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.


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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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Most resistant cell lines have been characterized by a complex phenotype of crossresistance to antineoplastic agents (Gottesman and Pastan 1993 ; Simon and Schindler 1994 ). The "multidrug resistance" (MDR) phenotype was correlated with (a) gene amplifications, (b) the overexpression of a 170-180-kD glycoprotein, the P-glycoprotein (Ling 1992 ), (c) a deficient accumulation of the cytotoxic drug, and (d) an enhanced drug efflux function. More recently, a shift of the subcellular partitioning/compartmentation of fluorescent anthracyclines away from their intracellular target has been displayed in MDR cells (Hindenburg et al. 1987 ; Coley et al. 1993 ; Meschini et al. 1994 ; Simon et al. 1994 ). Sensitive cells demonstrate a predominant nuclear accumulation of anthracyclines, whereas resistant cells show a loss of this nuclear fluorescence and an intense fluorescence in cytoplasmic perinuclear vesicles. This sequestration appears to affect drug accumulation and therefore conditions for it to reach critical concentrations at intranuclear sites, and to limit its cytotoxic effect in resistant cells.

It has been suggested that a pH shift in various cytoplasmic organelles might contribute to this intracellular redistribution of anticancer drugs (Thiebault et al. 1990 ; Schindler et al. 1996 ). Owing to their positive electric charge at weak pH, most anticancer drugs (vinca alkaloids, anthracyclines) accumulate in their protonated form on the side of a membrane at which the pH is lower. This suggests that cationic molecules become "acid-trapped" in acidic cytoplasmic vesicles (Warren et al. 1991 ). For identification and a better understanding of the MDR phenomenon, we aimed in this study to describe alterations of the pH gradient between acidic organelles and the cytosol that are correlated with multidrug resistance.

The most common method to study vacuolar acidification is based on the use of lipophilic weak bases, such as monoamines and diamines. These agents are membrane-permeant in their neutral form and relatively membrane-impermeable once protonated. If a pH gradient is established, they would accumulate in their protonated form on the side of the membrane at which the pH is lower. In quantitative terms, the degree of accumulation depends on the transmembrane pH gradient and on the total internal volume of the acidic vesicles. Among cationic dyes, acridine orange (AO) has been described to be trapped in acidic vesicles where high concentrations would be achieved (Barash et al. 1991). Alteration of pH gradients modifies the local accumulation of AO in acidic vesicles (Busch et al. 1994 ). Moreover, AO shows characteristic alterations of absorbance and fluorescence properties that result from a concentration-dependent increase in resonance energy transfer among individual AO molecules. Whereas a green emission is observed from the monomeric form of AO, a red emission has been attributed to the stacking of AO in oligomeric structures. Consequently, we have monitored the local accumulation of AO and therefore the vacuolar acidification through analysis of intracellular emission spectra of AO. A confocal laser scanning microspectrofluorometry (Millot et al. 1994 ; Sharonov et al. 1994 ) has been applied to allow both the spatial discrimination of acidic organelles by confocal analysis and the spectral (red/green) discrimination of AO emission. We describe here a higher red emission of AO from resistant cell organelles, interpreted as a more important local accumulation of the dye that would derive from higner acidity in these organelles.


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Cell Lines and Culture Conditions
K562 is a human erythroleukemic cell line established from a patient with chronic myelogeneous leukemia in blast transformation. Multidrug-resistant K562-R cells were obtained from the parental cell line by continuous exposure to gradually increasing doxorubicin concentrations up to 10-7 M. K562-R cells were grown in the presence of 10-7 M doxorubicin. To test the influence of the continuous presence of doxorubicin, K562-R cells were cultured without doxorubicin up to 3 months, and were designated as K562-RW. K562-R and K562-RW cells expressed the membrane P-glycoprotein. IC50 for doxorubicin was 80 ± 20 nM for K562, 2000 ± 500 nM for K562-R, and 1600 ± 400 nM for K562-RW, as determined after 4 hr in the presence of drug and 3 days in drug-free medium. MCF-7 is an adherent human breast cancer cell line cultured in Lab-Tek culture chambers (Nunc; Naperville, IL)

Cells were grown in a 5% CO2 atmosphere at 37C in RPMI-1640 medium (Gibco-BRL; Paisley, UK) supplemented with 10% fetal calf serum (Gibco) and 200 mM L-glutamine (Boy; Reims, France). Cell growth and viability were determined using the trypan blue (Sigma; St Louis, MO) exclusion technique. Cells were routinely examined for mycoplasma contamination.

