Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo cells

Regulation of BCRP/ABCG2 Expression By Progesterone And 17β-Estradiol in

Human Placental BeWo Cells

Honggang Wang, Lin Zhou, Anshul Gupta, R. Robert Vethanayagam, Yi Zhang,

Jashvant D. Unadkat, and Qingcheng Mao

Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle,

WA 98195-7610

Running title: Regulation of BCRP by progesterone and 17β-estradiol in BeWo cells

Corresponding author:

Dr. Qingcheng Mao

Department of Pharmaceutics

University of Washington

Seattle, WA 98195-7610

Phone: (206) 685-0355

Fax: (206) 543-3204

Email: [email protected]

1

Abstract

The breast cancer resistance protein (BCRP) is abundant in the placenta and

protects the fetus by limiting placental drug penetration. We hypothesize that pregnancy-

specific hormones regulate BCRP expression. Hence, we examined the effects of

progesterone (P4) and 17β-estradiol (E2) on BCRP expression in the human placental

BeWo cells. P4 and E2 significantly increased and decreased BCRP protein and mRNA,

respectively. Likewise, treatment with P4 and E2 respectively increased and decreased

fumitremorgin C-inhibitable mitoxantrone efflux activity of BeWo cells. Reduction in

BCRP expression by E2 was abrogated by the estrogen receptor (ER) antagonist ICI

182,780. However, the progesterone receptor (PR) antagonist RU 486 had no effect on

P4-mediated induction of BCRP. P4 together with E2 further increased BCRP protein and

mRNA, compared with P4 treatment alone. This combined effect on BCRP expression

was abolished by RU 486 or ICI 182,780 or both. Further analysis revealed that E2

significantly decreased ERβ mRNA, and strongly induced PRB mRNA in a dose-

dependent manner, but had no effect on PRA and ERα. P4 alone had no significant effect

on mRNA of ERα, ERβ, PRA and PRB. E2 in combination with P4 increased PRB mRNA,

but the level of induction was significantly reduced compared with E2 treatment alone.

Taken together, these results indicate that E2 by itself likely down-regulates BCRP

expression through an ER, possibly ERβ. P4 alone up-regulates BCRP expression via a

mechanism other than PR. P4 in combination with E2 further increases BCRP expression,

presumably via a non-classical PR and/or E2-mediated synthesis of PRB.

Keywords: BCRP, hormonal regulation, BeWo cells, progesterone, and 17β-estradiol

2

Introduction

The breast cancer resistance protein (BCRP) is the second member (gene symbol

ABCG2) of the subfamily G of the large ATP-binding cassette (ABC) transporter

superfamily (1, 9, 25). BCRP is highly expressed in many normal tissues, including the

epithelium of the small intestine and the liver canalicular membrane (22). Therefore, in

addition to conferring resistance in cancer cells to chemotherapeutic agents such as

mitoxantrone, topotecan and methotrexate (8, 9, 25, 36), BCRP has been shown to

mediate apically directed drug transport, and play a significant role in absorption,

distribution, and elimination of BCRP substrates (4, 19, 21, 32, 35). Of interest is that

BCRP is also abundantly expressed in the apical membrane of placental

syncytiotrophoblasts (22). Whereas the precise physiologic role of BCRP in the placenta

is still unclear, existing data suggest that BCRP may protect the fetus against toxic

substances/drugs and metabolites by extruding them across the placental barrier. For

example, Bcrp1, the murine homolog of BCRP, has been shown to significantly alter fetal

distribution of topotecan, a BCRP substrate. The fetus/plasma ratio of topotecan was

increased 2-fold in pregnant mice treated with the BCRP inhibitor GF120918 as

compared with the vehicle-treatment control (19).

Distribution of drugs that are BCRP substrates across the placenta therefore may

be altered by factors that can influence BCRP expression in the placenta. Several recent

studies have shown that pregnancy can affect expression and function of ABC

transporters. For instance, expression of P-glycoprotein (P-gp) protein in human placenta

at early gestational stages (13 – 14 weeks) was found to be 2 – 45 times higher than that

at late gestational stages (38 – 41 weeks) (13, 23). Expression and function of multidrug

3

resistance protein 2 (Mrp2) in the liver of pregnant rats decreased to 50% of that of non-

pregnant control rats (5). Thus, the protection of fetuses and drug disposition in general

can be influenced by pregnancy, through changing the expression and function of these

transporters. A recent study by Mathias et al. (23) showed that BCRP expression in

human placenta did not change significantly with gestational age. Since these studies

were preliminary with limited tissue samples, and substantial variation in BCRP

expression (mRNA and protein) was observed, more detailed analysis is needed.

To date, little is known about the molecular mechanism by which expression of

ABC transporters in the placenta is altered by pregnancy. Progesterone (P4) and 17β-

estradiol (E2) are the two most important steroid hormones produced by the human

placenta during pregnancy. Estrogens, including E2, play important roles in regulating the

growth, development, and differentiation of many reproductive tissues. P4 is believed to

be indispensable for the maintenance of pregnancy. Since the concentrations of E2 and P4

continuously increase throughout the course of pregnancy, we hypothesized that E2 and

P4 play a significant role in regulating expression of ABC transporters in human placenta.

