Introduction

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The multidrug resistance-associated protein-1 (MRP1) belongs to the adenosine triphosphate binding cassette superfamily of membrane transporters that derive their energy for transport from ATP hydrolysis. Other well known members of this family include P-glycoprotein (Pgp) and the cystic fibrosis transmembrane regulator (CFTR) protein encoded by the MDR1 and CFTR genes, respectively. MRP1 shares 15 and 19% homology to both these genes (Cole et al., 1992).

Since the cloning of the MRP1 gene, significant progress has been made in understanding the role of MRP1 in multidrug resistance (MDR). The observation that MRP1 transfected Hela cells exhibited increased doxorubicin resistance correlating to levels of MRP1 expression provided one of the earliest links between MRP1 efflux and multidrug resistance (Grant et al., 1994). Like Pgp, MRP1 recognizes a wide variety of structurally and pharmacologically diverse substrates. Substrates for MRP1 include uncharged hydrophobic molecules, lipid-soluble and hydrophilic anions, products of xenobiotic phase II metabolism (glutathione, glucuronide, and sulfate conjugates) as well as endogenous glutathione conjugates such as leukotriene C4 (Jedlitschky et al., 1994; Holló et al., 1996; Jedlitschky et al., 1996). Whereas Pgp has recently been proposed to possess multiple drug binding sites (Shapiro et al., 1999; Martin et al., 2000), at least two drug binding sites may exist on the protein (Stride et al., 1999).

Zaman et al. (1994) showed that MRP1 was a plasma-membrane drug efflux pump in transfected lung resistant cells. The advent of specific MRP1 antibodies has enabled further localization of MRP1 to be determined in both normal and malignant tissue. Strong staining of MRP1 has been shown in normal adrenal gland, lung, heart, and skeletal muscle (Flens et al., 1994) consistent with previous results obtained using in situ hybridization techniques (Thomas et al., 1994). Pgp is known to be localized to the apical side of most epithelia, however, recent evidence has shown that MRP1 is routed to the basolateral side of several cell types, such as kidney epithelia (Evers et al., 1996), choroid plexus (Nishino et al., 1999), and ciliated normal human bronchial epithelium (Bréchot et al., 1998).

The Calu-3 cell line represents an exciting alternative for existing in vitro models of the respiratory epithelium. We have recently proposed use of these cells as a tool to screen pulmonary drug delivery based on tight junction formation, permeability of low molecular weight compounds, asymmetric transferrin transport, and functional cytochrome P450 isoenzymes 1A1, 2B6, and 2E1 (Foster et al., 2000). Furthermore, we have also demonstrated that these cells possess functionally active Pgp consistent with an apical localization (Hamilton et al., 2001). In this report, we explore the contribution of MRP1 relative to Pgp to the efflux of model MDR substrates in the Calu-3 cell line. Specifically, the efflux of calcein, the hydrolysis product of calcein acetoxymethyl ester (C-AM), and the anticancer agent etoposide, was investigated.

	Materials and Methods

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Cell Maintenance. The Calu-3 cell line was obtained from the American Type Culture Collection (Rockville, MD) at passage 14 and used between passages 19 and 40. Cells were grown in 150-cm² culture flasks (Costar, Corning, NY) in a 95% humidified/10% CO₂ atmosphere at 37°C and maintained in a 1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 100 µg/ml penicillin G (Sigma), and 100 µg/ml streptomycin sulfate (Sigma). When the cells reached 90% confluence (approximately 4-5 days) they were subcultured at a 1:2 split ratio using 0.25% trypsin/0.1% EDTA (Sigma) in maintenance medium.