Chemicals
One-mM stock solutions of AO (Sigma) were prepared in ethanol. AO concentrations were determined by their absorbance at 495 nm ({varepsilon} = 63 103 M-1cm-1). Doxorubicin (Farmitalia; Milan, Italy) as hydrochloride was prepared as stock solutions in water (10-3 M) and stored at -20C. Monensine, nigericine, ammonium chloride, and methylamine were purchased from Sigma. Monensine (1 mM) and nigericine (1 mM) were dissolved in ethanol. Methylamine (10 mM) was dissolved in methanol. Verapamil was a clinical formulation (Isoptine; Laboratoires Biosedra, Malakoff, France). Double-labeled fluorescein-tetramethylrhodamine dextran (10,000 MW) (FRD) was purchased from Interchim (Paris, France).

Confocal Laser Scanning Microspectrofluorometry
x.y emission spectra from a confocal section within a living cell were recorded using a confocal laser scanning microspectrofluorometer (DILOR; Lille, France). An optical microscope (Olympus BH2) was equipped with a water immersion objective lens (x100, NA 0.95; State Optical Institute of St- Petersbourg). This lens was corrected for axial chromatic aberration so that both excitation and emission voxels were superposed. This allowed observation of the sample, focussing of a 4-µW laser beam emitting at 457 nm (2065A model; Spectra Physics), and collection of the fluorescence emission in the 500-700-nm range through the same optics. The required discrimination between optical sections was controlled by varying the aperture of a square pinhole from 50 to 1000 µm. For intracellular measurements of AO emission, the pinhole size was fixed to a diameter of 200 µm. Using this configuration, and by comparing with fluorescent beads of known diameter, confocal optical sections were estimated to be about 2 µm. The position of the sample was controlled by a motorized sample holder (Marzhauzer with increments of 0.1 µm), which enabled the y-scanning to be done. The x-axis scanning was achieved by a scanner that produced a periodic angular deflection of the beam. A second scanner oscillated in phase with the first scanner and deflected the emission from a line onto the entrance slit. A stigmatic spectrometer then projected the emission onto a two dimensional CCD detector.

Measurements of Intracellular Emission Spectra of AO
Cells in exponential growth phase were incubated at 5 x 104/ml density in RPMI containing AO for 30 min. For K562-R cells, doxorubicin was removed 1 day before incubation with AO. Cells were washed free of AO and seeded in Petri dishes containing RPMI without phenol red. Scanning of a cell was performed within an 18 x 18 µm2 zone in which 50 x 50 spectra were recorded. The total acquisition time was 1 sec for a line and 50 sec for the cell mapping. Repeated measurements from the same cell over 15 min did not change the intensity or the distribution of intracellular fluorescence.

To characterize the profile of AO emission spectra, the red band contribution (R%) within the whole emission spectrum has been calculated as follows :

R% = 100 . I655 / (I655 + I530)

where I530 and I655 are the green (520-540-nm) and the red (645-665-nm) integrated emission intensities, respectively.

Determination of Intranuclear Doxorubicin Concentration
Cells at 5 x 104/ml density were incubated for 2 hr in RPMI-1640 medium containing 2 µM of doxorubicin and nigericine. Cells were washed free of drug in PBS at 4C and were placed in a Petri dish containing PBS. The laser (457.9 nm, 4 µW) was focused for 1 sec on a nucleus to obtain fluorescence emission spectra. The emission arising from the nucleus can be expressed as a sum of spectral contributions of the free drug, DNA-bound drug, and intranuclear autofluorescence. Each of these contributions has a characteristic spectral shape. Then drug concentrations in living cell nuclei were obtained from the determined spectral contributions and by means of the corresponding fluorescence yields (Gigli et al. 1988 ).