Recent studies have indeed demonstrated that E2 is an important determinant in the

regulation of BCRP expression in cancer cells by transcriptional or post-transcriptional

mechanisms (10, 11, 17). The effects of P4 and, particularly, the combined effects of E2

and P4 on BCRP expression have not been reported.

In the present study, we have systematically analyzed the effects of P4 and E2 on

expression and efflux function of BCRP in the model human placental BeWo cells, which

express high levels of endogenous BCRP (2). We found that E2 by itself decreased BCRP

expression and P4 increased BCRP expression. P4 in combination with E2 further

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increased BCRP expression compared with P4 treatment alone. The effects of E2 and P4

on expression of progesterone receptor A (PRA), progesterone receptor B (PRB), estrogen

receptor α (ERα) and estrogen receptor β (ERβ) have also been investigated to explore

the possible contribution of these steroid hormone nuclear receptors in regulating BCRP

expression in BeWo cells. These studies found that some of the steroid hormone nuclear

receptors could be involved in the regulation of BCRP in BeWo cells. Our findings

provide new insights into the regulation of BCRP in the human placenta by pregnancy.

5

Materials and Methods

Materials. Progesterone (P-8783), 17β-estradiol (E-2758) and 17β-hydroxy-11β-[4-

dimethylamino phenyl]-17α-[1-propynyl] estra-4,9-dien-3-one (RU 486) were purchased

from Sigma (St. Louis, MO). 7a,17b-[9-[(4,4,5,5,5-pentafluoropentyl)-sulfinyl]-nonyl]-

estra-1,3,5(10)-triene-3,17-diol (ICI 182, 780) was from Tocris Cookson Inc (Ellisville,

MO). Fumitremorgin C (FTC) was a kind gift from Dr. Susan Bates (NCI, Bethesda,

MD). HPLC grade DMSO was from Fisher Scientific (Pittsburgh, PA) and used as the

solvent to dissolve the above compounds. [3H]-mitoxantrone ([3H]-MX) (1.5 Ci/mmol)

was purchased from Moravek Biochemicals (Brea, CA). The CompleteTM protease

inhibitor cocktail was obtained from Roche Molecular Biochemicals (Mannheim,

Germany). The Laemmli sample buffer and 2-mercaptoethanol were purchased from Bio-

Rad (Hercules, CA). DNase I was obtained from Sigma. BeWo cell line was from ATCC

(Manassas, VA). RPMI 1640 phenol-red free and Gibco Opti-MEM were from Gibco

(Grand Island, NY). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were

from Invitrogen (Carlsbad, CA). Charcoal/dextran-stripped fetal bovine serum was

purchased from HyClone (Logan, UT).

Cell Culture and Whole Cell Lysate Preparation. The BeWo cells were maintained in

RPMI 1640 phenol-red free medium supplemented with 10% FBS and 2 mM L-

glutamine at 37oC in a 5% CO2 humidified incubator. The medium was replaced with

fresh medium every other day. To examine BCRP expression in the BeWo cells treated

with E2 or P4 or both, the cells were first cultured in RPMI 1640 phenol-red free medium

supplemented with 5 % charcoal/dextran-stripped fetal bovine serum for at least 48 h to

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achieve 60 – 70 % confluence. The medium was then replaced with fresh medium, and E2

or P4 at various concentrations was then added into the medium. Cell culture was

continued for an additional 12 h to 72 h with replacement of medium after 48 h. For

studies in which cells were treated with a combination of E2 and P4, the cells were first

primed with E2 at various concentrations for 24 h. The medium was then replaced with

fresh medium, and the cells were incubated with E2 at the same concentrations in the

presence of P4 for 72 h. The cells were then harvested for immunoblotting, mRNA

isolation, or functional assays. Only cells within 8 passages after purchase were used in

these experiments. The concentration of DMSO used in all experiments was 0.1 % (v/v).

No effects of the vehicle on cell viability, BCRP protein and mRNA expression, the

plasma membrane localization of the transporter, and mitoxantrone efflux activity were

observed at this concentration.

For whole cell lysate preparation, the BeWo cells grown in 10-cm dishes were

washed once with ice-cold PBS after hormone treatment, and then harvested by scraping

the cell monolayer in ice-cold PBS. The suspended cells were centrifuged at 400 × g for 5

min at 4oC. The cell pellet was resuspended in 200 µl of lysis buffer (1 M Tris/HCl, pH

7.5, 10% SDS, 5 mg/ml DNase I, 1 M MgCl2, 50 mg/ml PMSF, and protease inhibitor

cocktail). The mixture was placed on ice for 1 h with gentle vortexing every 15 min, then

sonicated on ice using a tip-top sonicator for 20 s, and finally centrifuged at 15,100 × g

for 15 min at 4oC. The supernatant was immediately frozen in liquid N2 in aliquots and

stored at -80oC until use. Protein concentrations were determined by the Bio-Rad Dc

protein assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin as standard.

