Determinants of the disparate antitumor activities of (6 R)-5,10-dideaza-5,6,7,8-tetrahydrofolate and methotrexate toward human lymphoblastic leukemia cells, characterized by severely impaired antifolate membrane transport

Abstract

We previously reported (Matherly et al., J Biol Chem 267: 23253–23260, 1992) that impaired methotrexate transport in a drug-resistant CCRF-CEM variant (CEM/MTX) involved the synthesis of a structurally altered isoform of the “classical” carrier for methotrexate and related derivatives. Although CEM/MTX cells were highly resistant (162- to 300-fold) to assorted antifolate substrates for the classical transporter, including methotrexate, aminopterin, 10-ethyl-10-deazaaminopterin, ICI D1694, and 1843U89, they were only 3.6-fold resistant to (6 R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF)> These divergent antifolate sensitivities were not associated with appreciable differences in the levels of dihydrofolate reductase, thymidylate synthase, and 5′-phosphoribosylglycinamide (GAR) transformylase, or the expression of a high affinity membrane folate binding protein receptor in either line. The initial rate of [ 14C]DDATHF influx was increased 2.9-fold over that for [ 3H]metrotrexate in parental cells (at 2 μM). Whereas [ 14C]DDATHF initial uptake was, likewise, increased over [ 3H]methotrexate in CEM/MTX cells (5.3-fold), influx of both compounds was impaired substantially (95–97%). For the parent, influx of [ 14C]DDATHF was inhibited by substrates for the classical transporter including unlabeled DDATHF, methotrexate, (6 R)-5-formyl tetrahydrofolate, 10-ethyl-10-deazaaminoproterin, 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. Inspite of its impaired transport, [ 14C]DDATHF (at 2 μM), unlike methotrexate, continued to accumulate in CEM/MTX cells, eventually reaching 62% of the parental drug levels after 4 hr. At this time, 53% (parent) and 71% (CEM/MTX) of the intracellular radioactivity from [ 14C]DDATHF was identified as polyglutamates. DDATHF polyglutamates in CEM/MTX cells after 4 hr reached 90% of the levels measured in parental cells. While significant levels of methotrexate polyglutamates were detected in the parental line, methotrexate polyglutamylation was negligible in intact CEM/MTX cells. The specific activity of folypolyglutamate synthetase was measured in cell-free extracts from parental and CEM/MTX cells using aminopterin, methotrexate, and DDATHF as substrates; in each case, CEM/MTX cells showed 2-fold higher enzyme activity than parental cells. These data show that even for tumor cells with severely impaired antifolate transport, the extensive conversion of DDATHF to polyglutamyl forms required for GAR transformylase inhibition preserves high levels of antitumor activity.