Role of MDR1 and MRP1 in trophoblast cells, elucidated using retroviral gene transfer

Diane E. Atkinson,1 Susan L. Greenwood,1 Colin P. Sibley,1 Jocelyn D. Glazier,1 and Leslie J. Fairbairn2

1Academic Unit of Child Health, University of Manchester, St Mary's Hospital, Manchester M13 OJH; and 2Paterson Institute of Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom

Submitted 9 September 2002 ; accepted in final form 24 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [3H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus.

placenta; multidrug resistance; xenobiotic


NEARLY ALL DRUGS and other xenobiotics enter the fetus by transfer across the placenta. Prenatal exposure to such compounds is usually an unwanted consequence of maternal drug therapy, e.g., treatment of epilepsy with anticonvulsants (27), cyclosporin exposure in the case of maternal organ transplant (23), or, more catastrophically, treatment of morning sickness with thalidomide (22). Only a few drugs are considered to be unequivocally safe during pregnancy, which imposes severe limitations on maternal gestational drug therapy (21). Maternal exposure to environmental xenobiotics with potentially harmful effects on the fetus is less easy to control, and such compounds are less easy to identify. A few cases have, however, been reported in which a clear link has been established between the presence in the environment of a xenobiotic and deleterious effects in the fetus, e.g., the pesticide hexachlorobenzene (19). In addition to drug treatment and environmental xenobiotics, there is a wide range of "recreational" xenobiotics to which the fetus may be exposed, e.g., alcohol, nicotine, and drugs of abuse, all of which have potentially harmful effects.

From an evolutionary point of view, there will be a selective advantage in having the ability to maintain low levels of naturally occurring xenobiotics in the fetus, and, therefore, one would expect a mechanism to have evolved to achieve this. In other tissues, both P-glycoprotein (P-gp, encoded by the MDR1 gene) and MRP1 (multidrug resistance-related protein, encoded by the MRP1 gene) have been shown to transport a wide variety of natural product drugs (18). This transport is ATP-dependent, can occur against a concentration gradient, and is independent of electrochemical transmembrane potential or proton gradients. Substrates for these transporters show no structural similarity, but most are of natural origin (e.g., derived from plants, bacteria, fungi, sponges, etc.) or minor variants of natural products (24).

Overexpression of MDR1 in response to treatment of cancer with one cytotoxic agent leads to resistance, not only to the original agent but also to a wide range of structurally unrelated compounds. The presence of P-gp in a range of classic pharmacological barriers and excretory tissues, e.g., blood brain barrier and kidneys, together with its known vectorial transport capacity and the nature of the compounds transported, has led to the hypothesis that this protein may be involved in protection of the organism from environmental xenobiotics. The expression of MDR1 in placenta (17) may indicate such a role in protecting the fetus from potentially harmful environmental compounds. In mice, the absence of mdr1 has been linked to increased teratogenic effects of avermectin L-652,280 (14), and targeted disruption of the mdr1 gene results in increased fetal accumulation of MDR1 substrate drugs (25).

The discovery of multidrug resistance in cancer cells that did not overexpress P-gp led to the discovery of a second protein, MRP1 (4), that can handle a range of naturally occurring compounds similar to P-gp. This protein belongs to the ATP-binding cassette superfamily, along with P-gp, and has a widespread expression (8). In addition to handling a wide range of natural product drugs, it also has many endogenous substrates, e.g., cysteinyl leukotriene LTC4 and bilirubin (6). A major difference between P-gp and MRP is the involvement of glutathione in the transport of many MRP1 substrates. Like MDR1, the ability of MRP1 to transport a wide range of natural product drugs, coupled with its expression in placenta (8, 26), could indicate a role for this protein in protecting the fetus from environmental toxins.