Intracellular Emission Spectra of Fluorescein-Tetramethylrhodamine Dextran
MCF7 cells were incubated in 0.5 mg/ml double-labeled fluorescein-tetramethylrhodamine dextran (FRD) for 18 hr. Cells were washed three times in RPMI without phenol red and then were scanned with a laser beam (4 µW, 488 nm) over 50 sec to obtain a spectral image of 50 x 50 emission spectra. For each spectrum, the ratio of both emission intensities I580 nm/I520 nm was then determined.


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Emission Spectra of Acridine Orange in Sensitive and Resistant Cells
Typical emission spectra from confocal sections of K562 and K562-R cells treated with 10 µM AO are shown in Figure 1. Using a scanning laser, a set of 50 x 50 spectra was recorded at different locations from these cells (Figure 1B) after acquisition of the conventional images (Figure 1A). The integrated intensities of the emission bands centered at 530 nm and 655 nm allowed reconstruction of images that corresponded to green (Figure 1C) and red (Figure 1D) emission intensities, respectively. For both cell lines, all spectra from the nuclear compartment displayed a unique green band at 530 nm. Several spectra from the cytoplasm of the K562-R cell displayed an additional red band at 655 nm. This red emission component was very weak in the cytoplasm of the K562 cell (Figure 1D). From these red stained compartments, the means of emission spectra were significantly different between K562 and K562-R cells (Figure 1E). They displayed a more important red/green band intensity ratio for the K562-R than for the K562 cell line.



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Figure 1. Fluorscence spectral images of K562 and K562-R cells treated with 10 µM AO. (A) Photonic microscopy of K562 and K562-R cells; (B) emission spectra from a set of 50 x 50 intracellular positions. (C) Images of the green (520-540 nm) integrated emission intensity; (D) images of the red (645-665 nm) integrated emission intensity. (E) Mean spectra from the red-stained compartments (marked by arrows in D). Fluorescence emission intensities are in arbitrary units (a.u.). Bars = 4 µm.

Emission spectra from red cytoplasmic regions of K562-R cells showed two components whose maximal emissions are reproducibly centered at 655 nm and 530 nm. The red band (655 nm) has been compared with the emission spectra from the surface of AO microcrystals (spectrum b, Figure 2). On such crystals, the wavelength of the maximal emission varies from 650 to 670 nm according to the position on the crystal surface. Therefore, self-associated molecules of AO could be the origin of the red emission band from the cytoplasmic organelles. Spectra from the crystal and from cytoplasmic organelles, both recorded under the same experimental conditions (laser power, confocal microvolume size), displayed equivalent red emission intensities. Therefore, red-stained organelles should be the site of very important local concentrations of AO. In these cytoplasmic compartments, the maximal emission of the green band is about 530 nm. In aqueous solutions, the maximal emission of AO is dependent on the pH value and varies from 542 nm (pH 10) to 530 nm (pH 4), as shown in Figure 2. Therefore, the 530-nm maximum from red stained organelles is compatible with a low pH.



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Figure 2. Emission spectra of AO from (a) a red-stained region of a K562-R cell treated with 10 µM AO; (b) the surface on an AO solid crystal. One µM AO in buffered solutions at (c) pH 4, (d) pH 7, (e) pH 10. All spectra were recorded under the same experimental conditions.

The relation between the intracellular emission spectra of AO (green and red emission intensities) and incubation conditions was also investigated, and results are shown in Figure 3. For both cell lines, the green band emission intensities are equivalent. The increase of the extracellular AO concentration from 2 to 10 µM induces an increase in intensity of only the red emission band (p < 0.01, Students' t-test). This observation is compatible with the model of a green emission band that would correspond to monomers, whereas the red emission band would originate from self-associated molecules. The increase of extracellular AO concentration would induce the increase of intracellular concentration. In acidic vesicles, the local concentration of AO would be so important that the additional AO molecules associate between themselves in oligomeric structures, thus increasing the red luminescence intensity.