Kinetic Analysis of Calcein Accumulation. For kinetic studies, cells were plated onto 96-well plates (0.32-cm² growth area; Corning) at a seeding density of 5 × 10⁵ cells/cm² and experiments were conducted once the cells formed confluent monolayers (day 2). The time-dependent accumulation of calcein was monitored using a microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT). Experiments were performed at 37°C in pH 7.4 PBSA (phosphate-buffered saline, PBS, supplemented with 0.63 mM CaCl₂, 0.74 mM MgSO₄, 5.3 mM glucose, and 0.1 mM ascorbic acid) or PBSA (containing glucose or 2-deoxy-D-glucose) plus Na azide for energy-dependent determinations. All reagents were obtained from Sigma. The growth medium was first aspirated off and then the cells were rinsed three times with prewarmed (37°C) PBSA. The monolayers were allowed to equilibrate in buffer solution for 1 h at 37°C, followed by a 1-h preincubation with the appropriate inhibitors (5 µM CsA, 1 mM probenecid, 10 µM indomethacin, 10 mM Na azide, 10 mM Na azide/6 mM 2-deoxy-D-glucose, all obtained from Sigma). For the energy-dependent experiments, all steps were performed in PBSA containing azide, glucose or 2-deoxy-D-glucose as appropriate. Calu-3 cells were incubated with 1 µM C-AM (1 mg/ml stock in dry dimethyl sulfoxide; Molecular Probes, Eugene, OR) in the appropriately modified PBSA solution and continuous fluorescent monitoring of calcein accumulation was assessed every 2.5 min for a total of 60 min in the presence or absence of inhibitor. At the end of the kinetic run, the extracellular medium was removed, the monolayers rinsed three times with ice-cold PBSA then solubilized in lysing solution (2% v/v Triton X-100 in PBS). The protein content of each monolayer was determined using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) and fluorescence of the cell lysates was corrected for autofluorescence of untreated cells. Calcein fluorescence was measured at excitation/emission wavelengths of 485/530 nm. The rate of calcein accumulation was determined from the slope of the kinetic curves and expressed as millifluorescence units of calcein per minute per microgram of protein. Intracellular calcein concentration was quantified against a standard curve of calcein (Sigma) in lysing solution. Calcein accumulation was expressed as femtomoles of calcein per microgram of cellular protein.

Efflux Studies. Cells were seeded onto 12-well cluster dishes and used upon formation of confluent monolayers (by day 4). All experiments were performed with gentle agitation (~30 rpm) at 37°C in PBSA. Monolayers were rinsed then equilibrated for 1 h in PBSA for control experiments or for 30 min in PBSA alone, followed by another 30-min preincubation with the appropriate inhibitors (5 µM CsA, 1 mM probenecid, 10 µM indomethacin in PBSA, or some combination of these). Calu-3 cells were then loaded with 1 µM C-AM for 60 min in the presence or absence of inhibitor solution. The extracellular medium was then aspirated and the monolayers rinsed three times with ice-cold PBSA. Prewarmed (37°C) PBSA was then added back to the monolayers to initiate active drug efflux. At frequent time intervals (up to 2 h) the PBSA was removed, analyzed for fluorescent content, and replaced with fresh, warm PBSA containing the appropriate inhibitors. At the end of the efflux period, the monolayers were rinsed three times in ice-cold stop solution then solubilized in lysing solution. This lysate represented residual accumulation of calcein following efflux. The protein content of each monolayer was then determined using a bicinchoninic acid protein assay reagent kit. Calcein concentration was quantified against a standard curve of calcein in PBSA or lysing solution for efflux or residual accumulation, respectively, as described above. The fluorescence of the cell lysates was again corrected for autofluorescence of untreated cells. Cumulative calcein efflux was expressed as nanomolar calcein, and residual accumulation expressed as femtomoles of calcein per microgram of protein.