We hypothesized that the products of both the MDR1 and MRP1 genes contribute to xenobiotic handling by the trophoblast and that the net effect will be determined by their localization. To test this hypothesis, we have exploited the natural differences in expression of these two drug-transporting proteins in the choriocarcinoma, trophoblast-derived cell lines BeWo, JAr, and JEG and primary cytotrophoblast cells isolated from normal term placenta to elucidate the relative contribution of both proteins to xenobiotic handling in trophoblast cells. In addition, we have used retroviral gene transfer of MDR1 to BeWo cells, which show little or no natural expression of P-gp, to further establish the role of this protein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Cytotrophoblast Cells

The syncytiotrophoblast is the multinucleated transporting epithelium of the placenta with microvillous (maternal facing) and basal (fetal facing) plasma membranes differentially expressing transport proteins, which effect vectorial transcellular transport. Cytotrophoblast cells isolated from the placenta are used as models of syncytiotrophoblast because they multinucleate after 66 h in culture (1113).

The method of Kliman et al. (13), as adapted in this laboratory (11, 12), was used to isolate cytotrophoblast cells from normal term placenta. Placentas were obtained after uncomplicated pregnancies and were processed within 40 min of delivery. Placental villous material was dissected and subjected to three sequential 30-min trypsin digests (0.25% trypsin; Boehringer Mannheim, Lewes, UK). After each digest, the supernatant was removed, layered over heat-inactivated newborn calf serum (Sigma, Poole Dorset, UK), and centrifuged at 1,100 g for 10 min. The resultant pellets were resuspended in Dulbecco's modified Eagles medium (DMEM; Sigma), layered on to discontinuous Percoll gradients (10–70%), and centrifuged at 1,800 g for 30 min. Cytotrophoblast cells band between 35 and 55% Percoll, and these bands were collected from each gradient, pooled, and centrifuged at 1,800 g for 10 min. The cytotrophoblast pellet was then resuspended in 2 ml of culture medium [50:50 DMEM-Ham-F12 (Sigma), 10% fetal calf serum (Invitrogen, Paisley, UK), 0.12% benzyl-penicillin, 0.2% streptomycin, 0.6% glutamine, and 0.005% gentamycin], and the cells were plated out in P35 culture dishes at ~4–5 x 106 cells per dish. Cells were maintained at 37°C in 5% CO2 and 95% humidity, and the medium was changed every day. The cells were used at either 18 (mononuclear) or 66 h, at which time they had formed fully differentiated, multinucleated cell islands (12).

Cell Culture

The three choriocarcinoma cell lines BeWo, JAr, and JEG and T47D cells (obtained from ECACC, Salisbury Wiltshire, UK) were maintained in 75-cm2 culture flasks under the same conditions of culture as described above. On reaching confluency, cells were passaged by treating with 0.05% trypsin-EDTA solution (Invitrogen) and resuspended in culture medium before transfer of an aliquot to a new flask. For accumulation experiments, cells were plated out into P35 culture dishes and used 2 days after subculturing.

The retroviral packaging cell lines GPAM MDR (gene accession no. M-14758) and GPAM GFP (gene accession no. M-62653) were obtained from Dr. Leslie Fairbairn. These were maintained in 25-cm2 culture flasks in DMEM (Sigma) with 10% new born calf serum (Invitrogen), 0.12% benzyl-penicillin, 0.2% streptomycin, 0.6% glutamine, and 0.005% gentamycin. When cells reached confluency, they were treated with 0.1% trypsin (Roche Diagnostics, Lewes, UK) and resuspended in medium, and an aliquot was transferred to new culture flasks.

RT-PCR

Total RNA was extracted using the solvent method described by Chomzynski and Sacchi (3) from at least three passages of each choriocarcinoma cell line and from cytotrophoblast cells maintained in culture for 18 and 66 h (isolated from three placentas in each case). RNA was also extracted from T47D cells, a breast cancer cell line that overexpresses MRP1, used here as a positive control for this gene. Integrity of the RNA was confirmed by the presence of 28S and 18S ribosomal RNA bands on electrophoresis through 1.2% agarose/6.3% formaldehyde gels. Placental RNA was a pooled sample from four placentas. Kidney total RNA (used as a positive control) was obtained commercially from Clontech (Basingstoke, Hants, UK).