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Figure 3. Fluorescence emission of AO from red-stained organelles of K562 (-----), K562-R (--------), K562-RW (- - - -) after 30 min of incubation. (A) Green (520-540 nm) integrated intensity; (B) red (645-665 nm) integrated intensity (*p<0.01); (C) contribution of the red band (R%) in the emission spectrum (#p<0.001). Error bars are standard deviations. A total of 25 cells were analyzed for each condition.

For K562-R-resistant cells, the intensity of the red emission band was twice that from sensitive cells, indicating a more important accumulation of AO in such cytoplasmic structures. When resistant cells were deprived of doxorubicin, the spectral features from K562-RW were maintained. We have calculated the contribution of the red emission within the whole spectrum to minimize the influence of the size of each red organelle. In K562-R- and K562-RW-resistant cells, these red band contributions are 72 ± 10% (mean ± SD) and 70 ± 12%, respectively, and are significantly more important than the calculated value (48 ± 8%) from K562-sensitive cells (p<0.001) (n = 25, Student's t-test).

Co-localization of Low-pH Compartments and the Red Stained Organelles
To evaluate pH values in the organelles that display the red emission of AO, MCF-7 cells were first incubated for 18 hr with FRD to generate a spectral image of the pH-sensitive dye. After this acquisition, the same adherent cells were incubated at 37C in 10 µM AO for 30 min to generate an image of the R% parameter. For an equivalent excitation power (10 µW, 488 nm), the emission intensity of FRD was 20- to 40-fold lower than that of AO and became negligible.

The fluorescein emission (520 nm) was highly pH-dependent (pK = 6.4), whereas the rhodamine emission at 580-nm was relatively insensitive to pH in the physiological range. The 580 nm emission intensity was relatively homogeneous throughout the whole cell, suggesting a redistribution of the probe after endocytosis. The image of the 580 nm/520 nm intensity ratio of FRD clearly shows more important values (0.85 ± 0.05), which corresponds to intracellular sites with the lowest pH values (Figure 4B). An equivalent ratio of 0.82 was obtained from a pH 5.5 buffered solution (Table 1). After the second staining with AO, areas that display a red emission of AO display also a more important 580 nm/520 nm intensity ratio from the FRD staining (Figure 4). Therefore, these red-stained organelles with AO clearly included low pH values.



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Figure 4. Successive staining of MCF7 cells with 0.5 mg/ml double-labeled fluorescein-tetramethylrhodamine dextran (18 hr) followed by 10 µM AO (30 min). (A) Photonic microscopy image of the cell. (B) Image of the I580nm/I520nm intensity ratio after FRD staining. (C) Image of R% (red band contribution within the whole spectrum) after AO staining.


 
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Table 1. Emission intensity ratios (580 nm/520 nm) of double-labeled fluorescein-tetramethylrhodamine dextran (FRD) in buffered solutions at different pH

Inhibition of Vacuolar Acidification
The effect of NH4Cl on intracellular AO red emission has been followed as a function of time. The addition of 1 mM NH4Cl induced an important and rapid decrease in the red emission intensity (Figure 5) to a stable value, whereas the green emission band intensity was not significantly modified. This observation should be linked with the alkalinization of acidic organelles. The equilibration of the pH gradient through these organelles should induce leakage of AO from this compartment, partial dissociation of self-associated structures, and finally a decrease of the red emission intensity.



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Figure 5. Time course of R% in a red-stained organelle from a 10 µM AO-treated K562-R cell after addition of 1 mM NH4Cl (time 0).

Equilibration of the pH gradient through acidic organelles has been performed by the use of different concentrations of weak bases and carboxylic ionophores (Figure 6). In resistant cells, 0.1 mM methylamine, 0.1 mM NH4Cl, 10 µM monensine, 0.3 µM nigericine, or 50 µM verapamil all induced a significant decrease in the red emission component (p<0.001; n = 25, Student's t-test), whereas a less marked effect was observed in sensitive cells. For these concentrations, the initial difference of red emission contributions between both cell lines was suppressed. Higher concentrations of these agents induced a larger decrease in the red emission that became equivalent in acidic organelles for both cell lines.