7

SDS-Polyacrylamide Gel Electrophorsis and Immunoblotting. The protein samples of

whole cell lysates (20 µg each lane) were subjected to immunoblotting using BXP-21

(1:500 dilution), a BCRP-specific monoclonal antibody (mAb) (Kamiya Biomedical,

Seattle, WA) as previously described (15) with the exception that the secondary antibody,

goat anti-mouse HRP-conjugated antibody (Bio-Rad) was used at 1:5,000 dilution. For

detection of β-actin, a mAb specific for human β-actin (Sigma) was used as primary

antibody at 1:50,000 dilution and the goat anti-mouse HRP-conjugated antibody (Bio-

Rad) was used as secondary antibody at 1:25,000 dilution. Relative BCRP protein levels

were determined by densitometric analysis of the immunoblots using the NIH Scion

Image software (Scion Corp., Frederick, MD). β-actin was used as an internal control.

Confocal Microscopy. BeWo cells were seeded at approximately 5 × 104 cells/well in a

four-chamber glass slide (Falcon; BD Biosciences Discovery Labware, Bedford, MA).

Cells were grown and treated with 10-5 M P4 or 10-7 M E2 or vehicle control (0.1% (v/v)

DMSO) for 72 h as described. After treatment, cells were washed twice with PBS at

room temperature. Cells were then fixed with 4% paraformaldehyde in PBS for 30 min,

washed twice with PBS, and incubated in permeabilization buffer (0.2% Triton X-100 in

PBS) at room temperature for 10 min. Cells were then blocked for 90 min in blocking

solution (0.1% Triton X-100/2% FBS) and incubated with BXP-21 (1:250 dilution in

blocking solution) for 1 h at room temperature. After the cells were washed with blocking

solution twice, Alexa Flour 488-conjugated goat anti-mouse IgG (H + L) (Fab’)2

fragment (Molecular Probes) was added (1:1,000 dilution in blocking solution) and

incubated in the dark for 1 h. Cells were then washed twice with PBS and mounted in

8

Fluoromount G (Southern Biotechnology Associates, Birmingham, AL) and observed at

488-nm excitation and 519-nm emission wavelengths using a Leica TCS SPI MP

multiphoton confocal microscope (Leica Microsystems, Exton, PA). The concentration of

DMSO used in all experiments was 0.1 % (v/v).

Total RNA Isolation and Quantitative Real-Time TaqMan RT-PCR Analysis. The

effects of hormone treatment on mRNA expression of BCRP, PRA, PRB, ERα or ERβ

were quantified by TaqMan real-time reverse transcription-polymerase chain reaction

(RT-PCR) as follows. After treatment of the BeWo cells with P4, E2, RU 486 or ICI 182,

780, total cellular RNA was isolated from the cells using the Trizol® reagent (Invitrogen)

according to the manufacturer’s instructions. To eliminate contamination of genomic

DNA, all RNA samples were treated with DNase I (Promega, Madison, WI) and purified

by ethanol precipitation prior to RT-PCR. The concentration of RNA was determined by

measuring optical density at 260 nm. The OD260nm/OD280nm ratios of all RNA samples

were determined to be between 1.7 and 2.0, to ensure that all RNA samples are highly

pure. RNA integrity was examined by agarose gel electrophorsis. Single-strand cDNA

used for analysis of BCRP was then synthesized from 0.5 µg of purified total RNA using

a TaqMan® reverse transcription kit (Applied Biosystems, Branchberg, NJ) and single-

strand cDNA used for analysis of PRA, PRB, ERα or ERβ was synthesized from 2.5 µg of

purified total RNA using a high capacity cDNA archive kit (Applied Biosystems, Foster

City, CA), all in a volume of 25 µl. The synthesized cDNA was further purified by

ethanol precipitation and dissolved in 25 µl of pure H2O. Real-time PCR reactions were

then performed using a TaqMan® universal PCR master mix on the ABI Prism 7000

9

Sequence Detection System (Applied Biosystems, Foster City, CA). All the primers and

specific probes were synthesized by Applied Biosystems. Reactions were carried out in

quadruplicates in a MicroAmp optical 96-well plate in a total volume of 20 µl. Each

reaction mixture contained 10 µl of 2 × TaqMan® universal PCR master mix, 6.1 µl of

sterile Millipore water, 0.47 µl of forward primer (235 nM), 0.47 µl of reverse primer

(235 nM), 0.47 µl of probe (118 nM) and 2.5 µl of reverse-transcription products. PCR

conditions were as follows: 50°C for 2 min; 95°C for 10 min; 95°C for 15 s, 60°C for 1

min (40 cycles). Quantification of relative mRNA levels was carried out by determining

the threshold cycle (CT), which is defined as the cycle at which the 6-carboxyfluorescein

reporter fluorescence exceeds 10 times the standard deviation of the mean baseline

emission for cycles 3 to 10. β-actin was used as an internal control. The mRNA levels of

BCRP, PRA, PRB, ERα or ERβ were normalized to those of β-actin according to the

following formula: CT(BCRP, PRA, PRB, ERα or ERβ) - CT(β-actin) = ∆CT. Thereafter,

the relative mRNA levels of these genes after hormone treatment were calculated using

the ∆∆CT method: ∆CT (test hormone) - ∆CT (vehicle) = ∆∆CT (test hormone). The fold-

changes of mRNA levels of BCRP, PRA, PRB, ERα or ERβ in BeWo cells upon treatment

with respective hormones were expressed as 2-∆∆CT. The primer pairs and probes for