Calcein and [³H]Etoposide Transport across Polarized Monolayers of Calu-3 Cells. Calu-3 cells were seeded at a density of 5 × 10⁵ cells/cm² onto 12-well polycarbonate Transwells (0.4-µm pore size; Costar) that had previously been coated with type IV human placental collagen (0.75 mg/ml, 70 µl/insert; Sigma). The cells were allowed to attach for 24 h, and then the medium in the apical compartment was removed. Thereafter, the monolayer was allowed to differentiate under air-interface feeding conditions by replenishing the culture medium in the basolateral compartment every other day (Shen et al., 1994). Under these conditions, tight junction formation was evident once culture medium ceased to diffuse back into the apical chamber. Polarized monolayers of Calu-3 cells were used for transport studies between days 13 and 15 postseeding. Following equilibration of Calu-3 monolayers in PBSA, the transepithelial electrical resistance of each monolayer was measured using EVOM Chopstick electrodes (World Precision Instruments, Sarasota, FL). Only monolayers with transepithelial electrical resistance values 350  · cm² were used. A 30-min preincubation step with inhibitor applied to both apical and basolateral compartments was performed if necessary. Vectorial transport of calcein (added to cells as 1 µM C-AM) and [³H]etoposide (5 µM) was investigated in the presence or absence of inhibitor with probe added to either apical (A, air facing) or basolateral (B, blood facing) side of cell monolayers. Inhibitors used were 5 µM CsA (Pgp), 200 µM quinidine (mixed Pgp/MRP1, Sigma) and 5 µM MK571 (MRP1; BIOMOL, Plymouth Meeting, PA). At various time intervals, 100 µl of the receiver solution was removed and immediately replaced with an equal volume of PBSA with or without inhibitor. The transport of these probes was performed for 1 h. Monolayer integrity was assessed using [¹⁴C]mannitol flux (0.25 µCi/ml; Amersham Pharmacia Biotech UK, Buckinghamshire, England) for an additional hour following calcein transport, or simultaneously with [³H]etoposide transport. Calcein fluorescence in receiver solutions was quantified against a standard curve of calcein in PBSA. For etoposide quantitation, 5 ml of Scintiverse cocktail (Fisher Scientific, Fairlawn, NJ) was added to receiver samples in scintillation vials. Radioactive content was assessed by scintillation counting. Apparent permeability coefficients (P_app) were calculated according to the following equation:

(1)

where V is volume of the receiver chamber, A is area available for diffusion, C_o is initial donor concentration, and dC/dt is flux of test agent. Net secretion was defined as the ratio of B to A versus A to B permeability coefficients.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Total RNA was isolated from Calu-3 cell lysates using the RNeasy Mini kit (Qiagen, Valencia, CA). RNA quantitation and purity were determined from spectrophotometric absorbance at 260 nm. First strand cDNA synthesis and RT-PCR were performed using Ready To Go RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ). A 348-bp fragment specific for human MRP1 mRNA sequence (GenBank accession number L05628) was selected for cDNA first strand synthesis and amplification. Oligonucleotide primers corresponding to nucleotides 252-271 (upstream, 5'-ATGTCACGTGGAATACCAGC-3') and 581-600 (downstream, 5'-GAAGACTGAACTCCCTTCCT-3') were obtained from Sigma-Genosys (The Woodlands, TX). First strand cDNA synthesis was performed in 50-µl total volume containing 10 pmol each of upstream and downstream primer and 1 µg of template RNA, added to Ready To Go RT-PCR beads according to the manufacturers instructions. RT-PCR conditions were as follows: 4 min at 95°C then 34 cycles of 95°C for 1 min (denaturation), 56°C for 1 min (annealing), and 72°C for 1 min (elongation). The integrity of the PCR product was confirmed on a 1.2% agarose/TAE gel followed by band excision and purification (Qiaquick Spin kit; Qiagen). RT-PCR with omission of the template RNA served as the negative control reaction. Amplification of -actin from total RNA of human placenta and Calu-3 cells was used as a positive control reaction. -Actin primer sets were obtained from CLONTECH (Palo Alto, CA).

Immunolocalization of Pgp and MRP1. Membrane localization of Pgp and MRP1 was performed using indirect immunofluorescent staining and analyzed by confocal laser scanning microscopy. Calu-3 cells were grown on polyester clear Transwells (0.4-µm pore size; Costar) to aid in microscopic visualization of the monolayers. The monolayers were rinsed three times in PBS+ (PBS, pH 7.4 + 0.63 mM CaCl₂ + 0.74 mM MgSO₄) then fixed for 10 min at room temperature in 4% paraformaldehyde in PBS. Cells were permeabilized for 5 min at room temperature in 1% v/v Triton X-100 followed by blocking of nonspecific binding sites for 1 h in blocking solution [1% w/v bovine serum albumin (Sigma) in PBS+]. The monolayers were incubated for 1 h in primary antibody solution in blocking solution, rinsed three times in blocking solution, and then incubated for an additional hour in secondary antibody solution in PBS+ containing 1 µg/ml propidium iodide (Molecular Probes) for counterstaining of nucleic acids. The cells were then rinsed three times in PBS+ and the inserts removed and mounted with Advantage Permanent mounting medium (Accurate Chemical & Scientific, Westbury, NY). The Pgp rabbit polyclonal antibody mdr (Ab-1) was obtained from Oncogene Research Products (Cambridge, MA) and used at 5 µg/ml. The MRP1 mouse antibody mrpm6 was obtained from Kamiya Biomedical (Seattle, WA) and used at 8 µg/ml. All manipulations were performed at room temperature with antibody solutions being applied to both the apical and basolateral compartments. Alexa-Fluor 488-labeled secondary antibodies (rabbit and mouse, for Pgp and MRP1, respectively) were used at 20 µg/ml (Molecular Probes). Imaging was performed using a Nikon inverted fluorescence microscope equipped with a Bio-Rad MRC 1000 confocal unit. The green (Alexa-Fluor 488) and red (propidium iodide) fluorescent probes were excited at 488 and 568 nm, respectively. Horizontal sections were taken at 3.5-µm intervals through the cell depth beginning from either the apical or basolateral side of the monolayer to obtain z-scan images. This was done to show that collected images reflected a true MDR pump membrane polarization rather than mere bleaching of the fluorescent label during laser scanning. Images were processed using Scion Image Beta 4.0.2.