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Figure 6. Effects of inhibitors of vacuolar acidification on AO emission spectra in red-stained organelles. K562-R and K562 were incubated for 30 min in 10 µM AO and (A) nigericine, (B) monensine, (C) verapamil, (D) methylamine, and (E) NH4Cl. The contribution of the red band within the whole spectrum (R%) is shown. Error bars are standard deviations. A total of 25 cells were analyzed for each condition. *Significant decrease in R% (p<0.001) for treated K562-R cells.

Intranuclear Doxorubicin Concentrations
We have determined that the resistant line K562-R exhibited an altered doxorubicin uptake compared to the sensitive cells (Figure 7). Intranuclear concentrations of doxorubicin have been also determined as a function of extracellular nigericine doses to determine whether co-incubation with nigericine produces an enhancement of intranuclear doxorubicin concentration (Figure 7). After 2 hr of incubation with doxorubicin and nigericine, accumulations of doxorubicin in nuclei of K562-R cells increased as a function of the extracellular nigericine dose. In contrast, for the sensitive line, the doxorubicin uptake was found to be independent of the nigericine dose in the medium.



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Figure 7. Doxorubicin uptake in the nucleus of K562 ({square}) and K562-R ({blacksquare}) cells as a function of extracellular concentration of nigericine. Cells were exposed simultaneously to doxorubicin (2 µM) and nigericine. After 2 hr, intranuclear concentrations of doxorubicin were determined from 30 cells. Error bars are standard deviations.


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Staining of Cytoplasmic Acidic Organelles by Acridine Orange
AO is an optical probe that shows various different absorption and fluorescence properties in its different forms. The monomeric dye in solution and its complex with double-stranded nucleic acids exhibit a green fluorescence ({lambda} = 530 nm). In a solution, the increase in AO concentration induces progressive quenching of the green AO emission. This phenomenon has been attributed to resonance energy transfer among individual molecules. Note that none of the molecules used to cancel the pH gradient will change the emission spectrum of AO in a buffered aqueous solution. In addition, the stacking of AO molecules, either as crystals or in the presence of single-stranded nucleic acids, produces an additional red emission (Kapuscinski et al. 1982 ).

In acidic vesicles, both green and red emissions of AO were observed simultaneously. Owing to the important local accumulation of this weak base, both monomers (green emission) and stacked structures of AO (red emission) could coexist. We have induced the decrease of the AO accumulation inside acidic vesicles either by lowering extracellular AO concentrations or by suppressing the pH gradient through the vesicle membrane. The decrease in red/green emission intensity ratio was interpreted as the result of the dissociation of oligomeric self-associated molecules of AO into monomers. Therefore, the determination of this emission ratio can be directly related to the ratio of stacked/monomers molecules of AO, which depends on the local concentration of AO and consequently on the pH gradient through a membrane vesicle. Therefore, we show in this work that the intracellular distribution of the red/green intensity ratio of AO is in total agreement with the emission spectra of a pH-sensitive dye. Emission spectra of AO cannot be converted into actual pH values in acidic compartments. Indeed, the emission spectra from AO in acidic organelles are dependent on extracellular AO concentrations (Figure 3). Moreover, it has been shown that, in a membrane-free system, different anions could modify to different extents the formation of dimers and excimers, which in turn could alter the fluorescence emission of AO (Palmgreen 1991 ).

Finally, the AO staining can provide information to compare the potency of weak base sequestration by acidic compartments. The main interests of AO consist of the important spectral discrimination between red and green emissions and the important sensitivity of the red/green intensity ratio to slight pH variations in acidic organelles. For example, the addition of 1 mM NH4Cl, which has been described to increase the pH value by only 1 unit in isolated lysosomes (Ohkuma and Poole 1978 ), induces, in our model, the total inversion of both red and green bands.