BCRP were 5’-CAGGTCTGTTGGTCAATCTCACA-3’ (forward), 5’-

TCCATATCGTGGAATGCT GAAG-3 (reverse), and 5’-

CCATTGCATCTTGGCTGTCATGGCTT-3’(probe); the primer pairs and probe for PRA

were 5’-AGAGCACTGGATGCTGTTGCT-3’ (forward), 5’-

TGGCTTAGGGCTTGGCTTT-3’ (reverse), and 5’-

CCACAGCCATTGGGCGTTCCAA-3’(probe); the primer pairs and probe for PRB were

10

5’-GCCAGACCTCGGACACCTT-3’(forward), 5’-CAGGGCCGAGGGAAGAGTAG-

3’(reverse), and 5’-CCTGAAGTTTCGGCCATACCTATCTCCCT-3’ (probe); the

primer pairs and probe for ERα were 5’-AGCACCCAGTGAAGCTACT-3’ (forward),

5’-TGAGGCACACAAACTCCT-3’ (reverse), and 5’-

TGGCTACATCATCTCGGTTCCGCA-3’ (probe); the primer pairs and probe for ERβ

were 5’-AAGAATATCTCTGTCAAGGCCATG-3’ (forward), 5’-

GGCAATCACCCAAACCAAAG-3’(reverse), and 5’-

TTGCTGAACGCCGTGACCGATG-3’ (probe). The primer pairs and probe for human

β-actin were purchased from Applied Biosystems (Foster City, CA). The concentration of

DMSO used in all experiments was 0.1 % (v/v).

Intracellular Mitoxantrone (MX) Accumulation Assay. Transport studies using [3H]-

MX were performed to examine whether treatment with P4 and E2 affects MX efflux

activity of the BeWo cells. Briefly, the BeWe cells were seeded at a cell density of

approximately 2 × 105 per well in 6-well plates and treated as described with P4 and/or E2

in the presence and absence of RU 486 and ICI 182,780 at concentrations indicated in

Table 1. After 72 h treatment, cells grown on the cell culture plates as a monolayer were

washed once with pre-warmed PBS and incubated in 1 ml per well of Opti-MEM for 30

min. In inhibition experiments, cells were first incubated with 10 µM FTC for 1 h. The

experiments were then started by the addition of [3H]-MX (20 nM) in the presence and

absence of 10 µM FTC in 1 ml of Opti-MEM and incubation was continued for 30 min to

90 min. The MX efflux was then stopped by washing the cells three times with ice-cold

PBS. The cell monolayer was suspended in 1 ml of 2% (w/v) SDS for whole cell lysate

11

preparation. The whole cell lysates (900 µl) were subjected to counting in a scintillation

counter. Counts were normalized to the protein concentration that was measured by the

Bio-Rad Dc protein assay using the remaining lysates. The intracellular MX

concentrations were calculated based on radioactivity associated with the cells and

presented as pmol of [3H]-MX per mg protein. The difference in intracellular MX

concentrations in the presence and absence of FTC was used as a measure of FTC-

inhibitable MX efflux activity of the BeWo cells. This FTC-inhibitable MX efflux

activity should be attributable to BCRP. Only cells within 8 passages after purchase were

used in the experiments. The experiments were performed in triplicate at 37oC in a

humidified incubator.

Statistical Analysis. Data were analyzed for statistical significance using one-way

ANOVA analysis or Student’s t test. Differences with p-values of < 0.05 were considered

statistically significant.

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Results

Progesterone (P4) Stimulates BCRP Protein Expression. We first examined whether

treatment with P4 or E2 can affect membrane localization of BCRP in BeWo cells with

immunofluorescent confocal microscopy using the BCRP-specific mAb BXP-21. We

found that BCRP was predominantly expressed on the plasma membrane of untreated

BeWo cells with some intracellular expression (Fig. 1A). Treatment with 10-5 M P4 or 10-

7 M E2 for 72 h had no qualitative effect on the plasma membrane localization of the

transporter versus the intracellular compartments (Fig. 1B and Fig. 1C). Thus the levels

of BCRP protein determined in the whole cell lysates should reflect the levels of BCRP

protein on the plasma membrane. We therefore determined BCRP protein expression

using whole cell lysates in all of the subsequent immunoblotting experiments.

To investigate the effect of P4 on BCRP protein expression, the BeWo cells were

treated with P4 over a range of concentrations (10-9 M - 10-5 M) for 48 h. BCRP protein

expression was then analyzed by immunoblotting of whole cell lysates using mAb BXP-

21. Densitometric analysis of the immunoblots revealed that P4 at 10-6 M only slightly

increased BCRP protein expression; however, P4 at 10-5 M significantly increased BCRP

protein approximately 2-fold compared with the vehicle control (Fig. 2A). P4 at

concentrations below 10-6 M had no significant effect on BCRP expression. In contrast,

the expression of β-actin (internal control) was not significantly affected by P4 at any of

the concentrations used. When the effect of P4 on BCRP expression was analyzed at

different treatment times, P4 at 10-5 M was found to increase BCRP expression at 48 and

72 h (Fig. 2B). The inductive effect of 10-5 M P4 on BCRP protein expression could not

be reversed by the addition of the PR antagonist RU 486 at 10-5 M (Fig. 2C). RU 486

13

itself at 10-5 M had no significant effect on BCRP protein expression (data not shown).