Expression of MDR1 and MRP1 was determined by RT-PCR as described by Mylona et al. (16). Reverse transcription was followed by 30 cycles of PCR using gene-specific, intron-spanning primers: 5' (5'-GCTCAGAGTTTGCAGGTACC-3'; bases 2,761–2,780): 3' (5'-TCCTTCCAATGTGTTCGGCA-3'; bases 3,080–3,099) of the human MDR1 gene (2, 16), and 5'(5'-AACTGCCTTGGGATTTTTGC-3', bases 418–437): 3'(5'-CAGCCACAGGAGGTAGAGAG-3', bases 1,557–1,576) of the human MRP1 gene (4). Annealing temperatures of 59 and 57°C and Mg2+ concentrations of 1.5 and 2.0 mM were used for MDR1 and MRP1, respectively. cDNA was replaced by water in negative control. Restriction enzyme digests confirmed the identities of the PCR products.

Western Blot Analysis

Protein extraction from cells. Four 75-cm2 flasks of each choriocarcinoma cell line or all cells obtained from a single cytotrophoblast cell preparation were used to extract protein for Western blotting. Two different extraction protocols were employed. First, a simple cell lysate was prepared: cells were washed in ice-cold PBS without Ca2+ or Mg2+, placed on ice in PBS for 1 h, scraped, and then homogenized by being passed through a 25G needle. The homogenate was then centrifuged at 4,000 g for 10 min. The pellet was resuspended in PBS and stored at –80°C. Second, a magnesium precipitation step was included in the extraction protocol. Again, the cells were washed in ice-cold PBS without Ca2+ or Mg2+ and placed in mannitol buffer (300 mM mannitol, 10 mM HEPES-Tris, 1 mM Mg SO4) on ice for 1 h. The cells were then scraped and homogenized by being passed through a 25G needle. MgCl2 (1 mM) was added to a final concentration of 10 mM and stirred on ice for 10 min. Centrifugation was then performed at 3,000 g for 15 min. Both the supernatant and the pellet were retained. The supernatant was recentrifuged at 60,000 g for 30 min, and the resultant pellet was retained. Both pellets were then resuspended in mannitol buffer and stored at –80°C until used for immunoblotting.

Microvillous and basal plasma membranes used for localization of the two proteins in placenta were prepared in this laboratory by Dr. Paul Speake according to the methods of Glazier et al. (9, 10). Membranes prepared from four different placentas were used in each case. Purity of the membranes was assessed according to established criteria (9, 10). Alkaline phosphatase enrichment in the microvillous preparations used in this study was 16.2 ± 1.3, whereas DHA (di hydroalprenolol) binding enrichment for the basal preparations used was 27.7 ± 1.2. The degree of cross contamination obtained in this laboratory is typically <5% as estimated by alkaline phosphatase enrichment in basal membranes and DHA binding enrichment in microvillous membranes (Speake P, personal communication).

The protein concentration of all cell lysates was determined using a Bio-Rad Protein assay (Bio-Rad, Richmond, CA), whereas protein concentration of the membrane preparations was established using the Lowry method.

Gel electrophoresis and probing. Placental membranes (20 µg) and 10 µg of cell protein were mixed with a loading buffer containing 8 M urea, 5% SDS, 0.04% bromphenol blue, and 455 mM DTT in 50 mM Tris · HCl (pH 6.9). These were resolved on 6.5% polyacrylamide gels and transferred to Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes. The membranes were blocked for 1 h using 3% dried milk powder in 0.05% Tween TBS (Blotto). They were then incubated for 1 h with primary antibody, i.e., 1:500 clone F4 (Sigma) or 1:50 C219 (Alexis, Nottingham, UK) for P-gp and 1:250 MRPm6 (Chemicon, Harrow, UK) or 1:20 MRPr1 (Alexis) for MRP1. All antibodies were prepared in Blotto. For clone F4 and MRPm6, three 10-min washes were followed by incubation with a horseradish peroxidase (HRP)-conjugated sheep anti-mouse secondary antibody (1:1,000 Amersham Life Science) for 1 h. After three more washes in TBS, the membranes were developed for 1 min in ECL reagent (Amersham Pharmacia Biotech, Little Chalfont, UK). For the C219 and MRPr1 antibodies, an avidin biotin amplification step was included using an ABC kit (Vector Laboratories, Burlingame, UK) before development of the membranes with ECL.