Sequestration Models of Weak Bases in Acidic Organelles of Resistant Cells
The major result of this study concerns the more important ratio of red/green emission intensities of AO in acidic compartments of K562-R cells compared with the sensitive ones. This spectral change has been interpreted as a more important accumulation of this weak base in such compartments. The same model of sequestration of other weak bases, such as anthracyclines, has been previously investigated by confocal microscopy (Hindenburg et al. 1989 ; Gervasoni et al. 1991 ; Meschini et al. 1994 ). In resistant cells, anthracyclines are redistributed from the nucleus into cytoplasmic vesicles, thereby assuming a punctate pattern. Moreover, various unrelated drugs known to overcome resistance can also revert the pattern of intracellular anthracycline distribution in resistant cells back to the distribution found in sensitive cells (Hindenburg et al. 1987 ; Simon et al. 1994 ).

This study addressed the question of the identity of intracellular acidic organelles that are implicated in drug redistribution. Ultrastructural investigations have previously revealed additional osmium-stained cytoplasmic vesicles in K562-R compared to K562 cells (Bobichon et al. 1992 ). Lysosomes have been first considered, since drug resistance is partly circumvented by lysosomotropic agents. Indeed, among various MDR revertant molecules, chloroquine, verapamil, and propranolol delayed the degradation of low-density lipoproteins in lysosomes (Akiyama et al. 1985 ). The weak-base anticancer drugs would be expected to be protonated and sequestrated in these organelles (Shiraishi et al. 1986 ; Zamora and Beck 1986 ). However, this hypothesis supports the notion that an equilibrium would be reached quickly, limiting the utility of a static mechanism for effluxing drugs. In addition, confocal imaging of anthracyclines has shown intense staining in granules located in the perinuclear cytoplasm of MDR cells, which supports the hypothesis of drug trapping within the Golgi complex. Different organizations of the trans-Golgi network have been also evoked to account for intracellular drug redistribution. Stainings with bodipy ceramide have shown dispersed vesicular distribution in sensitive MCF7 cells, wheareas compact pericentriolar structures appeared in resistant MCF7-R cells (Schindler et al. 1996 ). It should be noted that size and density increases of organelles towards condensed structures could also increase the red/green intensity ratio of AO. Indeed, the green emission (monomers) from the cytosol may contribute to the signal of small and dispersed organelles (less than 1 µm3), whereas this influence becomes less important for high-density structures. By sequential fluorescence imaging and scanning electron microscopy, vesicle formation (red colored by doxorubicin) appeared first in the perinuclear region and was then followed by a unidirectional transport to the periphery of the cell (Seidel et al. 1995 ). The enhanced dynamic turnover of the luminal content of their trans-Golgi network to the cell surface would then contribute to the continuous efflux pathway of anticancer drugs.

Two models can be proposed to account for the more important accumulation of weak bases in vesicles of MDR cells than in sensitive ones: (a) direct interaction between the P-glycoprotein and weak bases pumps them into subcellular organelles; or (b) a shift of local subcellular pH modifies the intracellular distribution of weak bases.

Pgp Hypothesis. A number of proteins are overexpressed in MDR cell lines. The 170-kD transmembrane glycoprotein (P-glycoprotein), the product of the mdr-1 gene, is strongly homologous to a family of protein membrane transporters and is linked to the drug efflux from MDR cells. In the first model, it could be suggested that the P-glycoprotein of vesicle membranes interacts directly with weak bases. Then, they may be pumped directly into subcellular organelles and can be relocalized intracellularly (Schurrhuis et al. 1989). However, as mentioned by Roepe 1994 , it is difficult to explain how one enzyme can specifically interact with hundreds of structurally divergent molecules. Moreover, although Pgp has been found both in plasma membranes and on the luminal side of Golgi stacks, this protein is not present in endocytic vesicles and lysosomes (Willingham et al. 1987 ; Molinari et al. 1994 ). Therefore, the direct participation of Pgp in the drug sequestration phenomenon is not clear. We suggest that the intracellular sequestration of weak bases and the enhanced outward transport should be distinct mechanisms. Indeed, it has been shown previously that the resistant K562-R cells present an enhanced doxorubicin efflux out of the nucleus compared with K562 cells (Millot et al. 1989 ). However, we have determined that, for resistant cells, the efflux of AO was not enhanced (data not shown), although this molecule was intracellularly redistributed. In addition, other overexpressed glycoproteins in some MDR cells, the 180-kDa (Cole et al. 1993; Slovak et al. 1993 ) and 110-kDa (Scheper et al. 1993 ), were present predominantly in intracellular organelles and in lysosomes, respectively, and may be implicated in the mechanism of intracellular weak base redistribution.

