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Abstract

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I_550nm/I_600nm) ratio was 0.50 ± 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I_550nm/I_600nm ratio was 0.85 ± 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and selfoligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipyceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THPDOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

Keywords

multidrug resistance anthracyclines microspectrofluorometry reversal cytoplasmic environment

Studies of chemoresistance in tumor cells have revealed the existence of several mechanisms for cell survival in the presence of cytostatic drugs (Gottesman and Pastan 1993; Simon and Schindler 1994). One of the most thoroughly investigated mechanisms is the expression of a transmembrane glycoprotein (P-glycoprotein; Pgp) which serves as an efflux pump to remove several types of cytostatic drugs from multidrugresistant (MDR) tumor cells (Bradley et al. 1988). One model stemming from the observation that P-glycoprotein-positive MDR cells have elevated intracellular pH (cytosol) suggests that Pgp may also act as an intracellular pH regulator, possibly sequestering drug molecules in specific subcellular compartments to mediate efflux or affecting their pH-dependent binding to cellular targets (Thiebaut et al. 1990; Roepe 1992). P-glycoprotein is also found in the Golgi apparatus (Molinari et al. 1994) and nucleus (Baldini et al. 1995) of MDR tumor cells.

Nucleo-cytoplasmic distribution and compartmentalization of anthracyclines have already been investigated, using confocal fluorescence microscopy, on both sensitive and resistant tumor cells (Schuurhuis et al. 1993; Rhodes et al. 1994; Seidel et al. 1995). This technique does not take into account the modifications that can affect the fluorescence profile of the drug, such as quenching and spectral shift of a few nanometers due to different molecular environments or specific interaction with the target.

In our previous works, using confocal laser microspectrofluorometry (Gigli et al. 1988; Morjani et al. 1992) and by measuring nuclear concentrations of anthracyclines on single living cells, we were able to acquire information on the mechanisms of drug resistance related to drug transport (Gigli et al. 1989; Pelletier et al. 1990; Pignon et al. 1995; Morjani et al. 1997). We could also show that this accumulation increased when MDR modulators were used in combination with the anthracyclines (Sebille et al. 1994). By using confocal fluorescence spectral imaging, Millot and co-workers have described a specific environment of rhodamine 123 (Millot et al. 1994) and acridine orange (Millot et al. 1997) in the cytoplasm of MDR cells.

Here we report on the spectroscopic evidence of a specific interaction of pirarubicin (THP-DOX) in cytoplasmic organelles of MDR cells. This specific spectral pattern disappeared in resistant cells incubated simultaneously in the presence of the drug and MDR modulators. Data obtained on growth inhibitory effect and nuclear accumulation of THP-DOX in the presence of MDR modulators, particularly quinine, poses intriguing questions regarding the role of the cytoplasmic environment of anthracyclines in MDR cells.

Materials and Methods

Chemicals

Anticancer Drugs. Pirarubicin or tetrahydropyranyl-doxorubicin (THP-DOX) was provided by Laboratoires Bellon (Neuilly, France). Doxorubicin (DOX) was purchased from Farmitalia (Milan, Italy). Vinblastine (VLB) was purchased from Sigma (St Quentin Fallavier, France). THP-DOX and DOX are prepared in PBS at a concentration of 1 mM and stored at -20C.

Resistance Modulators. S9788, a new triazinoaminopiperidine derivative, was generously given by Institut de Recherche Internationale Servier (Paris, France). Verapamil (VPL) and quinine (QUI) were purchased respectively from Biosedra (Malakoff, France) and Sigma. S9788 and QUI were prepared respectively in water and methanol at a concentration of 10 mM. VPL was provided in aqueous solution form at 5 mM. All stock solutions of modulators were kept at -20C.

Other Chemicals. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) or MTT and trypsin were purchased from Sigma. The trans-Golgi probe DMB-ceramide (C₅-DMB-Cer) was purchased from Molecular Probes (Eugene, OR).

Cell Lines

LR73 Chinese hamster ovary-sensitive carcinoma cells were transfected with the mammalian expression plasmid pDREX4 containing the biologically active cDNA insert of phagel DR11 (LR73R) (Gros et al. 1986). LR73D cells were selected by exposure of LR73 cells to continuously increasing doxorubicin concentrations. The two MDR cell lines LR73D and LR73R overexpress the P-glycoprotein at the same level as determined by immunolabeling using FITC-conjugated C219 antibody or RT-PCR (Belhoussine et al. 1997). Sensitive and resistant LR73 cells were grown as monolayer cultures in RPMI 1640 medium (Gibco; Paris, France) supplemented with 10% fetal calf serum (Gibco) at 37C in a humidified chamber containing 5% CO₂ in air. Resistant cell lines were maintained in the presence of 200 nM doxorubicin.