We found that the viability of BeWo cells was significantly reduced in the presence of

RU 486 at 5 × 10-5 M or higher concentrations (data not shown). To further clarify the

role of PR on BCRP expression, we performed experiments to examine the effect of RU

486 on BCRP expression at 10 times molar excess to P4. Thus, BeWo cells were treated

for 72 h with 2.5 × 10-6 M P4 in the presence and absence of 2.5 × 10-5 M RU 486.

Similarly, P4 at 2.5 × 10-6 M increased BCRP protein and mRNA expression

approximately 1.7-fold and 1.5-fold, respectively, and the addition of 10 times molar

excess RU 486 had no significant effect on P4-mediated induction of BCRP expression

(Fig. 3). The immunoblots sometimes showed double bands (Fig. 2B). We treated the

protein samples with PNGase F (New England Biolabs, Beverly, MA). This treatment led

to an approximately 10 kDa reduction in the apparent molecular mass of BCRP and the

disappearance of double bands (data not shown). These data suggest that the double

bands are most likely caused by multiple glycosylation on BCRP. Hence, the upper bands

were always included in the densitometric analysis of BCRP expression.

17β-Estradiol (E2) Decreases BCRP Protein Expression. To examine the effect of E2

on BCRP protein, BeWo cells were treated with E2 at various concentrations (10-11 M -

10-7 M) and for the duration of treatment up to 72 h. E2 at 10-8 and 10-7 M significantly

decreased BCRP protein expression after 48 h treatment by approximately 60% and 70%,

respectively; however, β-actin expression was not affected by the same experimental

conditions (Fig. 4A). E2 only slightly decreased BCRP protein expression at

concentrations below 10-9 M. In time course studies, E2 at 10-7 M was found to

14

significantly decrease BCRP protein expression by approximately 60% and 75%, at 48

and 72 h, respectively, but had no effect at 12 and 24 h (Fig. 4B). The inhibitory effect of

E2 at 10-7 M after 72 h treatment was significantly reversed by the addition of 10 times

molar excess (10-6 M) of the ER antagonist ICI 182, 780 (Fig. 4C). ICI 182,780 itself at

10-6 M had no significant effect on BCRP protein expression (data not shown).

BCRP Protein Expression Was Further Increased by P4 in Combination with E2. To

examine the combined effects of P4 and E2 on BCRP expression, BeWo cells were first

primed with E2 at various concentrations (10-9 M - 10-7 M) for 24 h. The cells were then

switched to fresh medium and incubated with E2 at the same concentrations and P4 at 10-6

M or 10-5 M for 72 h. P4 at 10-5 M in combination with E2 at 10-9 M or 10-8 M further

increased BCRP protein expression compared with P4 treatment alone (Fig. 5B). For

example, P4 alone at 10-5 M stimulated BCRP protein expression approximately 2-fold,

whereas P4 at the same concentration with 10-8 M E2 increased BCRP protein

approximately 3-fold (Fig. 5B). Although not statistically significant, this further

stimulation of BCRP protein by 10-8 M E2 was greater than that by 10-9 M E2. However,

further increase of E2 concentration to 10-7 M decreased rather than increased BCRP

expression. Similar effects of the combination of P4 with E2 on BCRP protein were

observed for P4 at 10-6 M (Fig. 5A). The expression of β-actin was not influenced by any

of these treatments. We then explored the combined effects of P4 and E2 on BCRP

expression in the presence of 10-6 M ICI 182,780 and/or 10-5 M RU 486. The further

stimulation of BCRP protein by the combination of 10-5 M P4 and 10-8 M E2 was

completely abrogated by the addition of either ICI 182,780 or RU 486 or both. Treatment

15

with ICI 182,780 or RU 486 decreased BCRP protein expression to 1.9-fold and 2.0-fold

of the vehicle controls, respectively, and brought it down to the same level as for P4 (10-5

M) treatment alone (Fig. 5C). Likewise, treatment with ICI 182,780 and RU 486

decreased BCRP protein expression to 1.7-fold of the vehicle controls (Fig. 5C).

Effects of P4 and E2 on BCRP mRNA. We then examined whether the effects of P4 and

E2 on BCRP protein expression were due to changes on BCRP mRNA levels.

Endogenous BCRP mRNA in BeWo cells could be readily detected by real-time RT-PCR

at approximately 23 cycles (data not shown). Quantitative real-time RT-PCR analyses

revealed that treatment of BeWo cells with P4 alone at 10-5 M significantly increased

BCRP mRNA approximately 1.5-fold compared with the vehicle control (Fig. 6). Similar

results were obtained after treating the BeWo cells for 24 h (Fig. 6A) and 72 h (Fig. 6B).

Although not statistically significant, 10-5 M P4 in combination with 10-8 M E2 further

increased BCRP mRNA compared with P4 treatment alone. After 24 and 72 h treatment,

E2 by itself at 10-7 M significantly reduced BCRP mRNA by approximately 40%. While

the ER antagonist ICI 182, 780 at 10 times molar excess completely reversed the

inhibitory effect of E2 on BCRP mRNA, the PR antagonist RU 486 at the same

concentration (10-5 M) as P4 did not significantly affect the P4-mediated induction of

BCRP mRNA (Fig. 6). RU 486 at 10 times molar excess to P4 (2.5 × 10-5 M RU 486

versus 2.5 × 10-6 M P4) also did not influence the inductive effect of P4 on BCRP mRNA

(Fig. 3C). RU 486 and ICI 182, 780 themselves had no effect on BCRP mRNA

expression (data not shown).