Statistical Analysis. All experiments were performed at least in quadruplicate. Data is presented as mean ± standard deviation. Statistical significance was performed using the unpaired Student's t test (Sigma Plot, version 4.01) and determined at the 95% confidence limit.

	Results

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MRP1 Gene Expression. Initial RT-PCR analysis revealed a single band of approximately 400 bp (Fig. 1a). However, upon reamplification, this band was resolved into two distinct gene fragments of approximately 400 and 350 bp (Fig. 1b). DNA sequencing confirmed that the lower size fragment corresponded to the partial mRNA sequence for MRP1 with the higher size band being an unidentified gene product.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   RT-PCR of MRP1 in Calu-3 cells. a, MRP1 cDNA resolved on 1.2% TAE agarose. Lanes 1 and 2, MRP1 cDNA; lane 3, negative control; lane 4, molecular weight markers; lane 5, -actin from Calu-3 cells; lane 6, -actin from total human placenta RNA. b, Resolution of 400-bp amplification product yielded a 350-bp gene fragment corresponding to MRP1. Lane 1, MRP1; lane 2, molecular weight markers; lane 3, unidentified gene fragment. Arrows show positions of molecular weight markers given in base pair units.

Immunolocalization of Pgp and MRP1. Progressive sectioning through polarized monolayers of Calu-3 cells allowed membrane localization of Pgp and MRP1 efflux pump proteins to be determined. Black-and-white images are shown in Fig. 2. Pgp-labeled monolayers revealed more intense staining at the apical surface of the monolayers compared with a more basolateral intense staining of MRP1-labeled monolayers. Furthermore, the relative intensity of staining of Pgp was greater than that observed with MRP1 compared with propidium iodide counterstaining of nucleic acids (data not shown). For both proteins, a concentric staining pattern around the cell periphery indicating membrane versus cytoplasmic staining was observed.

View larger version (82K): [in this window] [in a new window]   Fig. 2.   Confocal laser scanning micrographs of polarized Calu-3 monolayers using indirect immunofluorescent staining of Pgp and MRP1. Images were retrieved from progressive scans taken in the horizontal plane. a, Pgp rabbit polyclonal antibody mdr (Ab-1) was used at 5 µg/ml followed by detection with Alexa-Fluor 488-labeled anti-rabbit IgG (20 µg/ml). b, MRP1 mouse monoclonal antibody mrpm6 was used at 8 µg/ml followed by detection with Alexa-Fluor 488-labeled anti-mouse IgG (20 µg/ml). i, direct view of fluorescently labeled efflux pump proteins. ii, stacked images rotated at 180° to the y-axis to show preferential localization within the cell monolayer. iii, Representative histogram of ii to show intensity of labeling at either the apical or basolateral membrane. Arrow, the apical and basolateral membrane for Pgp- and MRP1-stained monolayers, respectively. Horizontal bar, the cumulative width of stacked horizontal sections.