K562 is a human erythroleukemic cell line established from a patient with chronic myelogenous leukemia in blast transformation (Lozzio and Lozzio 1975). K562R cells were obtained by continuous exposure of parental cells to stepwise increasing concentrations of DOX in the medium until 100 nM (Tsuruo et al. 1986).

CEM is a human leukemic lymphoblastic cell line (Beck et al. 1979). CEMR cells have been selected for resistance to 500 nM of VLB (Beck 1983). Sensitive and resistant CEM and K562 cells were maintained in suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum at 37C in a humidified chamber containing 5% CO₂ in air. The drug was removed from the culture medium 2 days before the experimentation. Northern blot, RT-PCR, and immunolabeling analyses have shown that resistant K562 and CEM cell lines overexpress the P-glycoprotein (Morjani et al. in press).

Cytotoxic Effect of THP-DOX

LR73 cells were maintained at 37C in the absence of THPDOX in a 96-multiwell dish (10³ cells/well). After 1 day, THP-DOX (0.1 nM-100 nM) was added to each sample in the presence or absence of different concentrations of each modulator. After 48 hr, LR73 cells were washed and incubated for 2 days in fresh medium before MTT assay measurements. Cell viability was then determined by addition of 20 μl of 2.5 mg/ml MTT for 3 hr at 37C. Then the medium was discarded and 200 μl DMSO was added to each well. Optical densities were measured at 540 nm using a Series 750 microplate reader (Cambridge Technology; Watertown, MA). IC₅₀ (concentration that induces 50% inhibition of cell growth) values were calculated from the ratio of optical densities of treated cells to that of control cells (Sebille et al. 1994).

Confocal Laser Scanning Microspectrofluorometry

This technique allowed the acquisition and analysis of fluorescence signals from a microvolume of living cancer cells treated with the anthracycline. The original microspectrofluorometer M51 (Dilor; Lille, France) is equipped with an ionized argon laser (2065; Spectra Physics, Les Ulis, France). For our measurements the 457.9-nm excitation line was used. The optical microscope BX40 (Olympus; Tokyo, Japan) equipped with a X 100 phase contrast water-immersion objective UVFL-100PL permitted a micrometer spatial resolution (Sharonov et al. 1994). The microscope was coupled to a single-grating (300 grooves mm^-1) spectrograph through the confocal entrance chamber. Two synchronized scanners realized the line illumination along the X axis in the plane of the sample, and the motorized stage translates the sample along the Y axis. The fluorescence emission of treated cells was analyzed with an air-Peltier-cooled CCD detector (Wright; Stonehouse, UK) supplied with an 1125 X 298 pixel sensor element and optically coupled to an image intensifier (Sharonov et al. 1994).

Confocal Spectral Image Acquisition

Confocal spectral imaging analysis is an effective probe for antitumor drug distribution in single living cells. This approach is based on a line illumination system that employs a two-dimensional detector and a system of two synchronized scanning mirrors, which provides simultaneous spectral accumulation from hundreds of points on the sample in real confocal mode for each point. While the laser beam is being moved by the first scanning mirror along the X axis in the plane of the cell, the emitted light from the line is timeencoded on the first scanning mirror and transmitted to the pinhole diaphragm for confocal filtration. After passing through the diaphragm, the signal is decoded by the second scanning mirror vibrating in phase with the first one. Then the light is dispersed into spectra by the grating spectrograph and focused onto a two-dimensional CCD detector. The sample holder is moved with an automatic scanning stage along the Y axis with a minimal step size of 0.1 μm. A set of 50 X 50 spectra are recorded at different locations of the cell and a whole spectral image is generated by the line-by-line scanning system. The scanning of the sample stage and mirrors of the optical scanners and all operations connected with recording of spectra are computer-controlled. All treatment and analysis of spectral images were acquired with an RISC 6000 model 520 workstation (IBM).