Immunohistochemistry. Placental tissue was collected and small pieces were dissected within 30 min of delivery. These were fixed overnight in 4% formaldehyde in PBS and then paraffin embedded. Sections (4 µM) were dewaxed in xylene, and endogenous peroxidase activity was blocked using 0.5% hydrogen peroxide in methanol. Sections were then blocked using DAKO protein block (DAKO, Ely, UK) for 10 min and incubated with primary antibody overnight at 4°C (MDR1 clone F4 1:20, MRPm6 1:20).

The next day, a biotinylated secondary rabbit anti-mouse antibody was applied for 1 h at room temperature (1:200, DAKO), followed by 0.5 µg/ml avidin peroxidase in 0.125M TBS. The sections were then placed in a bath of 0.05% diaminobenzidine tetrahydrochloride dihydrate (DAB), with 0.015% hydrogen peroxide for 5 min. Methyl green (0.25%) was used to counterstain. Sections were then viewed using a Leitz Dialux 22 microscope fitted with a Sony DXC930P camera at a magnification of x400. Negative controls were processed in exactly the same way, but TBS replaced the primary antibody during the 4°C incubation.

Functional Studies

[3H]vinblastine accumulation. Intracellular accumulation of [3H]vinblastine, a substrate of both MDR1 and MRP1 in the presence and absence of cyclosporin A [an inhibitor of both transporters (5, 28)], was investigated in all cell types. The cells were initially washed in Tyrode solution [135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 (6 H2O), 10 mM HEPES, 5.6 mM glucose, pH 7.4, 300 mosmol/kgH2O] at 37°C and allowed to equilibrate for 30 min. Accumulation was initiated by applying 1 ml of Tyrode solution containing 0.2 µCi/ml (17.7 µM) [3H]vinblastine (Amersham Pharmacia Biotech), either with or without 20 µM cyclosporin A (Sigma). This dose of cyclosporin A had been previously determined in a preliminary series of experiments to give a maximal inhibition (1). Accumulation was stopped at either 5, 15, 30, 60, or 120 min by removal of extracellular radiolabeled vinblastine by washing with 25 ml of ice-cold Tyrode solution over 30 s. Duplicate determinations were made at each time point. One milliliter of 0.3 M NaOH was then added to lyse the cells. Radioactive content of the lysate was measured using liquid scintillation, and protein content was assayed using the Bio-Rad protein assay. Accumulation was then calculated as picomoles per milligram of protein.

Retroviral gene transfer of MDR1 to BeWo cells. A retroviral vector based on the Moloney murine leukemia virus was used to achieve a stable transduction of the BeWo choriocarcinoma cell line with MDR1 (7). The packaging cell line for the MDR1 vector, GPAM-MDR, was seeded into the lower compartments of a Transwell plate (Costar, High Wycombe, UK) and cultured for 4 days to allow cell numbers to increase. BeWo cells were then seeded into the Transwell inserts and cocultured for a further 4 days until confluent. The pore size of the Transwells (0.4 µm) allows virus particles to pass through from the lower compartment without cellular contamination. Once the BeWo cells were confluent, they were removed from the Transwell insert using 0.05% trypsin EDTA and placed in a 75-cm2 culture flask and cultured as described above.

To establish a culture with a high proportion of MDR1-transduced cells, the BeWo MDR culture, when confluent, was treated with 100 µg/ml of colchicine. This concentration had previously been established by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay to give a 10% survival rate of nontransduced BeWo cells. Having selected for transduced cells, the surviving cultures were then maintained as described for the choriocarcinoma cell lines.

Statistics

Data are shown as means ± SE; n values for the choriocarcinoma cell lines refer to determinations performed in separate passages of cells and for cytotrophoblast cells refer to isolates of cells from separate placentas. Statistical analysis of differences between cell types was performed using a two-way ANOVA.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
mRNA Expression

In placenta, cytotrophoblast cells, and JAr choriocarcinoma cells, RT-PCR with MDR1 specific primers showed a cDNA product of 340 bp, as expected for this transcript. However, no product was detected in BeWo or JEG cells after 30 cycles of PCR (Fig. 1A).



