Flow Cytometric Analysis of P-Glycoprotein Function Using Rhodamine 123

Flow cytometric analysis of P-glycoprotein function using rhodamine 123

JPe´triz and J Garcı´a-Lo´pez

Departament de Criobiologia i Tera`pia Cel·lular, Centre de Refere`ncia de Citometria Izasa-Coulter, Institut de Recerca Oncolo`gica, Hospital Duran i Reynals, Gran Via Km 2,7, L’Hospitalet de Llobregat, 08907, Barcelona, Spain

The MDR1 gene product, P-glycoprotein (P-gp), works as a ing its functional activity.24–26 The detection of P-gp activity transmembrane efflux pump for several cytotoxic products, in human tumor samples is of great clinical interest, parti- representing a major cause for cancer treatment failure. Rhoda- cularly for investigating certain agents that are known to mine 123 (Rh123), a low toxic fluorescent probe commonly 27,28 used to assess mitochondrial bioenergetics in living cells, has reverse the multidrug resistant phenotype. Functional also been used to measure the efflux activity of P-gp in both analysis of the P-gp can be accomplished using diverse normal and malignant cells. Analysis of variation in cellular techniques: (1) transport experiments with radiolabeled fluorescence by measuring the rates of Rh123 influx and efflux, substrates29 (ie 3H vinblastine); (2) analysis of drug transport together with the effect of mdr reversing agents, allows the kinetics using P-gp substrates attached to fluorescent com- investigation of drug-resistant phenotypes in cancer samples. pounds30 (ie coumarin-vinblastine, photoactivatable taxol); We have studied the functional activity of P-gp in human leu- 31 kemic cell lines using flow cytometry, taking into consideration and (3) flow cytometric efflux assays with fluorescent dyes. that variables such as Rh123 cytotoxicity, culture conditions, For this last purpose, and since certain anticancer agents such cell membrane integrity, as well as the effect of specific P-gp as anthracyclines32 (doxorubicin, daunorubicin) have fluor- modulators, can impair the resolution of the Rh123-efflux escent properties, they can be used to quantitate their cellular measurements. The studies show that: (1) optimal non-cyto- influx, retention and efflux in tumor cell populations. Some toxic concentrations of Rh123 which allow appropriate color other relatively non-toxic dyes are well known substrates for compensation are in the range of 50–200 ng/ml; (2) life-gating 33 allows accurate measurement on the 50% average rate of P-gp, such as rhodamine-123 (Rh123), bisbenzimidazole 34 Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC- dyes (Hoechst 33342), 3,3′-diethyloxacarbocyanine iodide Ͼ Ͼ ′ 833 cyclosporin A verapamil; and (4) the presence or (DiOC2(3)), 3,3 -diethyloxadicarbocyanine iodide (DODC absence of fetal calf serum had no effect on the bioavailability iodide),35 thiazole and oxazole derivatives36 (SY-38 and SY- of chemosensitizer agents, with the exception of serum-free 3150 respectively), and fluorescent calcium probes37 (ie indo- experiments, which showed a significant decrease in P-gp Hence, we 1AM, fluo-3AM). These substrates can also be used, together .(0.05 ؍ activity under the presence of PSC-833 (P recommend this experimental strategy for clinical practice bet- with P-gp inhibitors, to study MDR functionality. Rh123 is a ␭ = ter to study the cellular drug resistance phenotype. cationic and lipophilic fluorescent dye ( ex/em 505/534 nm) Keywords: P-glycoprotein; multidrug resistance; rhodamine 123; more photostable than fluorescein, pH insensitive under flow cytometry physiological conditions, and membrane permeable, which shows a rapid uptake by mitochondria in living cells.38 It has been used in diverse studies, particularly in the investigation Introduction of drug resistance,39 assessment of mitochondrial bioenergetics in living cells40 (ie enzymatic activity and mitochondrial Multidrug resistance (MDR) in tumor cells is associated with transmembrane potential assessment); isolation of primitive the overexpression of mdr encoding genes, whose expression bone marrow hematopoietic progenitor cells,41 