Determination of Nuclear Concentration of THP-DOX

The fluorescence emission spectrum originating from nuclei of LR73-treated cells F(λ), can be expressed as a sum of the spectral contributions of free THP-DOX, DNA-bound THPDOX, and signal of nuclear autofluorescence (Gigli et al. 1988)

where F_f and F_b are the fluorescence spectra of free and bound drug referred to a unitary concentration. Taking this concentration into account, C_f and C_b represent intranuclear concentrations of free and bound drug, respectively. C_n is the contribution of autofluoresence responsible for the intrinsic nuclear spectrum F_n. In aqueous solution, each of these contributions has a characteristic spectral shape. The fluorescence yield in the free form was 40 times higher than that of the bound-DNA form. These spectral contributions lead to the concentrations of free and DNA-bound THPDOX. The sum of the values obtained gives the total nuclear concentration of THP-DOX (Gigli et al. 1988,1989).

Co-localization of THP-DOX and DMB-Ceramide in LR73D Cells

Cells were incubated in the presence of C5-DMB-Cer complexed with defatted BSA (DF-BSA) for 30 min at 2C, washed, and incubated with 5 μM THP-DOX in HMEMB for 2 hr at 37C. The complex contained 5 μM of both the fluorescent lipid and DF-BSA and was prepared in 10 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid-buffered Eagle's MEM, pH 7.4, containing 0.5 mM choline chloride, ethanolamine, serine, and (myo)inositol (HMEMB) (Pagano et al. 1991).

Results

Cytoplasmic Localization of THP-DOX in MDR Cell Lines

For spectral measurements, cells at 100,000 cells/ml density were incubated in the presence of 1 μM THPDOX for 2 hr at 37C. Figure 1A shows the spectra recorded from the cytoplasmic region of K562 and K562R cells. After subtraction of autofluorescence, analysis of the spectral contributions of THP-DOX showed that, in the cytoplasm of K562 cells, THPDOX emission was similar to that in PBS. In this case, the intensity ratio of the 550- and 600-nm bands (I_550nm/I_600nm) was 0.5 ± 0.1 in K562 cells and 0.5 in PBS. However in the cytoplasm of K562-resistant cells, the I_550nm/I_600nm was 0.85 ± 0.2, which was significantly different from the ratio in sensitive cells (p<0.01). We observed the same spectral profile in CEMR, LR73R, and LR73D cells. It is important to note that the LR73R cell line was transfected with the mdr1 gene. Data on the emission intensity ratios (I_550nm/I_600nm) corresponding to sensitive and resistant K562, CEM, and LR73 cells are summarized in Figure 2A.

Figure 1

(A) Fluorescence emission spectra of THP-DOX from selected microvolumes within the cytoplasm of parental K562 cells (2), K562R cells (1) treated with 1 μM THP-DOX for 2 hr, and untreated cells (3). (B) THP-DOX in PBS (1) and THP-DOX complexed to sphingomyelin (2). The THP-DOX-sphingomyelin complex was prepared as described by Schwartz and Kanter (1979).

Using the scanning system, a set of 50 X 50 spectra were recorded at different locations in the tumor cells and the integrated intensities of spectra allow a fluorescence spectral image equivalent to that expected with a conventional microscope to be obtained. In each of the 50 X 50 spectra, the contribution of the model spectra, i.e., (a) fluorescence spectrum of untreated cells, (b) fluorescence spectrum of THP-DOX in the cytoplasm of a sensitive cell, and (c) fluorescence spectrum of THP-DOX in the cytoplasm of resistant cells, were determined (Figure 1).

Taking into account the scale and by employing a pseudocolor intensity representation, the integrated intensities of these three contributions allowed us to obtain three images: (a) autofluorescence of the cell (data not shown), (b) THP-DOX fluorescence in a sensitive-like environment, and (c) THP-DOX fluorescence in a resistant-like environment. We investigated the distribution of THP-DOX in the resistant cell lines K562R, CEMR, LR73D, and LR73R. As expected, Figure 3 shows that in K562R cells the contribution of THP-DOX in a resistant-like environment was more important than the contribution of the free drug. In contrast, the images from K562 cells showed a significant contribution of free THP-DOX in the cytoplasm and no spectral contribution of THP-DOX as recorded in a resistant-like environment. A similar pattern of THP-DOX distribution was observed in the sensitive and resistant CEM and LR73 cells.

