Metabolism of methotrexate and gamma-tert-butyl methotrexate by human leukemic cells in culture and by hepatic aldehyde oxidase in vitro.
The cellular uptake and metabolism of methotrexate (MTX) and gamma-tert-butyl methotrexate (TBM) were compared in CEM human leukemic lymphoblasts and a highly MTX-resistant subline (CEM/MTX) in which MTX uptake is defective. The CEM/MTX cells were found previously to be as sensitive as the parent line to TBM. While MTX was polyglutamylated extensively in the CEM cells, giving abundant levels of non-effluxing conjugates, polyglutamylation in CEM/MTX cells was reduced severely, even after exposure to a high MTX concentration (100 microM) in the medium. This treatment provided free intracellular MTX in greater than 100-fold excess over the dihydrofolate reductase level. In contrast to MTX, the ester TBM was unmetabolized in either cell line. Uptake levels after incubation of CEM and CEM/MTX cells with 2 microM TBM for 24 hr were 17 and 15 pmol/mg protein respectively. Thus, TBM accumulated equally in both cells and was well retained despite the lack of polyglutamylation. These results, together with the previously observed affinity of the drug for dihydrofolate reductase, provide a plausible rationale for the comparable sensitivity of CEM and CEM/MTX cells to TBM. Experiments were also performed to determine the susceptibility of TBM to metabolic detoxification by hepatic aldehyde oxidase. Km values were 8-fold lower for TBM than for MTX in assays using an enzyme preparation from rabbit liver, and Vmax values were 8-fold higher. Neither MTX nor TBM was oxidized to its 7-hydroxy derivative in intact CEM or CEM/MTX cells. Because TBM is capable of overcoming at least one of the modalities of MTX resistance, defective polyglutamylation, and may be more efficiently detoxified than MTX by the action of hepatic aldehyde oxidase, it has the potential to be a useful agent for the treatment of MTX-resistant tumors.
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011817:Rabbits; D013601:T-Lymphocytes
D011099:Polyglutamic Acid; C045204:gamma-tert-butyl methotrexate; C014085:methotrexate polyglutamate; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase; D008727:Methotrexate