the study of confers cellular drug resistance against some specific anti- cell-cycle processes,42 and the staining of living micro- tumor agents.1–4 P-glycoprotein (P-gp), the product of the mdr organisms.43 With regard to drug resistance assessment, con- gene, is a Mr 170 000–180 000 energy-dependent transmem- ditions for the action of P-gp depend on the mitochondrial brane pump5–7 which extrudes a diverse group of unrelated membrane potential and cell membrane potential, as well as compounds, such as the Vinca alkaloids, anthracyclines, epi- on the time and temperature of exposure, and drug concen- podophyllotoxins, taxol, and certain protein synthesis inhibi- tration. Since living cells are sensitive to these changes,44 pre- tors,8–11 resulting in a decreased, and less toxic, intracellular liminary studies of the transporter activity are needed in order concentration of anticancer agents.12,13 However, increased accurately to design and interpret future experiments. This levels of P-gp are not the only cause of MDR; other mech- paper focuses on the study of the P-gp function, as determined anisms can be involved, such as the overexpression of the with Rh123 and flow cytometry, as well as on the effect of drug resistant MRP (MDR-associated protein) gene, which has diverse efflux blockers on Rh123 retention by tumor cells. been observed in non-P-gp cell lines selected for resistance to cytotoxic drugs.14,15 P-gp is also expressed in a wide variety of normal cells, Materials and methods specially in secretory tissues, adrenal cortex, liver, jejunum, colon, kidney, certain capillary endothelia, peripheral blood Cell cultures lymphocytes, and hemopoietic precursor cells.16–21 P-gp levels can be analyzed by identifying the protein with specific KG1, KG1a, and K562 cell lines were obtained from the antibodies22 (ie immunohistochemistry, Western blot, flow American Type Culture Collection (Rockville, MD, USA) and cytometry), by measuring the MDR1 mRNA levels23 (ie reverse grown in RPMI 1640 nutrient mixture (Imperial, Andover, transcriptase-PCR, Northern blot), and, in addition, by analyz- UK), supplemented with 2 mML-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, 100 mg/ml streptomycin (Biological Industries, Kibbutz Beth Haemek, Israel), and 10% Correspondence: J Pe´triz fetal calf serum (FCS). Cells were cultured at 37°C in a humidi- Received 3 October 1996; accepted 20 December 1996 fied atmosphere of 5% CO2, in air. Exponentially growing Functional expression of P-glycoprotein JPe´triz and J Garcıá-Lo´pez 1125 cells with an optimal density of 5 × 105 cells/ml were prepared to the culture medium, in the presence or absence of either for uptake and retention experiments. Cell lines were period- Vpl, CsA, or PSC-833. After 1 h of incubation, cells were ically tested for Mycoplasma infection and found to be nega- washed and fed with Rh123-free culture medium, and cul- tive. Cell viability was determined by trypan blue exclusion tured for 90 min at 37°C, again in the presence or absence of using a hemocytometer. the MDR reversing agents, to evaluate their effect on Rh123 retention. Afterwards, cells were incubated in Rh123-free medium. Life-gate was based in light scatter parameters and in Clinical samples simultaneous staining with 5 ␮g/ml propidium iodide. Cellular efflux of Rh123 was measured by monitoring its fluorescence Peripheral blood and bone marrow specimens from healthy decrease at 525 nm emission wavelength. Analysis of 104 cells donors were collected in heparinized tubes. Mononuclear per sample was carried out in the Rh123/count four decades cells were prepared by Ficoll–Hypaque density gradient cen- log histogram, collecting the autofluorescence signal in the trifugation (Pharmacia, Uppsala, Sweden) according to the first decade. All analyses were performed in triplicate in three manufacturer’s recommendations, washed, and resuspended separate experiments, and the results were expressed as the in phosphate-buffered saline (pH = 7.4). In all cases viability mean of fluorescence intensity. was Ͼ90% by trypan blue exclusion.