To identify the organelle in which THP-DOX is localized in MDR cells and especially the 550-nm emission band, LR73D cells were incubated in the presence of C5-DMB-Cer and THP-DOX (see Materials and Methods). The results in Figure 4 show that THPDOX fluorescence emission in both the sensitive- and resistant-like environments (Figure 1A, Spectrum 1) appears to be co-localized. This co-localization (cf. Figures 4E and 4F) is probably due to the existence of an equilibrium between the free and bound forms of the drug. Distribution of the monomeric form of C5-DMB-Cer (green fluorescence) appears to be more diffuse in the cytoplasm, whereas the oligomeric form (red fluorescence) is more localized in the trans-Golgi apparatus. In fact, DMB-ceramide in the cis-Golgi (monomer) is converted to DMB-sphingomyelin, which accumulates in the trans-Golgi. This red fluorescence was partially co-localized with THP-DOX fluorescence in a resistant-like environment (cf. images 4D and 4E). Figure 1B shows that this specific pattern of fluorescence emission (Figure 1A, Spectrum 1) was similar to that obtained when THP-DOX is complexed to sphingomyelin (Figure 1B, Spectrum 1). THP-DOX complexed to sphingomyelin was prepared as described by Schwartz and Kanter (1979).

Figure 2

Emission intensity ratio (I_550nm/I_600nm) of THP-DOX. (A) Cells were treated with 1 μM THP-DOX for 2 hr and washed with PBS. (B) Cells were treated simultaneously with 1 μM THP-DOX and 5 μM VPL, S9788, or QUI.

Figure 3

Scanning microspectrofluorometric images of THP-DOX in K562 (A-D) and K562R (E-H) cells. Photonic microscopic images of cells (A,E); total fluorescence intensity (B,F); contribution of THPDOX in resistant-like (C,G) and sensitive-like (D,H) environments. Cells were treated with 1 μM THP-DOX for 2 hr and washed with PBS. Taking into account the scale, acquisitions were recorded employing the pseudocolor intensity representation.

Figure 4

Co-localization of THP-DOX and C₅-DMB-Cer in an LR73D cell. (A) Photonic microscopic image; (B) model spectra of C5-DMB-Cer (1, nonspecific fluorescence emission; 2, fluorescence emission in Golgi apparatus). Fluorescence spectral images showing contribution of nonspecific fluorescence of C5-DMB-Cer (C); contribution of fluorescence in Golgi apparatus (D); fluorescence emission of THP-DOX in a resistant-like environment (E) and fluorescence of THP-DOX in a sensitive-like environment (F).

Effect of the Reversing Agents on THP-DOX Distribution in LR73 Cells

To study the effect of MDR modulators on the subcellular distribution of THP-DOX in MDR cells, LR73R and LR73D at 100,000 cells/ml were incubated simultaneously with 1 μM of THP-DOX and 5 μM QUI, VPL, or S9788, maintained for 2 hr at 37C, and then washed with PBS at 4C. The effect of VPL, S9788, and QUI on emission intensity ratio (I_550nm/I_600nm) of THPDOX are summarized in Figure 2B. The results show that modulators were able to decrease the fluorescence intensity ratio (I_550nm/I_600nm).

We have evaluated the cytotoxic effect and nuclear accumulation of THP-DOX. For this, LR73D and LR73R cells were incubated with THP-DOX and reversing agents (S9788, QUI, and VPL) as described in Materials and Methods. The results obtained are summarized in Table 1. At modulator concentrations that induce less than 10% cell death, S9788 and verapamil appear more potent than QUI. Verapamil and S9788 decreased the IC₅₀ to the same level as that in sensitive cells. The reversing activity of QUI was different in the two cell lines. Treatment with 20 μM QUI decreased the IC₅₀ of THP-DOX in LR73D cells to the same level as that obtained in LR73 cells (the reversing factor was 43), whereas in LR73R cells the reversing factor was 14.4.

To determine if co-incubation of MDR cells in the presence of THP-DOX and S9788, QUI, or VPL induces an alteration in the nucleo-cytoplasmic distribution of THP-DOX, we measured the nuclear concentration of THP-DOX by confocal laser microspectrofluorometry. In this study, LR73 cell lines were incubated simultaneously with 1 μM THP-DOX and an appropriate modulator for 2 hr at 37C. It should be noted that, for all experiments performed on all cell lines, the intercell variations were small, and for each experiment the nuclear drug concentration was measured in 20-30 nuclei. Table 2 shows that VPL and S9788 were able to restore drug accumulation in nuclei of resistant cells, whereas QUI was unable to significantly increase nuclear THP-DOX accumulation even at a much higher concentration (20 μM). In addition, LR73D and LR73R cells were not affected in the same manner in the presence of VPL. Nuclear accumulation of THP-DOX in LR73D cells appeared to be less affected by VPL than in the transfected cells, whereas S9788 increased drug accumulation in the same manner in both resistant cell lines.