16

Effects of P4 and E2 on BCRP-mediated MX Efflux Activity. To further examine

whether the function of BCRP in BeWo cells is affected by treatment with P4 and/or E2,

we investigated the effects of hormone treatment on MX efflux by the BeWo cells, using

a MX accumulation assay. MX, a high affinity BCRP substrate (29), was used as a model

substrate to measure BCRP transport activity of the BeWo cells. To eliminate possible

contribution of endogenous efflux transporters like P-gp, a relatively specific BCRP

inhibitor FTC was used to determine FTC-inhibitable MX efflux activity. Since 10 µM

FTC used in the assay is sufficient to fully inhibit BCRP (29), the portion of MX efflux

which can be inhibited by 10 µM FTC is attributable to BCRP expression. Similar FTC

modulation of MX efflux has been used to detect BCRP expression in clinical leukemia

samples (33). We first performed time course studies to find the optimal accumulation

time for the efflux assay. The baseline FTC-inhibitable MX efflux activity of BeWo cells

treated with the vehicle control after 60 min accumulation was slightly greater than the

activity after 30 min accumulation; however, further increase of accumulation time to 90

min did not increase the activity (Fig. 7). With all three accumulation times, treatment

with 10-5 M P4 or 10-7 M E2 significantly increased or decreased the FTC-inhibitable MX

efflux activity of the BeWo cells, respectively (Fig. 7), and accumulation for 60 min

seems to produce the most significant difference, as compared with the vehicle control.

Therefore, an accumulation time of 60 min was used in all of the subsequent efflux

experiments. As shown in Table 1, the FTC-inhibitable MX efflux by BeWo cells treated

with 10-7 M E2 was significantly reduced, by approximately 30%, compared with the

vehicle control cells. This reduction of MX efflux by E2 treatment was completely

reversed by the addition of 10-6 M ICI 182,780. Treatment with 10-5 M P4 or 10-5 M P4 in

17

combination with 10-8 M E2 resulted in an approximately 1.2-fold and 1.4-fold increase in

MX efflux, respectively. In particular, the MX efflux activity of the cells treated with 10-5

M P4 in combination with 10-8 M E2 was significantly greater than the activity of the cells

treated with 10-5 M P4 alone. These findings are consistent with the protein and mRNA

data (Fig. 5 and Fig. 6). The addition of 10-5 M RU 486 had no effect on P4-mediated

stimulation of MX efflux (Table 1).

Effects of E2 and P4 on mRNA of PRA, PRB, ERα and ERβ. To investigate the possible

mechanisms by which BCRP expression is regulated by P4 and/or E2 in BeWo cells, we

examined the effects of P4 and/or E2 on mRNA levels of PRA, PRB, ERα and ERβ using

quantitative real-time RT-PCR. To perform this study, BeWo cells were treated with E2

or P4, or a combination of both, the same as described for determining protein expression.

Endogenous expression of PRA, PRB, ERα and ERβ mRNA in untreated BeWo cells was

detected by real-time PCR at 37.1 ± 0.7, 37.6 ± 0.5, 35.0 ± 0.6, and 35.6 ± 0.4 cycles,

respectively, under the current assay conditions. The relative changes of mRNA levels of

these receptors upon hormone treatment were then determined and are summarized in

Table 2. First, E2 by itself at 10-7 and 10-8 M significantly decreased ERβ mRNA by

approximately 40% compared with the vehicle control, but had no significant effect on

ERα mRNA. E2 at 10-7 M did not significantly influence PRA mRNA and increased PRB

mRNA by 60%. However, E2 at 10-8 M strongly induced PRB mRNA approximately 7.5-

fold, but had no significant affect on PRA mRNA. These effects of E2 on mRNA levels of

ERβ and PRB were abrogated by the addition of 10-6 M ICI 182,780. For instance,

addition of ICI 182,780 almost completely reversed E2-mediated reduction of ERβ

18

mRNA. In addition, the 7.5-fold increase in PRB mRNA by 10-8 M E2 was significantly

reduced to only 2-fold by the addition of ICI 182,780. Second, when the effect of P4

alone was measured, P4 at 10-5 M was found to slightly decrease mRNA levels of PRA

and PRB but had no effect on mRNA levels of ERα and ERβ. The addition of 10-5 M RU

486 did not influence the effect of P4 on mRNA levels of these receptors (data not

shown). Third, we measured the combined effects of P4 and E2 on mRNA of PRA, PRB,

ERα and ERβ. A combination of 10-5 M P4 with 10-8 M E2 did not decrease ERβ mRNA,

although E2 alone significantly attenuated ERβ mRNA. The combination of P4 and E2

also had no significant effect on ERα mRNA, but slightly decreased PRA mRNA, and

significantly increased PRB mRNA approximately 2.2-fold.