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Fig. 1. Expression of MDR1 (A) and MRP1 (multidrug resistance-related protein) (B) mRNA in T47D (T), JEG (J), BeWo (B), JAr (R), 18-h cytotrophoblast (E), and 66-h cytotrophoblast (S) cells and in human placenta (P) and kidney (K). One-hundred base pair ladder (L) and negative control (–ve) are shown.

 

RT-PCR using MRP1 specific primers showed a cDNA product of the expected size, 1,159 bp, in placenta and in all cell types studied (Fig. 1B).

Protein Expression

Western blot analysis. Protein expression, as detected by Western blot analysis, followed the patterns observed for mRNA expression (Fig. 2). MRP1 was found to be ubiquitously expressed (Fig. 2B), whereas P-gp was expressed strongly in JAr and cytotrophoblast cells, with only a very faint signal observed in BeWo and no detectable signal in JEG cells (Fig. 2A).



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Fig. 2. Western blots showing expression of (A) P-gp and (B) MRP1 in 18 (18 cyt)- and 66-h cytotrophoblast (66 cyt), JAr, JEG, and BeWo cells and microvillous (MVM) and basal membranes (BM).

 

To establish where the two proteins are expressed in the placental trophoblast, Western analysis was performed on purified microvillous and basal membranes isolated from term placenta. As shown in Fig. 3A, a clear polarization of P-gp expression between the two syncytiotrophoblast membranes was noted, with P-gp being localized predominantly on the microvillous membrane. In contrast, MRP1 (Fig. 3B) expression in basal membranes was stronger than that in microvillous membranes. To establish the significance of these differences, densitometry was performed using a Bio-Rad model G5700 imaging densitometer running Molecular Analyst version 1.5. MDR1 expression on MVM and BM gave optical densities of 9.12 ± 1.8 and 1.065 ± 0.04, respectively (P < 0.005, t-test), whereas MRP1 expression on MVM and BM gave optical densities of 2.3 ± 0.3 and 8.8 ± 1.4, respectively (P < 0.005, t-test).



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Fig. 3. Western blots showing localization of P-glycoprotein (P-gp) to the microvillous plasma membrane (MVM) (A) and MRP1 to the basal plasma membrane (BM) (B) in human placenta. Four different membrane preparations are shown in each case.

 

Immunohistochemistry. To further investigate placental localization of the two proteins, immunohistochemistry was performed on fixed placental sections taken from four separate placentas. As shown in Fig. 4A, P-gp is predominantly expressed on the microvillous membrane of the term human placenta. In comparison, MRP1 (Fig. 4, B and C) is less discretely localized, showing a diffuse distribution throughout the syncytium and fetal endothelium. No staining was apparent in negative controls (Fig. 4D)



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Fig. 4. Immunohistochemistry showing expression of P-gp (MDR clone F4 1:20) (A) and MRP1 (MRPm6 1:20) (B and C) in term human placenta. Negative controls are shown in D. FE, fetal endothelium; BM, basal membrane; MVM, microvillous membrane.

 

Functional studies. P-gp and MRP1 are efflux pumps, which function to reduce the intracellular accumulation of a wide range of substrates. Inhibition of such multidrug efflux pumps would therefore result in an increased intracellular accumulation of such compounds. In all cell types studied here, accumulation of [3H]vinblastine increased toward a steady state at 120 min (Fig. 5), and cyclosporin A significantly increased accumulation, i.e., inhibited efflux, in all cases (P < 0.0001, two-way ANOVA).



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Fig. 5. Accumulation of [3H]vinblastine by 66 h cytotrophoblast cells (A), JAr (B), BeWo (C), and JEG (D) cells in the presence ({blacktriangleup}) and absence ({blacksquare}) of 20 µM cyclosporin A. Data are means ± SE of at least 3 passages of cells. Cyclosporin A, which inhibits the efflux pumps, was associated with a significantly higher accumulation of 3[H]vinblastine in all cell types (P < 0.0001, two-way ANOVA).