Calcein Accumulation and Efflux. The extrusion of the fluorescent MRP1 substrate calcein was used to investigate functional MRP1 activity in Calu-3 cells (Fig. 3). The Pgp inhibitors CsA and vinblastine, as well as conditions of energy depletion (azide with glucose or 2-deoxy-D-glucose) all caused an increase in the rate of calcein accumulation following loading of cells with 1 µM C-AM. While no effect of indomethacin was observed in the experiment shown, subsequent experiments revealed that this MRP1 inhibitor caused a slight increase (up to 2-fold) in calcein accumulation.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Rate of accumulation of calcein, loaded as 1 µM C-AM in Calu-3 cells over a 1-h period, in the presence or absence of selected Pgp inhibitors (5 µM CsA, 10 µM vinblastine), MRP1 inhibitor (10 µM indomethacin), and conditions of energy depletion (10 mM Na azide + 6 mM glucose/6 mM deoxy-D-glucose). *p < 0.05 compared with control.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Efflux (a and b) and residual uptake (c) of calcein, previously loaded as 1 µM C-AM for a 1-h period in Calu-3 cells in the presence or absence of selected Pgp inhibitors (5 µM CsA, 10 µM vinblastine), MRP1 inhibitors (10 µM indomethacin, 1 mM probenecid) and various combinations of these.

MDR Inhibition of Polarized Transport of Calcein and Etoposide across Calu-3 Monolayers. When the transport of calcein was performed in cells exposed to C-AM, we observed a 2-fold decrease in net secretion (i.e., net transport in the basolateral to apical direction) of the measured species, calcein, following Pgp inhibition with CsA. The MRP1 inhibitors probenecid and indomethacin resulted in a 3.5- and 2.4-fold increase in calcein net secretion, respectively (data not shown). These results should be interpreted with caution, however, because the permeability coefficients varied considerably between experiments (between 10⁵ and 10⁷ cm/s). In all cases, however, the effect on net secretion was consistent. The observed variability in permeabilities could also be due to differences in ester hydrolysis between different batches of cells.

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	Discussion

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Pgp and MRP1 Gene Expression and Membrane Localization. The apical localization of Pgp suggested in Calu-3 cells is consistent with Pgp expression in normal human bronchus and submucosal glands from nasal epithelium (Henriksson et al., 1997; Lechapt-Zalcman et al., 1997). Our findings are also consistent with basolateral localization of MRP1 in normal human bronchial epithelium (Bréchot et al., 1998) and with the suggested excretory role of MRP1 for xenobiotic conjugates following phase II metabolism (Borst et al., 1999). High levels of MRP mRNA were reported in normal bronchial epithelial cells compared with weak staining in the epithelium of lung tumors (Thomas et al., 1994). The staining of MRP1 was relatively less intense than that of Pgp in our studies in spite of a higher concentration of MRP1 antibody used. Therefore, our studies would suggest that while Calu-3 cells express higher levels of Pgp than MRP1, they possess a normal phenotype to that found in the lung with respect to both efflux pump proteins.

Accumulation and Efflux of Calcein from Calu-3 Cells. The accumulation and efflux of calcein in Calu-3 cells is the result of several kinetic processes, shown schematically in Fig. 5. A similar scheme has been reported previously (Holló et al., 1994; Essodïagui et al., 1998) and is modified here to include proposed rate constants. The highly lipophilic, nonfluorescent C-AM ester enters the cell by passive diffusion (k₁). Within the cell, C-AM is converted enzymatically and irreversibly to fluorescent calcein (k₂). Since hydrophilic calcein is not a substrate for Pgp (Holló et al., 1994; Feller et al., 1995), the only route of removal of this charged molecule (net 4 at physiological pH) is via MRP1 (k₃). In contrast, C-AM is known to be a substrate for both Pgp and MRP1 (Holló et al., 1994); rates of C-AM efflux by these two routes are indicated by the rate constants k₁ and k₂.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Schematic representation of C-AM diffusion, enzymatic conversion and efflux via Pgp and MRP1 in the Calu-3 cell system.

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Polarized Transport Studies. An increase in calcein net secretion with MRP1 inhibitors is consistent with inhibition of calcein efflux by a basolaterally localized efflux pump. On the other hand, CsA inhibition of Pgp-mediated C-AM efflux resulted in a decrease in calcein net secretion. For these studies, Calu-3 cells were not preloaded with C-AM, but were continuously exposed to parent compound over a 2-h transport and sampling period. Under these conditions, accumulated intracellular calcein would be expected to be lower due to simultaneous C-AM efflux during transepithelial transport. This might explain the smaller differences observed between calcein transport with Pgp and MRP1 inhibition compared with the efflux experiments. Collectively, however, the transport data support inhibition of one or more efflux pumps with opposing membrane localization.