Characterization of intracellular pH gradients in human multidrug-resistant tumor cells by means of scanning microspectrofluorometry and dual-emission-ratio probes

Multidrug-resistant cells are believed to contain a plasma-membrane-efflux pump which is hypothesized to expel anticancer drugs from the cytosol to th e cell exterior. Many of these drugs are classified as weak bases whose bin ding to intracellular targets is pH-dependent. Slight alterations in intrac ellular pH gradients have been shown to affect accumulation, endocytosis an d secretion of drugs. In this study, we developed a new method based on con focal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells. Fluorescein isothiocyanate (FITC) and tetram ethylrhodamine conjugated to dextran (FRD) and SNAFL-calcein-AM were used t o determine pH in acidic compartments. Carboxy-SNARFI-AM was used to examin e cytosolic pH. We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that correspondin g to drug-resistant derivatives. Moreover, results obtained with carboxy-SN ARFI-AM show that resistant cells display a more alkaline cytosolic pH, Thi s results in a considerably larger pH gradient between the vesicular compar tments and the cytosol of resistant cells than of sensitive cells. The lowe r 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 microscopi c analysis of cells labelled with FRD and SNAFL-calcein showed that sensiti ve cells contain a lower number of acidified vesicles. This suggest a dimin ished 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. (C) 1999 Wiley-Liss, Inc.