Multicolor ﬂow cytometric analysis Source of drugs and chemicals Single suspensions of Rh123 loaded cells were incubated in Rhodamine 123 (Rh123) was obtained from Lambda Fluoresz- the presence of 20 ␮g/ml of CD34 monoclonal antibody in enztechnologie, (Graz, Austria); propidium iodide (Pl) from PBS-BSA (HPCA-2 PE; Becton Dickinson, San Jose, CA, USA). Sigma Chemical (St Louis, MO, USA); 7-aminoactynomycin After incubation, cells were stained with either 5 ␮g/ml of Pl D from Molecular Probes Europe (Leiden, The Netherlands); or 2 ␮g/ml of 7-AAD, 10 min prior to analysis. verapamil (Vpl) from Laboratorios Knoll, Spain; Cyclosporin A (CsA) and PSC-833 from Sandoz Pharma (Basel, Switzerland). Flow cytometry

Rhodamine 123 uptake/retention assays Flow cytometric data were collected on an EPICS Elite ESP cell sorter and on an EPICS XL-MCL (Coulter Electronics, In order to achieve optimal growth conditions, cells were Hialeah, FL, USA). All studies were carried out using an air- seeded 12 h before the Rh123 uptake/retention experiments cooled 15 mW argon laser (Cyonics, Hialeah, FL, USA), in 6-well tissue culture plates (Costar, Cambridge, MA, USA), operating at a 488 nm wavelength. Forward scatter and side at a final concentration of 5 × 105 cells/ml, in 3 ml of RPMI scatter were collected on linear scale and were used to 1640 media (Gibco, Life Technologies, Gaithersburg, MD, exclude dead cells and cell aggregates either with logarithmi- USA). Rh123 uptake assays were performed by adding cally amplified Pl fluorescence (collected through a 620 nm

200 ng/ml of the dye (stock solution 1 mg/ml in distilled H2O) bandpass filter) or with 7-AAD fluorescence (collected through a 675 nm bandpass filter). Analyses were carried out using the Elite Workstation Software Ver. 4.01 and the WinMDI Ver 2.1.3. Software.

Results and discussion

Rh123 uptake and retention depends on both the mitochondrial and the cell membrane potential. Therefore, the integrity of the cell membrane must be assessed in Rh123 experiments by parameters other than light scatter (life-gate). Rh123 charged cells are excited with a 488 nm wavelength and analyzed through a band pass (BP) filter of 525 nm (green fluorescence). Then, simultaneous staining with propidium iodide (red fluorescence), a non-permeant dye that cannot stain living cells, is used to overcome artifactual uptake and retention phenomenon results.18 In addition, normal and tumor cells must be characterized taking into consideration that forward scatter vs Rh123 fluorescence histograms can indicate heterogeneity of Rh123 retention, and show the effect of inhibitors or MDR indicator dyes on scatter and viability.33 Since one of the major limiting points in Rh123 uptake and retention experiments is that dye-efflux is affected by Rh123 concentration, optimal Rh123 concentration, time, and tem- Figure 1 Concentration, time, and temperature dependence of perature of incubation must be tested prior to its experimental rhodamine 123 uptake. Separate cultures of leukemic cells initially containing 5 × 105 cell/ml were incubated in the indicated Rh123 use. Rh123 has anticarcinoma activity in vivo and is selec- concentrations, time, and temperature. These data were obtained from tively toxic to cancer cells in vitro. The cytotoxicity of Rh123 two separate experiments. directly correlates with its intracellular concentration: from Functional expression of P-glycoprotein JPe´triz and J Garcıá-Lo´pez 1126

