates for the classical transporter, in cluding methotrexate, aminopterin, 10-ethyl-10-deazaaminopterin, ICI D 1694, and 1843U89, they were only 3;6-foId resistant to (6R)-5,10-dide aza-5,6,7,8-tetrahydrofolate (DDATHF). These divergent antifolate sens itivities were not associated with appreciable differences in the leve ls of dihydrofolate reductase, thymidylate synthase, and 5'-phosphorib osylglycinamide (GAR) transformylase, or the expression of a high affi nity membrane folate binding protein receptor in either line. The init ial rate of [C-14]DDATHF influx was increased 2.9-fold over that for [ H-3]methotrexate in parental cells (at 2 mu M). Whereas [C-14]DDATHF i nitial uptake was, likewise, increased over [H-3]methotrexate in CEM/M TX cells (5.3-fold), influx of both compounds was impaired substantial ly (95-97%). For the parent, influx of [C-14]DDATHF was inhibited by s ubstrates for the classical transporter including unlabeled DDATHF, me thotrexate, (6R,S)-5-formyl tetrahydrofolate, 10-ethyl-10-deazaaminopt erin, ICI D1694, 1843U89, and folic acid. The synthesis of a modified transporter in CEM/MTX cells was accompanied by significant changes in the binding of all these transport substrates. In spite of its impair ed transport, [C-14]DDATHF (at 2 mu M), unlike methotrexate, continued to accumulate in CEM/MTX cells, eventually reaching 62% of the parent al drug Levels after 4 hr. At this time, 53% (parent) and 71% (CEM/MTX ) of the intracellular radioactivity from [C-14]DDATHF was identified as polyglutamates. DDATHF polyglutamates in CEM/MTX cells after 4 hr r eached 90% of the levels measured in parental cells. While significant levels of methotrexate polyglutamates were detected in the parental l ine, methotrexate polyglutamylation was negligible in intact CEM/MTX c ells. The specific activity of folylpolyglutamate synthetase was measu red in cell-free extracts from parental and CEM/MTX cells using aminop terin, methotrexate, and DDATHF as substrates; in each case, CEM/MTX c ells showed 2-fold higher enzyme activity than parental cells. These d ata show that even for tumor cells with severely impaired antifolate t ransport, the extensive conversion of DDATHF to polyglutamyl forms req uired for GAR transformylase inhibition preserves high levels of antit umor activity.