Table 1

MDR-reversing effect of modulators a

		THP-DOX IC₅₀ in the presence of modulators (reversing factor)
Cell lines	THP-DOX IC₅₀ (nM) (resistance factor)	Verapamil (50 μM)	Quinine (20 μM)	S9788 (5 μM)
LR73	1.16
LR73R	46 (40)	1.2 (38)	3.2 (14.4)	0.95 (48)
LR73D	43 (37)	1.1 (39)	1 (43)	1.2 (35.8)

Comparative effect of the different modulators on THP-DOX IC₅₀ in both MDR LR73R qnd LR73D cell lines. Cells were incubated simultaneously with increasing concentration of THP-DOX and modulator for 2 days, washed with fresh medium free of drugs, and incubated for 2 days at 37C before MTT test. Results are mean ± SD of three independent experiments, each one performed in octaplates. Resistance factor, IC₅₀ of MDR cells/IC₅₀ of sensitive cells; reversing factor, IC₅₀ without modulator/IC₅₀ in the presence of modulator.

Discussion

Cytoplasmic Interaction of THP-DOX in MDR Cells

We report here that the specific fluorescence spectral shape of THP-DOX recorded in the cytoplasm is different in MDR cells (I_550nm/I_600nm = 0.85 ± 0.2) compared to sensitive ones (I_550nm/I_600nm = 0.5 ± 0.1). Our spectral imaging data show that the observed phenomenon in MDR cells does not depend on the origin of the MDR cell line (K562, CEM, and LR73 cells) and on the drug used for resistance selection (doxorubicin or vinblastine). Moreover, transfection of LR73 cells with the mdr1 gene leads to the observation of this fluorescence spectral pattern. Bodipy-ceramide (green fluorescence), which is converted to bodipy- sphingomyelin in the cis-Golgi and accumulated in the trans-Golgi (red fluorescence), was co-localized with the typical fluorescence emission peak at 550 nm relative to the anthracycline in the cytoplasm of MDR cells. Neutral red, a vital probe of lysosomes, did not have the same cytoplasmic distribution as THP-DOX in resistant cells (data not shown). The same pattern of fluorescence emission at 550 nm was obtained in solution when THP-DOX was complexed with sphingomyelin (Figure 1B). In this case, the complex THPDOX- sphingomyelin was prepared as described by Schwartz and Kanter (1979). The three modulators (verapamil, quinine, and S9788) were able to decrease fluorescence intensity of the 550-nm band, especially quinine and S9788. The relationship between the disappearance of the typical fluorescence emission peak at 550 nm of THP-DOX relative to the cytoplasm of MDR cells in the presence of VPL, S9788, and QUI and the capacity of these modulators to restore nuclear THP-DOX accumulation and sensitivity was studied. All modulators were able to restore THPDOX sensitivity in the resistant cell lines. Our previous data using short time incubation (Belhoussine et al. 1997), and the present work shows that VPL and S9788, which compete with azidopine labeling of P-glycoprotein (Bennis et al. 1995), were able to restore nuclear THP-DOX in both resistant cell lines. However, quinine was unable to significantly increase nuclear THP-DOX accumulation. We have obtained similar results on MDR K562 and CEM cells (Morjani et al. in press).

Models of THP-DOX Interaction in the Cytoplasm of MDR Cells

Phospholipid Hypothesis. Results obtained on THPDOX when complexed to sphingomyelin suggest that a lipidic environment of MDR cells could play an important role in THP-DOX distribution. Sweet and Hume (1996), have shown that bacterial lipopolysaccharide treatment of a murine macrophage cell line conferred on it a transient resistance to the cytotoxic drugs taxol and doxorubicin. Fluorescence properties of THP-DOX and other anthracyclines when incorporated into phospholipids in vitro have been reported by other workers and are similar to those of THPDOX in the cytoplasmic structures of MDR cells (Schwartz and Kanter 1979; Samuni et al. 1986). Results from Samuni and co-workers (1986) have indicated that exposure of vesicular stomatitis virus or E. coli to DOX resulted in the gradual generation of the typical fluorescence emission peak at 550 nm. Lavie et al. (1996) have reported an important accumulation of glucosyl-ceramides in MDR cells and that MDR modulators block glycosphingolipid metabolism by inhibiting ceramide glycosylation in MDR cells (Lavie et al. 1997). A recent study has shown that ratios of fatty acids (methylene/methyl and methyl/choline) are much lower in MDR cells (Le Moyec et al. 1996). Jaffrézou and co-workers (1992) have characterized laminated inclusions in the cytoplasm of MDR cells. These lipidic structures are similar to those observed in Niemann-Pick disease, which is described as a sphingomyelin lipidosis. Other close structures are the lipidic lamellar bodies specific to alveolar Type II pneumocytes, in which H⁺-ATPase has been functionally demonstrated (pH 5.5-6.0) (Fabisiak et al. 1987). In this case, the weak base propanolol accumulated specifically in these organelles.