Figure 2 Simultaneous three color flow cytometric analysis showing the rapid accumulation of Rh123 in human CD34+ living cells. Mobilized peripheral blood from patients undergoing autologous stem cell transplantation was stained with 7-AAD (far red fluorescence) for dead-cell discrimination (a), with the 8G12·PE monoclonal antibody (orange fluorescence) for progenitor cells (b), and with Rh123 (green fluorescence) for mitochondrial activity assessment. (c) A two parameter dot plot of 7-AAD negative cells showing green vs orange fluorescence compensation. (d) Kinetics of Rh123 accumulation in CD34+ living cells. (e) Characterization of the intracellular accumulation of Rh123 in CD34-positive/7-AAD-negative cells by means of a time-slice gating technique. Functional expression of P-glycoprotein JPe´triz and J Garcıá-Lo´pez 1127 50 ␮g/ml up to 100 ␮g/ml, Rh123 can be toxic to entire cells exposed over 24 h at 37°C, while using isolated mitochondria, 5 ␮g/ml Rh123 inhibit ATP synthesis.40 Rh123 acts inhibiting mitochondrial ATP synthesis,45,46 mitochondrial protein synthesis,47 mitochondrial transhydrogenase,48 protein kinase C, and calmodulin activities.40,45,49 As shown in Figure 1, the accumulation of Rh123 in living cells is concentration, time and temperature dependent. Leukemic cells at 4°C, 21°C and 37°C show differential Rh123 accumulation, which is higher as the temperature increases, probably as a consequence of the variation in the metabolic rate and in the mitochondrial membrane potential. Similarly, increasing the Rh123 concentration from 100 to 200 ng/ml, leads to approximately twice the fluorescence intensity. Since the fluorescence intensity depends on the Rh123 concentration, in the majority of cases, adjustment of the concentration of dye may facilitate simultaneous flow cytometric multicolor analysis. The rapid accumulation of Rh123 by living cells can be demonstrated by a simple and rapid kinetic assay, using a histogram with time as a cytometric parameter (X-axis) vs the logarithmic green fluorescence signal (Y-axis). This assay allows the quantitation of the initial rate of Rh123 uptake by Figure 3 Two parameter plot illustrating intracellular accumu- cell suspensions.50 The concentrations of Rh123 used for this lation of Rh123 in leukemic cells. Cells were loaded with Rh123 assay are in the range of 50–200 ng/ml for 5 × 105 cells, (200 ng/ml) and analyzed at 5 min time intervals to show the amount of cellular fluorescence. Data were obtained from two separate allowing an appropriate electronic signal compensation when experiments. overlapping spectra occur, as in the case of simultaneous staining with certain antibodies and fluorescent probes. Auto- fluorescence needs to be adjusted near the first decade of the levels and to evaluate the changes in the fluorescence intensity. green log fluorescence and registered for 100–150 s. Then, Decreased drug accumulation in cancer cells can be acquisition is paused for approximately 10 s in order to add reversed by using modifiers of multidrug resistance.52–57 Cer- Rh123 and reach the appropriate fluorochrome concentration tain P-gp competitive inhibitors, such as the calcium channel to the cell suspension. Following this, and after continuous blocker verapamil, cyclosporin A, and the cyclosporin deriva- fluorescence registering, sample acquisition is stopped after tive PSC-833 act by blocking the Rh123 transport. Figure 4 5–10 min, the time commonly needed for cells to reach a plateau in the presence of Rh123. Figure 2 describes a typical experiment using mobilized peripheral blood cells from patients undergoing autologous stem cell transplantation. A multicolor triple cell staining is performed to show simul- taneously the kinetics of Rh123 uptake (Rh123, green fluorescence), the presence of cell progenitors (CD34 monoclonal antibody, 8G12·PE, orange fluorescence), and the assessment of cell membrane integrity. The latter parameter was achieved by means of life-gating with 7-aminoactyno- micin D (far red fluorescence), an alternative DNA dye which can be used for the exclusion of non-viable cells.51 Usually, the time needed to obtain a complete fluorescence register, or the time needed to perform Rh123 uptake and retention experiments, can surpass this 5–10 min of continuous acquisition. As shown in Figure 2d, Rh123 uptake seems to reach a plateau within 5–10 min. However, when longer time-points of Rh123 uptake are analyzed (Figure 1), we observe that the fluorescence intensity increases with time. To overcome this discrepancy, we have used the time–interval acquisition technique. This technique involves quantitation of the changes in the fluorescence intensity for up to 3 h, depending on the drug used after the dye accumulation. First, the living cells are kept at 37°C in the presence of 200 ng/ml of Rh123 for 60 min and then analyzed to evaluate the intracellular concentration of Rh123 at 5–10 min intervals (see Figure 3). Efflux experiments combining simultaneous antibody staining must include control samples for adequate color Figure 4 Mean fluorescence intensity of KG1, KG1a, and K562 compensation, such as a blank and Rh123 loaded cells cells incubated with or without verapamil (10 ␮g/ml) in 200 ng/ml of stained with the corresponding isotypic control. In addition, Rh123 for 90 min at 37°C. Analysis of rhodamine efflux was carried a blank sample is used in order to set the autofluorescence out as described in Material and methods. Functional expression of P-glycoprotein JPe´triz and J Garcıá-Lo´pez 1128 shows the in vitro effect of MDR reversing agents on Rh123 transport in leukemic cells. These experiments were performed using myeloblastic P-gp-positive KG1 and KG1a cells and erythroleukemic P-gp-negative K562 cells, either in the presence or absence of 10 ␮g/ml verapamil, as described in the Materials and methods section. After cold washes with buffered solutions with or without the specific inhibitor, cells were re-seeded in Rh123-free medium, cultured at 37°C, and analyzed for Rh123 fluorescence at 30, 60, 120, 180 and 240 min. In KG1 and KG1a cells, the decrease in Rh123 flu- oresence observed is prevented when Vpl is added, showing its capacity to reverse P-gp activity. However, K562 cells were not able to extrude Rh123 in the same conditions as myeloblastic cells. When Vpl is not present in the medium, the differences observed in the mean intensity of fluorescence, up to five-fold in K562 cells, could be due to a decreased accumulation of Rh123 in these myeloblastic cell lines. Although it is therefore likely that the retention and distribution of cytotoxic agents might be related to the plasma membrane electrochemical potential, P-gp could also be involved in regulating the influx/efflux rate. In vitro kinetics of living cells can be accurately quantitated Figure 6 Flow cytometric analysis of rhodamine 123 efflux in either with liquid scintillation spectrometry29 (ie standard fast mdr1-positive cells showing the effect of increased concentrations of filtration techniques) or by continuous monitoring of fluor- PSC-833, cyclosporin A, and verapamil, in the presence (dashed lines) 30 or absence (dotted lines) of fetal calf serum. Data represent the mean escence, and both methods allow quantification of the effect of three independent experiments, each carried out in triplicate. of specific inhibitors on drug transport. Even though flow cytometric time–interval acquisition techniques achieve less pre- cise kinetic measurements, they offer advantages for studies to calculate the 50% decrease of fluorescence, the initial on the heterogeneity of drug retention by clinical samples, as 10 min time interval must be investigated. From the efflux well as for the different effects of P-gp modulators on specific curves obtained, it can be estimated that KG1 cells need 2– cell populations. As shown in Figure 5, myeloblastic KG1 and 3 min to achieve the 50% decrease of fluorescence, while