19

Discussion

The present study examines the effects of P4 and E2 on BCRP expression in

BeWo cells. We found that P4 significantly increased BCRP protein only at a relatively

high concentration 10-5 M (Fig. 2A). The plasma P4 concentration at term was reported to

be approximately 0.7 × 10-6 M and the intracellular P4 concentrations in placenta were

about 12 times greater than those in maternal plasma (20). Hence 10-5 M P4 could be

achieved in the placenta at term. This concentration is much greater than the binding

affinity of P4 to a classical PR (7). Moreover, RU 486, even at 10 times molar excess, did

not inhibit the inductive effect of P4 (Fig. 3). These results suggest that it is unlikely that

induction of BCRP by P4 is mediated by a classical PR. Several studies have

demonstrated novel, non-classical, membrane-bound forms of steroid receptors involving

non-genomic actions of hormones (12, 27). For example, P4 at µM concentrations

stimulated the expression of the steroidogenic acute regulatory protein in Leydig cells by

a non-classical PR (30). Therefore, up-regulation of BCRP in BeWo cells by P4 at µM

concentrations is possibly mediated by a non-classical PR pathway.

The plasma E2 concentration during pregnancy increases steadily to around 0.8 ×

10-7 M at term (3, 20). At concentrations observed during pregnancy (10-8 and 10-7 M), E2

significantly decreased BCRP protein (Fig. 4A). This decrease was abolished by ICI

182,780 (Fig. 4C), suggesting that down-regulation of BCRP by E2 is mediated by ER.

Male-predominant expression of Bcrp1 in rat kidney has been reported (34). The authors

showed that castration had no effect on Bcrp1 mRNA in rat kidney; however, Bcrp1

mRNA in the kidneys of ovariectomized female rats was significantly higher than that of

control females, indicating that male-predominant expression of Bcrp1 in rat kidneys is

20

likely caused by the absence of the suppressive effects of female-sex hormones such as

E2. Male-predominant expression of human BCRP and mouse Bcrp1 in liver has also

been demonstrated (24). These in vivo data seem to support our in vitro findings with

respect to down-regulation of BCRP by E2.

We demonstrated for the first time the combined effects of P4 and E2 on BCRP

expression. It is of considerable interest that E2 at subthreshold doses (10-9 and 10-8 M)

further increased P4-mediated induction of BCRP, even at 10-6 M P4, which by itself

showed little effect (Fig. 2A and Fig. 5A). This finding suggests that placental BCRP

expression could be affected by pregnancy, even at earlier gestational stages when the P4

concentrations are low. Since E2 at 10-8 M with 10-5 M P4 significantly induced PRB

mRNA 2.2-fold (Table 2), this further increase in BCRP expression is possibly mediated

by E2-induced synthesis of PRB. The combination of 10-7 M E2 with 10-5 M P4 decreased,

rather than increased, BCRP expression to the levels of P4 treatment alone (Fig. 5). This

could be explained by the fact that E2-induced synthesis of PRB was significantly

diminished by 10-7 M E2 compared with 10-8 M E2 (Table 2). Hence, both a non-classical

PR (when P4 was used alone) and a classical PR (when P4 and E2 were used together)

may be involved in P4-mediated up-regulation of BCRP. E2 at 10-8 M induced PRB

mRNA 7.5-fold. The decrease in E2-mediated induction of PRB in the presence of P4 is

likely due to the widely observed suppressive effect of P4 on E2 action (28). The addition

of RU 486 or ICI 182,780, or both, abolished this further increase in BCRP protein by the

combination of P4 and E2 (Fig. 5C), further suggesting that endogenous expression of

PRB in BeWo cells is low, and thus PRB exerts its function only after it is induced by E2

through ER.

21

The effects of P4 and E2 on BCRP mRNA in general corresponded well to the

effects on BCRP protein (Fig. 6), suggesting that P4 and E2 regulate BCRP expression, at

least in part, by a transcriptional mechanism. However, the possibility of a post-

transcriptional mechanism cannot be excluded (17). We consistently observed a

significant increase and decrease of the FTC-inhibitable MX efflux activity of BeWo

cells treated with 10-5 P4 and10-7 M E2, respectively, compared with the vehicle controls

(Fig. 7 and Table 1). ICI 182,780 completely reversed the inhibitory effect of E2 and RU

486 had no significant influence on the stimulatory effect of P4. The combination of P4

and E2 further increased MX efflux activity compared with P4 treatment alone. These

activity data in general well reflected the BCRP protein (e.g., 122% increase in activity

vs. 150 – 200% increase in protein by 10-5 M P4 and 30% decrease in activity vs. 60 -

70% decrease in protein by 10-7 M E2) and mRNA data (e.g., 122% increase in activity

vs. 150% increase in mRNA by 10-5 M P4 and 30% decrease in activity vs. 40% decrease

in mRNA by 10-7 M E2). Recently, Imai et al. reported expression of endogenous MX

efflux transporters other than BCRP in LLC-PK1 cells which can be inhibited by FTC

(16). We also noticed the existence of other endogenous efflux transporters for the anti-

HIV protease inhibitors ritonavir and saquinavir in HEK cells inhibited by FTC (15).

Therefore, the relatively smaller effects of P4 or E2 on MX efflux activity are most likely

attributable to the endogenous transporters other than BCRP in BeWo cells whose MX

efflux activity can also be inhibited by FTC. Such endogenous MX efflux transporters

would increase the background of the overall FTC-inhibitable MX efflux activity of the

BeWo cells (which do not have enforced BCRP expression by transfection) and mask the

changes in BCRP-specific MX efflux activity.