Table 2

Effect of modulators on nuclear THP-DOX accumulation at 1 μM a

		Nuclear THP-DOX accumulation (μM)
Cell lines	Control	Verapamil (50 μM)	Quinine (20 μM)	S9788 (5 μM)
LR73	252
LR73R	27	300	65	266
LR73D	18	98	51	258

Comparative study of nuclear accumulation of THP-DOX in both LR73-resistant cell lines (LR73D and LR73R) in the presence of VPL, QUI, and S9788. Cells were incubated simultaneously with 1 μM THP-DOX and modulators for 2 hr and washed with PBS at 4C. Nuclear accumulation of THP-DOX was evaluated by microspectrofluorometry. Results are means ± SD of three independent experiments, each one performed on 20-30 cell nuclei.

pH Hypothesis. pH could also play an important role in intracellular drug distribution. The pH-dependent character of anthracycline entrapping in unilamellar vesicles has been shown (Mayer et al. 1986,1990). Several investigators have reported an ATP-dependent uptake of anthracyclines by acidic organelles (Willingham et al. 1986; Moriyama et al. 1994). Weak bases are non-ionized and can cross the cytoplasmic membranes, whereas in acidic vesicles, these molecules bind protons and become membrane-impermeable, i.e., acidtrapping. Evidence of direct involvement of the positively charged amino group of doxorubicin (NH₃ ⁺) and the phospholipid phosphate PO₂ ^- group has been provided using infrared spectroscopy (Goormaghtigh et al. 1987). In the study reported by Schindler and coworkers, trans-Golgi and pericentriolar recycling compartments (Barasch et al. 1991) were suspected to be implicated in weak base sequestration (Schindler et al. 1996). On the basis of our results, it appears that THP-DOX is localized in the trans-Golgi. If we postulate that there are negative sites on the internal face of the membrane of this organelle (phospholipids) and that THP-DOX has high affinity for these sites, then the acid-trapping mechanism in MDR cells would induce an increase in binding of anthracycline to these sites. Thus, the spectral shape alteration observed in MDR cells might correspond to accumulation of the anthracycline in such acidic organelles (Schindler et al. 1996). P-glycoprotein, which has been reported to function as a Cl^- channel (Valverde et al. 1992), could be responsible for maintenance of acidic pH. This is necessary for trans-Golgi recycling function and for protecting cells from environmental xenobiotics, particularly antitumor agents (Schindler et al. 1996). More recently, we have identified acidic compartments in sensitive and MDR cells by monitoring the emission spectra of acridine orange. The acidification was suppressed by equilibrating the pH gradient through acidic vesicles by carboxylic ionophores (Millot et al. 1997). Therefore, we suggest that an acidic pH of organelles and a lipidic environment may prevent diffuse distribution of anthracyclines and protect MDR cells from their cytotoxic effect.

Footnotes

Acknowledgments

We are grateful to the Institut de Recherche International Servier and Laboratoires Bellon (Paris, France) for their kind gifts of S9788 and pirarubicin, respectively. We thank Dr G.D. Sockalingum for his assistance in the preparation of the manuscript.

References

Baldini

Scotlandi

Serra

Shikita

Zini

Ognibene

Santi

Ferracini

Maraldi

(1995) Nuclear immunolocalisation of P-glycoprotein in multidrug resistant cell lines showing similar mechanisms of doxorubicin distribution. Eur J Cell Biol 68:226–239.

Barasch

Kiss

Prince

Saiman

Gruenert

Al-Awqati

(1991) Defective acidification of intracellular organelles in cystic fibrosis. Nature 352:70–73.

Beck

(1983) Vinca alkaloid-resistant phenotype in cultured human leukemic lymphoblasts. Cancer Treat Rep 67:875–882.

Beck

Mueller

Tanzer

(1979) Altered surface membrane glycoproteins in Vinca alkaloid-resistant human leukemic lymphoblasts. Cancer Res 39:2070–2076.

Belhoussine

Morjani

Lahlil

Manfait

(1997) Evidence for reversal of multidrug resistance by quinine in LR73 cells without alteration of nuclear pirarubicin uptake and down-regulation of mdr1 gene expression. Int J Cancer 73:600–606.

