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Characterization of intracellular pH gradients in human multidrug‐resistant tumor cells by means of scanning microspectrofluorometry and dual‐emission‐ratio probes
Author(s) -
Belhoussine Rajae,
Morjani Hamid,
Sharonov Serguei,
Ploton Dominique,
Manfait Michel
Publication year - 1999
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19990331)81:1<81::aid-ijc15>3.0.co;2-p
Subject(s) - intracellular , cytosol , calcein , cytoplasm , secretion , biophysics , microbiology and biotechnology , organelle , confocal microscopy , cancer cell , flow cytometry , efflux , biochemistry , chemistry , golgi apparatus , biology , cell , membrane , cancer , genetics , enzyme
Multidrug‐resistant cells are believed to contain a plasma‐membrane‐efflux pump which is hypothesized to expel anti‐cancer drugs from the cytosol to the cell exterior. Many of these drugs are classified as weak bases whose binding to intracellular targets is pH‐dependent. Slight alterations in intracellular pH gradients have been shown to affect accumulation, endocytosis and secretion of drugs. In this study, we developed a new method based on confocal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine conjugated to dextran (FRD) and SNAFL‐calcein‐AM were used to determine pH in acidic compartments. Carboxy‐SNARF1‐AM was used to examine cytosolic pH. We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that corresponding to drug‐resistant derivatives. Moreover, results obtained with carboxy‐SNARF1‐AM show that resistant cells display a more alkaline cytosolic pH. This results in a considerably larger pH gradient between the vesicular compartments and the cytosol of resistant cells than of sensitive cells. The lower pH gradient observed in sensitive cells may be related to a disruption in the organization of the trans ‐Golgi network (TGN). In drug‐resistant cells, the organization of TGN appears compact. In addition, confocal microscopic analysis of cells labelled with FRD and SNAFL‐calcein showed that sensitive cells contain a lower number of acidified vesicles. This suggest a diminished capacity of these cells to remove protonated drugs from the cytoplasm to secretory compartments followed by their secretion through the activity of the secretory and recycling pathways. Int. J. Cancer 81:81–89, 1999. © 1999 Wiley‐Liss, Inc.