Multidrug Resistance-Associated Protein-1 Functional Activity in Calu-3 Cells

Materials and Methods

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 × 10⁵cells/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 × 10⁵cells/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 Ω · 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:Equation 1where 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 β-actin from total RNA of human placenta and Calu-3 cells was used as a positive control reaction. β-Actin primer sets were obtained from CLONTECH (Palo Alto, CA).

Gene amplification was performed using hot-start PCR. The reaction mix included 5 μl of purified DNA template, 1 μl 10 mM dNTP (Amersham Pharmacia Biotech), 5 μl of 10× OnePhorAll Buffer Plus (Amersham Pharmacia Biotech), 200 nM each gene-specific primer, and 5000 U ofTaq DNA polymerase (Promega, Madison, WI) made up to 50-μl total volume with RNase-free water. Reaction conditions were as follows: 5 min at 95°C (template denaturation) followed by addition of enzyme then 22 cycles of 95°C for 2 min (denaturation), 61°C for 1 min (annealing), and 72°C for 1 min (elongation). The reaction mix was held at 4°C after amplification was completed. MRP1 bands were identified following agarose electrophoresis under the conditions specified in the figures. Direct PCR fluorescent DNA sequencing was performed by Seqwright (Houston, TX) and bands confirmed as MRP1 gene product using the BLAST search program.

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

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.

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.

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.

To probe this mechanism further, the cells were loaded with C-AM and the efflux of fluorescent calcein was performed in the absence or presence of selected MDR inhibitors. Calcein retention inside the cells was measured following efflux. Calcein efflux in the absence of any MDR inhibitor appeared biphasic, exhibiting an apparent slow phase of efflux (0–30 min) followed by an apparent faster phase (60–120 min). Treatment with CsA, indomethacin, or probenecid alone resulted in linearization of these efflux profiles (R² > 0.990). Furthermore, a significant increase in calcein efflux was observed with CsA treatment up to 30 min, with no effect being observed between 60 and 120 min (Fig. 4a). Contrary to this, an approximate 4-fold increase in intracellular calcein retention postefflux was observed (Fig. 4c). While the MRP1 inhibitors probenecid and indomethacin decreased the efflux of calcein by 70 and 53%, respectively (120 min; Fig. 4a), calcein retention was increased by approximately 2-fold in both cases.

In an effort to achieve maximum inhibitory effects on the Pgp and MRP1 efflux systems with these agents, similar experiments were conducted using a combination of CsA, probenecid, and indomethacin. With CsA present in these mixed inhibition studies, calcein efflux was again increased up to 30 min, while a decrease in efflux was observed between 60 and 120 min (Fig. 4b). Mixed MRP1 inhibition gave similar efflux profiles to those observed with MRP1 inhibition alone. Probenecid in combination with CsA was the more effective MRP1 mixed inhibitor. Maximum residual calcein levels were observed with all three inhibitors present, while trapped calcein levels were similar to those observed with either probenecid or indomethacin alone (Fig. 4c).

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 10⁻⁵ and 10⁻⁷ 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.

The effect of various MDR inhibitors on the transport of the Pgp/MRP1 substrate etoposide was investigated. The transport of etoposide was asymmetric, favoring transport in the basolateral-to-apical direction (P_app A to B = 2.1 ± 0.2 × 10⁻⁶ cm/s,P_app B to A = 6.4 ± 0.5 × 10⁻⁶ cm/s). Quinidine (Pgp/MRP1 mixed inhibitor) and CsA (Pgp inhibitor) both increased A-to-B transport while decreasing B-to-A secretion of etoposide. These treatments resulted in an approximate 2-fold decrease in net secretion of etoposide. On the other hand, the specific MRP1 inhibitor MK571 (Gekeler et al., 1995) did not affect etoposide-polarized transport and hence, net secretion (Table 1). As expected, mannitol flux across Calu-3 cells was not polarized, typically giving <1%/h. It should be emphasized, however, that the transport of a single concentration of etopside was investigated, and a positive control for MK571 inhibition of MRP1-mediated efflux was not used.

Discussion

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; Essodı̈agui 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 (k₋₃). 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 constantsk₋₁ andk₋₂.

The results of Fig. 3 can be interpreted in light of this kinetic scheme. Calcein accumulation implies that the processes involved in the production of intracellular calcein (k₁,k₂) are more rapid than those that deplete intracellular calcein (k₋₃) or its precursor, C-AM (k₋₁,k₋₂). In the absence of MDR inhibitors, the observed rate of calcein accumulation (k_obs) is 1783 millifluorescence units per minute per microgram of protein. The Pgp inhibitors vinblastine and CsA increase k_obs 2- to 4-fold, consistent with a significant reduction in the rate of C-AM efflux via Pgp (k₋₁). No effect onk_obs was observed with indomethacin, suggesting either ineffective MRP1 inhibition by this compound or that Pgp efflux is the predominant route of C-AM removal. An increasedk_obs in energy-depleted cells indicates that some or all of the processes involved in calcein accumulation are energy-dependent (k₋₁,k₋₂,k₋₃). That calcein accumulation in these energy-depleted cells is not increased to the level of CsA-exposed cells may reflect the relative insensitivity of Pgp and/or MRP1 to azide inhibition compared with other ATPase inhibitors (e.g., vanadate), which have previously been reported (Al-Shawi and Senior, 1993; Senior et al., 1995). Decreased enzymatic conversion of C-AM to calcein (k₂) due to energy depletion may also be involved.