 

To compare between different cell types, the cyclosporin-sensitive accumulation of [3H]vinblastine was calculated for all cells as the difference between accumulation in the presence of cyclosporin A and that under control conditions. Accumulation under control conditions was not significantly different between the four cell types. However, the effect of cyclosporin A was significantly greater in cells with low expression of P-gp. Figure 6 shows data for 66 h cytotrophoblast and JAr cells, which express both MDR1 and MRP1, compared with BeWo and JEG cells, which express predominantly MRP1. There is a significant difference in cyclosporin-sensitive accumulation between the cell types (P < 0.03, two-way ANOVA), with cyclosporin-sensitive accumulation at 120 min in JEG cells being 1.8 times and in BeWo 1.4 times that in cytotrophoblast and JAR cells. This is consistent with the expression of both efflux pumps by the later cell types. It is pertinent to note that JEG cells, which have no detectable expression of P-gp, have the highest cyclosporin-sensitive accumulation, whereas cytotrophoblast and JAr cells, which express abundant P-gp, have the lowest accumulation. BeWo cells, in which low levels of P-gp have been detected at Western analysis, have an intermediate cyclosporin-sensitive accumulation. It can therefore be seen that there is a significant effect of having a reduced expression of MDR1, with cyclosporin-sensitive accumulation of [3H]vinblastine being higher in cells that have little P-gp.



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Fig. 6. Comparison of cyclosporin-sensitive accumulation of [3H]vinblastine by cells expressing both MDR1 and MRP1 (open symbols) with that by cells expressing predominantly MRP1 (closed symbols). Data are means ± SE of at least 3 passages of cells. Cyclosporin-sensitive accumulation was lower in 66-h cyts and JAr cells compared with BeWo and JEG cells (P < 0.03, two-way ANOVA).

 

Effect of MDR1 Transduction of BeWo Cells

Western blots carried out on protein extracted from BeWo cells that had been transduced with MDR1 (BeWo MDR) demonstrated that transduction was successful, because these cells expressed abundant P-gp by comparison to the native BeWo cells (Fig. 7A). In contrast, MRP1 expression was similar between the two cell types (Fig. 7B).



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Fig. 7. Western blots showing expression of P-gp (A) and MRP1 (B) in native BeWo cells and BeWo cells transduced with MDR1 (BeWo MDR).

 

BeWo MDR cells showed accumulation of [3H]vinblastine with the same time course as noted for all other cells included in this study. Cyclosporin A significantly increased accumulation, i.e., inhibited efflux as predicted (Fig. 8).



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Fig. 8. Accumulation of [3H]vinblastine into BeWo MDR cells in the presence and absence of cyclosporin A. Cyclosporin A significantly increased accumulation, i.e., reduced efflux (P < 0.001, two-way ANOVA)

 

Cyclosporin-sensitive [3H]vinblastine accumulation in the transduced, BeWo MDR cells was significantly lower than in native BeWo cells (Fig. 9), i.e., there was a significantly higher cyclosporin-sensitive efflux after transduction with MDR1 (P < 0.01, two-way ANOVA), which was comparable to that in JAr and cytotrophoblast cells.



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Fig. 9. Cyclosporin-sensitive accumulation of [3H]vinblastine into native BeWo cells and BeWo MDR cells. Data are means ± SE, n = 5. There is a significant difference between the 2 cell types in their response to cyclosporin A (P < 0.006, two-way ANOVA).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both MDR1 and MRP1 have been shown to confer multidrug resistance by extruding a wide range of structurally unrelated compounds from cells in which they are expressed (18, 24). Several reports have shown that both P-gp (1517) and MRP1 (8, 26) are expressed by the human placenta. Furthermore, recent reports have suggested that MDR1 in placental trophoblast has a functional role in xenobiotic handling (17, 29, 30). The data presented here support our hypothesis that both P-gp and MRP1 contribute to xenobiotic efflux by trophoblast cells.

At the mRNA level, MRP1 was expressed in placenta, BeWo, JEG, and JAr choriocarcinoma cell lines and in 18- and 66-h cultured cytotrophoblast cells. This pattern is different from that for MDR1, which was expressed in placenta, JAr, and cytotrophoblast cells but had a very low level of expression in BeWo and JEG cells. These differences in relative expression were also reflected at the protein level. The low level of expression of MDR1 in BeWo cells shown here contrasts with that reported by Ushigome et al. (29) and Utoguchi et al. (30), who have shown BeWo to have a similar level of expression as cytotrophoblast cells. This difference may well be explained by our use of a different clone of the BeWo cell line, illustrating the need to specifically characterize such clones in terms of their repertoire of transport proteins.