KG1a cells show very similar Rh123-efflux curves at 10 min KG1a cells need between 5 and 6 min (50%/t50 average rate time intervals over a period of 60 min. Since it may be very of Rh123 efflux, or time that P-gp takes for 50% of the Rh123 difficult to derive an equation to fit these curves, the measure- to be transported out of the cells). From these data we can ment of the initial reaction velocity should be used to calcu- show differences in the velocity of the transporter, considering late the first part of the curve and to draw a tangent at the that the causes contributing to the decrease in velocity with origin. The curves are usually virtual straight lines, as long as time, such as the level of saturation with Rh123, may not have the amount of change does not exceed 20% of the total. Thus, an effect within the linear phase. In order to analyse the effect of specific P-gp modulators on the Rh123 efflux, and to determine the relative concentrations at which Vpl, CsA, and PSC-833 begin to reverse Rh123 transport, cell cultures pre-loaded with Rh123 (200 ng/ml) and several concentrations of these inhibitors were tested. It has been described that MDR modulators have a decreased activity in the presence of serum due to high plasma protein binding, thus altering Rh123 experiments.58 We have studied the modulation of P-gp in both FCS-free medium and in medium supplemented with FCS, by means of a flow cytometric functional analysis in cells overexpressing the mdr1 gene. Firstly, as shown in Figure 6, the inhibition of Rh123 efflux was dependent on the P-gp reversing agent used, showing that the relative inhibitory efficiency for different modulators was PSC- 833 Ͼ CsA Ͼ Vpl. Secondly, regarding the presence of serum in the media, we were not able to demonstrate a significantly decreased activity of MDR modulators in FCS when compared with FCS-free medium, with the exception of PSC-833, which showed decreased potential under FCS-free conditions (P = 0.05). However, this reduced effect was probably due to a decreased efficiency in the efflux metabolism because of the presence of serum, rather than to an inherent effect of the inhibitor. Thus, the bioavailability and doses of chemosensiti- Figure 5 Flow cytometric analysis of rhodamine 123 efflux in KG1 zers should be carefully evaluated for each cellular experi- (circles) and KG1a (squares) cells. After incubation with or without verapamil (10 ␮g/ml) in 200 ng/ml of Rh123, cells were placed in mental model, particularly for functional analyses, bearing in Rh123-free medium at 37°C, in the presence or absence of the resist- mind that the pharmacokinetics of MDR modulators may vary ance modifier. 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