Bennis

Ichas

Robert

(1995) Differential effects of verapamil and quinine on the reversal of doxorubicin resistance in a leukemia cell line. Int J Cancer 62:283–290.

Bradley

Juranka

Ling

(1988) Mechanism of multidrug resistance. Biochim Biophys Acta 948:87–128.

Fabisiak

Vesell

Rannels

(1987) Interactions of beta adrenergic antagonists with isolated rat alveolar type II pneumocytes II. Receptors-independent accumulation of beta adrenergic antagonists and other cationic amphiphilic drugs in lamellar bodies. J Pharmacol Exp Ther 241:728–735.

Gigli

Doglia

Millot

Valentini

Manfait

(1988) Quantitative study of doxorubicin in living cell nuclei by microspectrofluorometry. Biochim Biophys Acta 950:13–20.

10.

Gigli

Rasoanaivo

TWD

Millot

Jeannesson

Rizzo

Jardillier

Arcamone

Manfait

(1989) Correlation between growth inhibition and intranuclear doxorubicin and 49-iododoxorubicin quantitated in living K562 cells by microspectroflurometry. Cancer Res 49:560–564.

11.

Goormaghtigh

Brasseur

Huart

Ruysschaert

(1987) Study of the adriamycin-cardiolipin complex structure using attenuated total reflection infrared spectroscopy. Biochemistry 26:1789–1795.

12.

Gottesman

Pastan

(1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385–427.

13.

Gros

Ben Neriah

Croop

Housman

(1986) Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 323:728–731.

14.

Jaffrézou

Levade

Chatelin

Laurent

(1992) Modulation of subcellular distribution of doxorubicin in multidrug-resistant P388/ADR mouse leukemia cells by the chemosensitizer 2-isopropyl-1-4-[3-N-methyl-N-(3,4-dimethoxy-b-phenethyl)amino]propyloxy}-benzenesulfonyl indolizine. Cancer Res 52:6440–6446.

15.

Lavie

Cao

Bursten

Guliano

Cabot

(1996) Accumulation of glucosylceramides in multidrug-resistant cancer cells. J Biol Chem 271:19530–19536.

16.

Lavie

Cao

Volner

Lucci

Han

Geffen

Giuliano

Cabot

(1997) Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 272:1682–1687.

17.

Le Moyec

Tatoud

Degeorges

Calabresse

Bauza

Eugene

Calvo

(1996) Proton nuclear magnetic resonance spectroscopy reveals cellular lipids involved in resistance to adriamycin and taxol by the K562 leukemia cell line. Cancer Res 56:3461–3467.

18.

Lozzio

(1975) Chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45:321–324.

19.

Mayer

Bally

Cullis

(1986) Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta 857:123–126.

20.

Mayer

Tai

LCL

Bally

Mitilenes

Ginsberg

Cullis

(1990) Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients. Biochim Biophys Acta 1025:143–151.

21.

Millot

Morjani

Desplaces

Manfait

(1997) Characterization of acidic vesicles in multidrug-resistant and sensitive cancer cells by acridine orange staining and confocal microspectrofluorometry. J Histochem Cytochem 45:1255–1264.

22.

Millot

Sharonov

Manfait

(1994) Scanning microspectrofluorometry of rhodamine 123 in multi-drug resistant cells. Cytometry 17:50–58.

23.

Molinari

Cianfriglia

Meschini

Calcabrini

Arancia

(1994) P-glycoprotein expression in the Golgi apparatus of multidrug resistant cells. Int J Cancer 59:789–795.

24.

Moriyama

Manabe

Yoshimori

Tashiro

Futai

(1994) ATP-dependent uptake of anti-neoplasic agents by acidic organelles. J Biochem 115:213–218.

25.

Morjani

Belhoussine

Lahlil

Manfait

(in press) Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug-resistance by quinine in human K562 and CEM leukemic cells. Eur J Haematol.

26.

Morjani

Millot

Belhoussine

Sebille

Manfait

(1997) Anthracycline subcellular distribution in human leukemic cells by microspectrofluorometry: factors contributing to drug-induced cell death and reversal of multidrug resistance. Leukemia 11:1170–1179.

27.

Morjani

Pignon

Millot

Debal

Lamiable

Potron

Etienne

Manfait

(1992) Intranuclear concentration measurements of doxorubicin in living leucocytes, from patients treated for lympho-proliferative disorder. Leukemia Res 16:647–653.

28.

Pagano

Martin

Kang

Haugland

(1991) A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol 113:1267–1279.

29.

