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Journal of Histochemistry and Cytochemistry, Vol. 45, 1255-1264, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Characterization of Acidic Vesicles in Multidrug-resistant and Sensitive Cancer Cells by Acridine Orange Staining and Confocal Microspectrofluorometry

Christine Millota, Jean-Marc Millotb, Hamid Morjanib, Andrée Desplacesa, and Michel Manfaitb
a Laboratoire de Physiologie Cellulaire, GIBSA, IFR 53 UFR de Pharmacie, Reims, France
b Laboratoire de Spectroscopie Biomoléculaire, GIBSA, IFR 53 UFR de Pharmacie, Reims, France

Correspondence to: Christine Millot, Laboratoire de Physiologie Cellulaire, Faculté de Pharmacie, 51096 Reims Cedex, France.

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 ± 10%) than the value (48 ± 8%) from K562-sensitive cells (p<0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 µM), or nigericine (0.3 µM) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 µM verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles. (J Histochem Cytochem 45:1255-1264, 1997)

Key Words: multidrug resistance, fluorescence, acridine orange, acidic organelles, microspectrofluorometry


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