Characterization by flow cytometry and fluorescein-methotrexate labeling of hydrophilic and lipophilic antifolate resistance in cultured mammalian cells

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Abstract
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 antlfolates [e.g. trimetrexate (TMTX)] in cultured mammalian cells using fluorescein-methotrexate (F-MTX) and flow cytometry. Toward this end an integrative F-MTX labeling and flow cytometry-based protocol is proposed here to facilitate the rapid identification of modes of antifolate resistance in a heterogenous drug-resistant cell population or in clonal derivatives. Following antifolate selection, drug-resistant cells are first labeled with F-MTX In order to saturate Intracellular dihydrofolate reductase (DHFR). F-MTX-labeled cells are then subjected to flow cytometric analysis and mean fluorescence/cell is determined. Thus, increased F-MTX staining is an indication of overproduction 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 altered DHFR displaying a decreased affinity for antifolates. Alternatively, antifolate-resistant cells frequently display wild-type F-MTX labeling; these cells are subjected to competition with hydrophilic and lipophilic antifolates in order to examine whether the process of antifolate accumulation is deficient. Cells that lose F-MTX labeling upon competition with lipophilic antifolates yet still retain it with hydrophilic antifolates, are likely to possess transport alteration(s) that Impair 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 antifolates. The importance of the antifolate concentration yielding 50% displacement of cellular F-MTX labeling in the quantitative assessment of the degree of DHFR overexpression and/or antifolate transport alteration is discussed. Thus, flow cytometric analysis of F-MTX-labeled cells following competition with hydrophilic and lipophilic antifolates provides 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.

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