CHARACTERIZATION BY FLOW-CYTOMETRY AND FLUORESCEIN METHOTREXATE LABELING OF HYDROPHILIC AND LIPOPHILIC ANTIFOLATE RESISTANCE IN CULTURED-MAMMALIAN-CELLS

The aim of this review is to summarize currently available information on the rapid screening and initial characterization of the different mechanisms of resistance to hydrophilic [e.g. methotrexate (MTX)] and lipophilic antifolates [e.g. trimetrexate (TMTX)] in cultured mammalia n cells using fluorescein-methotrexate (F-MTX) and flow cytometry. Tow ard this end an integrative F-MTX labeling and flow cytometry-based pr otocol Is proposed here to facilitate the rapid identification of mode s of antifolate resistance in a heterogenous drug-resistant cell popul ation or in clonal derivatives. Following antifolate selection, drug-r esistant cells are first labeled with F-MTX in order to saturate intra cellular dihydrofolate reductase (DHFR). F-MTX-labeled cells are then subjected to flow cytometric analysis and mean fluorescence/cell is de termined. Thus, increased F-MTX staining is an indication of overprodu ction of the target enzyme for antifolates, DHFR, as a result of DHFR gene amplification. in contrast, significantly reduced cellular F-MTX labeling could be an indication of the existence of a structurally alt ered DHFR displaying a decreased affinity for antifolates. Alternative ly, antifolate-resistant cells frequently display wild-type F-MTX labe ling; these cells are subjected to competition with hydrophilic and li pophilic antifolates in order to examine whether the process of antifo late accumulation is deficient. Cells that lose F-MTX labeling upon co mpetition with lipophilic antifolates yet still retain it with hydroph ilic antifolates, are likely to possess transport alteration(s) that i mpair or abolish the accumulation of hydrophilic but not of lipophilic antifolates. In contrast, cells that lose their F-MTX labeling after competition with hydrophilic antifolates but retain it with lipophilic antifolates, possess a deficient accumulation of lipophilic antifolat es. The importance of the antifolate concentration yielding 50% displa cement of cellular F-MTX labeling in the quantitative assessment of th e degree of DHFR overexpression and/or antifolate transport alteration is discussed. Thus, flow cytometric analysis of F-MTX-labeled cells f ollowing competition with hydrophilic and lipophilic antifolates provi des a useful tool for the rapid screening and assessment of the major modes of antifolate resistance that may appear as exclusive mechanisms or co-emerge in mammalian cells following antifolate selection.