This kinetic scheme can also be used to interpret the effects of inhibitors on calcein efflux (Fig. 4, a and b). The calcein efflux profile in the control study (C-AM) represents the summation ofk₋₁,k₋₂, andk₋₃, in addition to other secretory pathways that may exist in these cells. In the presence of CsA, higher levels of calcein efflux were observed during the first 30 min. This is unlikely explained by MRP1 activity because calcein efflux was unchanged in the presence of MRP1 inhibitors during this period. On the other hand, the efflux profile was similar to the control during the last 60 min of efflux, implying a lack of Pgp involvement in the latter part of efflux. These data are consistent with complete intracellular conversion of C-AM to calcein during the loading phase, so that only MRP1-mediated removal of calcein (k₋₃) was active during the efflux phase. Decreased rates of efflux in the presence of MRP1 inhibitors support the involvement of MRP1 in calcein efflux in these studies. Note that each of the combinations contains at least one MRP1 inhibitor, and combinations including the Pgp inhibitor CsA are comparable with that without CsA. Again, this is consistent with a lack of Pgp involvement in the efflux phase.

The increased residual calcein levels observed with CsA is consistent with the inhibition of C-AM efflux during the loading phase (k₋₁), resulting in greater conversion to calcein (k₂). Since MRP1 can be involved in the removal of both C-AM and calcein, increases in residual calcein are consistent with either decreased calcein loss during efflux or increased calcein loading. In the presence of both CsA and either MRP1 inhibitor, residual calcein levels are comparable with those observed in the presence of CsA alone. This suggests that greater calcein loading due to Pgp inhibition is the dominant effect, although a slight additional increase in loading and/or a decrease in efflux may be contributed by probenecid. Although MRP1 inhibitors in combination do not increase residual calcein trapping, calcein levels are greatest when CsA, indomethacin, and probenecid are used in combination. Interestingly, the cumulative efflux of calcein is greatest for this combination, suggesting that increased calcein loading is primarily responsible for the high residual calcein levels in this study.

Saturation of Pgp and/or MRP1 transporters may complicate the simple kinetic analysis presented above. A detailed kinetic analysis of C-AM and calcein efflux in both Pgp- and MRP1-expressing cells has been presented by Essodı̈agui et al. (1998). In our studies, the maximum achievable intracellular C-AM in our system was 1 μM (i.e., the loading concentration). Essodı̈agui et al. (1998) report aK_m value for MRP1 mediated C-AM efflux (k₋₂) of 0.05 μM, which suggests that saturation of this pathway could occur under the conditions of our experiments. On the other hand, a K_mvalue for Pgp-mediated C-AM efflux (k₋₁) of 0.12 μM was reported, while MRP1-mediated transport of calcein (k₋₃) occurs with a much lower efficiency (K_m = 268 μM). Assuming similar kinetics of both transporter proteins in the Calu-3 cell model, a saturating concentration at k₋₃ in our experiments could never be achieved, given the apparentK_m. Furthermore, they report the transport efficiency (k_a) of C-AM via Pgp to be approximately 3-fold greater than that of MRP1, while that for calcein via MRP1 is at least three orders of magnitude less than the k_a of C-AM via MRP1. Our observations following MRP1 inhibition are also consistent with transport efficiency values for both calcein species by Pgp and MRP1.

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.

The data also suggest that etoposide efflux in Calu-3 cells is primarily Pgp-mediated, with an apparent Pgp-sensitive/MRP-insensitive quinidine inhibitory component. Etoposide is a known substrate for both Pgp and MRP1 (Makhey et al., 1998) and has been shown to be a substrate for the basolaterally localized organic anion MRP3 efflux pump, but not MRP2 or MRP4 (Chen et al., 1999; Zeng et al., 1999; Lee et al., 2000). It remains to be determined whether any of these MRP1 analogs are expressed and functional in Calu-3 cells.

In conclusion, while the use of C-AM/calcein clearly shows the existence MDR pathways in Calu-3 cells, the presence of functional Pgp in these cells prevents a simple evaluation of the contribution of MRP1 in the efflux of these two molecules. Kinetic modeling of each pathway for the determination of individual rate constants in our cell system certainly warrants further investigation. The mixed Pgp/MRP1 substrate etoposide does not appear to exhibit MRP1 efflux in our system at the concentration tested, suggesting that Pgp dominates in the Calu-3 model. Together with our previous report on functional Pgp (Hamilton et al., 2001) and the basolateral membrane localization of MRP1 in polarized Calu-3 cells, these data would indicate that our cell model provides a useful in vitro system of normal bronchial cells for evaluating efflux pump activity of compounds that are both Pgp and MRP1 substrates. The contribution of other transporter proteins (e.g., MRP3, CFTR, and the lung resistance related protein) in limiting the uptake of MDR substrates in Calu-3 cells remains to be investigated.