Pelletier

Millot

Chauffert

Manfait

Genne

Martin

(1990) Mechanisms of resistance of confluent human and rat colon cancer cells to anthracyclines: alteration of drug passive diffusion. Cancer Res 50:6626–6633.

30.

Pignon

Morjani

Vilque

Millot

Simon

Lartigue

Etienne

Potron

Manfait

(1995) In vitro study of THPdoxorubicin retention in human leukemic cells using confocal laser microspectrofluorometry. Leukemia 9:1361–1367.

31.

Rhodes

Barrand

Twentyman

(1994) Modification by brefeldin A, bafilomycin, and 7-chloro-4-nitrobenzo-2-oxa-1,3- diazole (NBD) of cellular accumulation and intracellular distribution of anthracyclines in the non-P-glycoprotein-mediated multidrug resistant cell line COR-L23/R. Br J Cancer 70:60–66.

32.

Roepe

(1992) Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein. Biochemistry 31:12555–12564.

33.

Samuni

Chong

Barenholz

Thompson

(1986) Physical and chemical modifications of adriamycin-iron complex by phospholipid bilayers. Cancer Res 46:594–599.

34.

Schindler

Grabski

Hoff

Simon

(1996) Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7 adr). Biochemistry 35:2811–2817.

35.

Schuurhuis

Van Heiningent

THM

Cervantes

Pinedo

De Lange

JHM

Keizer

Broxtermann

Baak

JPA

Lankelma

(1993) Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance, in SW-1573 lung cancer multidrug resistant tumor cells. Br J Cancer 68:898–908.

36.

Schwartz

Kanter

(1979) Chemical interactions of cardiolipin with daunorubicin and other intercalating agents. Eur J Cancer 15:923–928.

37.

Sebille

Morjani

Poullain

Manfait

(1994) Effect of S9788, cyclosporin A and verapamil on intracellular distribution of THP-doxorubicin in multidrug-resistant K562 tumor cells, as studied by laser confocal microspectrofluorometry. Anticancer Res 14:2389–2394.

38.

Seidel

Hasmann

Löser

Bunge

Schaefer

Herzig

Steidtmann

Dietel

(1995) Intracellular localization, vesicular accumulation and kinetics of daunorubicin in sensitive and multidrug-resistant gastric carcinoma EPG85-257 cells. Virchows Arch 426:249–256.

39.

Sharonov

Chourpa

Valisa

Fleury

Feofanov

Manfait

(1994) Confocal spectral imaging analysis. Eur Microsc Anal 32:23–25.

40.

Simon

Schindler

(1994) Cell biological mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci USA 91:3497–3504.

41.

Sweet

Hume

(1996) Bacterial lipopolysaccharide confers resistance to G418, doxorubicin, and taxol in the murine macrophage cell line, RAW264. J Leukocyte Biol 59:280–286.

42.

Thiebaut

Currier

Whitaker

Haugland

Gottesman

Pastan

Willingham

(1990) Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye. J Histochem Cytochem 38:685–690.

43.

Tsuruo

Lida-Saito

Kawabata

(1986) Characteristics of resistance to adriamycin in human myelogenous leukemia K562 resistant to adriamycin and in isolated clones. Jpn J Cancer Res 77:682–692.

44.

Valverde

Diaz

Sepulveda

Gill

Hyde

Higgins

(1992) Volume-regulated chloride channels associated with the human multidrug resistance P-glycoprotein. Nature 355:830–833.

45.

Willingham

Cornwell

Cardarelli

Gottesman

Pastan

(1986) Single analysis of daunomycin uptake and efflux in multidrug-resistant and sensitive KB cells: effects of verapamil and other drugs. Cancer Res 46:5941–5946.

Confocal Scanning Microspectrofluorometry Reveals Specific Anthracyline Accumulation in Cytoplasmic Organelles of Multidrug-resistant Cancer Cells

Abstract

Keywords

Materials and Methods

Chemicals

Cell Lines

Cytotoxic Effect of THP-DOX

Confocal Laser Scanning Microspectrofluorometry

Confocal Spectral Image Acquisition

Determination of Nuclear Concentration of THP-DOX

Co-localization of THP-DOX and DMB-Ceramide in LR73D Cells

Results

Cytoplasmic Localization of THP-DOX in MDR Cell Lines

Effect of the Reversing Agents on THP-DOX Distribution in LR73 Cells

Discussion

Cytoplasmic Interaction of THP-DOX in MDR Cells

Models of THP-DOX Interaction in the Cytoplasm of MDR Cells

Footnotes

Acknowledgments

References