Using Western blot analysis of microvillous and basal plasma membranes, we found that P-gp showed a polarized distribution to the microvillous membrane of the syncytiotrophoblast. This confirms previous observations (26, 29). In contrast, using this technique, we found MRP1 to be localized predominantly on the basal plasma membrane of the syncytiotrophoblast. Immunohistochemical analysis confirms the localization of MDR1 on the microvillous membrane. The immunohistochemistry, however, suggests a less discrete distribution of MRP1 with staining throughout the syncytium and fetal endothelium. This is in good agreement with St-Pierre et al. (26), who reported MRP1 predominantly on the fetal endothelium, with weak staining in the syncytium. Expression of MRP1 on the basal plasma membrane of the syncytiotrophoblast is in good agreement with its expression on the basolateral membrane of other epithelia (20).

Although cyclosporin A is a potent inhibitor of P-gp, it has also been reported to inhibit MRP1 (5, 28). Using radiolabeled vinblastine as a substrate for both of these transport proteins and cyclosporin A as an inhibitor, we found multidrug resistance activity to be present in all trophoblast cell types studied, irrespective of the level of expression of MDR1/P-gp. Hence, cyclosporin A increases accumulation of vinblastine by inhibiting efflux via both P-gp and MRP1. Using the natural differences we observed in P-gp expression between BeWo and JEG (minimal P-gp expression) and JAr and cytotrophoblast cells (higher P-gp expression), we were able to study the contribution of MDR1 to xenobiotic efflux from trophoblast cells. Cyclosporin-sensitive accumulation of vinblastine by JAr and cytotrophoblast cells was lower than by both BeWo and JEG cells. We suggest that this was due to the lower expression of MDR1 in the latter cells, resulting in a lower capacity to efflux vinblastine. This hypothesis was confirmed by transduction of BeWo cells with MDR1, because this resulted in a cyclosporin-sensitive accumulation that was reduced to the level seen in both JAr and cytotrophoblast cells.

MRP1 is expressed in placenta as shown here and elsewhere (14) and, from its known properties, undoubtedly contributes to the vinblastine efflux seen here. However, other MRP genes have been shown to be expressed in placenta (14) and could also theoretically contribute to vinblastine efflux.

The data presented here suggest that in the normal syncytiotrophoblast, which is derived from cytotrophoblast cells, both P-gp and MRP1 can contribute to xenobiotic handling. However, the polarization of expression of these two drug-transporting proteins in the syncytiotrophoblast with P-gp on the maternal facing, microvillous plasma membrane of the trophoblast and MRP1 predominantly (though not exclusively) on the fetal facing, basal plasma membrane, as well as in the fetal capillary endothelium, suggests very different roles in vivo. P-gp is ideally situated to provide a protective role for the fetus. Xenobiotics in the maternal plasma will be prevented from reaching high concentrations in the syncytiotrophoblast due to active efflux by P-gp. Because MRP1 has a wide range of endogenous substrates, e.g., leukotrienes, etc., it is likely that normally its primary role in placenta involves these rather than xenobiotics. Importantly, though, in circumstances where xenobiotics have reached the fetal circulation, MRP1 would be likely to concentrate them in this compartment, exacerbating deleterious effects on placenta and fetus.


    DISCLOSURES
 
This work was funded by the Medical Research Council and Action Research.


    ACKNOWLEDGMENTS
 
We thank Dr. Paul Speake (Academic Unit of Child Health, University of Manchester, UK) for the donation of placental membranes, Milly Cretney for technical assistance in maintaining cell lines, and Gary Ashton for assistance in preparation of placental sections.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. E. Atkinson, Academic Unit of Child Health, Univ. of Manchester, St Mary's Hospital, Hathersage Rd., Manchester M13 OJH, United Kingdom (E-mail: diane.e.atkinson{at}man.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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