Open AccessIdentification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry.
Author(s) -
Yehuda G. Assaraf,
Robert T. Schimke
Publication year - 1987
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.84.20.7154
Subject(s) - methotrexate , dihydrofolate reductase , chinese hamster ovary cell , antifolate , flow cytometry , biology , antimetabolite , microbiology and biotechnology , chemistry , cell culture , biochemistry , immunology , genetics
We have studied the frequency of transport mutations in methotrexate-resistant Chinese hamster ovary cells using a rapid-flow cytometric technique. After saturating cells with fluoresceinated methotrexate, we examined the ability of hydrophilic and lipophilic antifolates to displace fluoresceinated methotrexate binding to dihydrofolate reductase. Cells with methotrexate transport deficiency are unable to take up methotrexate and thus retain the fluorescence, whereas the lipophilic antifolates displace fluoresceinated methotrexate equally well in sensitive and resistant cell lines. These resistant clones fail to take up methotrexate and occur with high frequencies upon single-step selections at methotrexate concentrations approximately equal to 7-fold the 50% killing concentration. The majority of such first-step resistant clones appear to derive their resistance solely from transport deficiency; they exhibit no overproduction of dihydrofolate reductase and no increase in either steady-state mRNA levels or gene copy number. Possible applications of the use of fluoresceinated methotrexate to the characterization of various mechanisms of methotrexate resistance in mixed cell populations are discussed.