pH Hypothesis. In the second model, it could be proposed that the higher accumulation of weak bases in acidic vesicles of resistant cells may be driven by a more important pH gradient through the membrane of these organelles. In acidic vesicles, weak bases bind H+ and become membrane-impermeable, known as "acid-trapping." Recently, {Delta}pH (acidic organelles/cytosol) of 1.2 and 0.3 have been shown in multidrug resistant and sensitive MCF7 cells, respectively (Schindler et al. 1996 ). To establish a pH shift in the lumen of intracellular organelles, a vacuolar type H+-ATPase has been described to drive the uptake of antineoplastic agents into acidic organelles (Marquart and Center 1991; Moriyama et al. 1994 ). An electrogenic H+-ATPase coupled to a Cl- conductance is responsible for maintaining low pH in endocytotic and secretory compartments (Barasch 1991; Van Dyke and Belcher 1994 ). Indeed, alkaline shifts of the luminal pH have been measured in the absence of chloride (Van Dyke and Belcher 1994 ) or for a decreased chloride conductance in the trans-Golgi compartment and recycling endosomes (Barasch et al. 1991 ). The Pgp overexpression may also cause perturbations both in the plasma membrane electrochemical potential and in luminal pH. Indeed, Pgp has also been reported to function as a Cl- channel (Valverde et al. 1992 ) or to alter the chloride conductance (Hardy et al. 1995 ). Therefore, the transport and the intracellular redistribution of drugs may be driven by Pgp through an indirect mechanism (Roepe 1994 ).

The pH model has been confirmed by the addition of agents (weak bases, carboxylic ionophores) that suppress the intracellular pH gradients (Mellman et al. 1986 ). After the addition of such agents, the red/green emission ratio of AO decreases in vesicles of resistant cells down to the value measured in the sensitive ones. For both cell lines, the AO concentration in acidic organelles and the pH gradient through their membranes become equivalent. For example, it has been reported that 0.1 mM NH4Cl induces a 0.2 pH unit increase (from 4.8 to 5) in isolated lysosomes (Okhuma and Poole 1978). In comparison, our data show that 0.1 mM NH4Cl suppresses the initial difference of AO emission between both cell lines. Agents such as NH4Cl and methylamine are relatively lipophilic in their unprotonated form and pass through membranes of vacuoles. In an acidic environment, these molecules become protonated and cannot escape from these organelles, which favors their accumulation and the increase in vacuolar pH. Carboxylic ionophores such as monensine and nigericine have been also used to elevate vacuolar pH. They intercalate into membranes and mediate exchange of monovalent cations through membranes (Pressman 1976 ). Owing to the high K+ concentration in the cytoplasm, the main effect of ionophores on acidic organelles is the exchange of H+ for K+, thereby effecting a rise in vacuolar pH (Tartakoff 1983 ). Previous reports have shown that weak bases, carboxylic ionophores, and verapamil are potential modulators of multidrug resistance (Shiraishi et al. 1986 ; Sehested et al. 1988 ). This suggests that these agents can reverse multiple drug resistance through alteration of the lysosomal activity and by enhancement of intranuclear drug accumulation.

In conclusion, the control of a high pH gradient through membranes of acidic vesicles in multidrug-resistant cells could significantly contribute to multidrug resistance by means of weak base sequestration inside these organelles. In the future, the major challenge will concern the identification of acidification mechanisms in organelles of resistant cells, to elucidate how the pH in these organelles is regulated.


  Acknowledgments

Supported by the Association de Recherche contre le Cancer.

Received for publication October 8, 1996; accepted March 13, 1997.


  Literature Cited
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Summary
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Materials and Methods
Results
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Literature Cited

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