22

ERα has been detected in BeWo cells (18). This study, to the best of our

knowledge, is the first to demonstrate expression of PRA, PRB, and ERβ in BeWo cells.

E2 at 10-8 and 10-7 M significantly decreased ERβ mRNA (Table 2). Several studies also

reported down-regulation of ERα and/or ERβ by E2 in various tissues and cell lines (6,

14, 26, 31). Since ICI 182, 780 completely reversed the inhibitory effect of E2 on ERβ

(Table 2), down-regulation of BCRP by E2 is possibly mediated by a transcriptional

mechanism via ERβ. A combination of 10-5 M P4 and 10-8 M E2 did not decrease ERβ

mRNA, although E2 alone significantly attenuated ERβ mRNA. This finding further

supports the notion that E2 alone suppresses BCRP, presumably via ERβ, and P4 in

combination with E2 induces BCRP, possibly via PRB. Thus, P4 and E2 seem to interact

through PRB and ERβ for regulation of BCRP. Studies are now in progress in our

laboratory to elucidate the molecular mechanisms by which P4 and E2 regulate BCRP

expression in BeWo cells through PRB and ERβ.

Similar to the findings of this study, Imai et al. demonstrated down-regulation of

BCRP by E2 in T-47D and MCF-7 breast cancer cells (17). In contrast, Ee et al. reported

stimulation rather than suppression of BCRP by E2 in T-47D and BeWo cells (10, 11).

The reason for this apparent discrepancy is currently unknown. Genetic alterations may

occur in cells after prolonged culture. Hence, BeWo cells only within 8 passages after

purchase were used in this study.

In summary, the present study suggests that 1) P4 and E2 respectively up-regulates

and down-regulates BCRP expression in BeWo cells; 2) The interaction between P4 and

E2, through PRB and ERβ, may play a significant role in the regulation of BCRP in BeWo

cells; 3) Steroid hormones, for example P4, may function through a classical or non-

23

classical PR, or both pathways, in response to specific endocrine status during pregnancy.

Further studies are needed to elucidate the molecular mechanisms by which BCRP

expression is regulated by P4 and E2 in BeWo cells. Such studies will help understand

how pregnancy affects drug distribution across the placenta. It should be pointed out that

the BeWo cell line is not exactly the same as the placental trophoblast with respect to the

expression of ABC transporters, and therefore, care should be taken when extrapolating

the data obtained in this cell line to in vivo human subjects.

24

Acknowledgment The authors thank Drs. Robert W. Robey and Susan E. Bates (NCI, Bethesda, MD) for

providing FTC. We acknowledge Dr. Douglas Ross (University of Maryland, Baltimore,

MD) and Dr. Virendra B. Mahesh (Medical College of Georgia, August, GA) for their

helpful comments on this study. We thank Dr. Ed Kelly, Dr. Carl Ton, Ms. Hiuxia Zhang

and the Center for DNA Sequencing and Gene Analysis (Department of Pharmaceutics,

University of Washington, Seattle, WA) for technical assistance in real-time PCR, and

Greg Martin (the Keck Imaging Center, Department of Pharmacology, University of

Washington, Seattle, WA) for technical assistance in immunofluorescent confocal

microscopy.

Grants:

We gratefully acknowledge financial support from NIH Grant HD044404 (to QM and

JDU) and from the Department of Pharmaceutics, University of Washington.

25

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31

Figure Legends

Figure 1. Confocal microscopy of BeWo cells. BeWo cells were treated with 0.1% (v/v)

DMSO (A), 10-5 M P4 (B), and 10-7 M E2 (C) for 72 h. BCRP protein was then detected

using mAb BXP-21 as described in “Materials and Methods”. Selected areas of BeWo

cells are shown, and BCRP protein expression is indicated in green. Images have been

enhanced for maximal contrast between the black background and green fluorescence and

were not intended for quantitative determination of BCRP expression.

Figure 2. Effects of progesterone (P4) on expression of BCRP protein in BeWo cells.

Relative BCRP protein levels normalized to β-actin were determined as described in

“Materials and Methods”. Data shown are mean ± S.E. from three independent

experiments. The immunoblots shown are the representative results obtained in typical

experiments. The differences in BCRP protein levels are statistically significant: * p <

0.05 as compared with vehicle controls using one-way ANOVA analysis. A, effects of P4

at various concentrations (10-9 M – 10-5 M) on BCRP protein after treatment for 48 h.

The relative BCRP protein levels associated with vehicle controls are set as 1; B, effects

of P4 at 10-5 M on BCRP protein at different treatment times (0 – 72 h). The relative

BCRP protein levels without P4 treatment (time, 0 h) are set as 1; C, effect of 10-5 M RU

486 on P4-mediated induction of BCRP protein after treatment with 10-5 M P4 for 72 h.

Relative BCRP protein levels associated with vehicle controls are set as 1.

32

Figure 3. Effects of RU 486 on P4-mediated induction of BCRP expression. Relative

BCRP protein or mRNA levels normalized to β-actin were determined as described in

“Materials and Methods”. Data shown are mean ± S.E. from three independent

experiments. The differences in BCRP protein or mRNA levels are statistically

significant: * p < 0.05 as compared with the vehicle controls using Student’s t-test

analysis. A, the representative results of immunoblots obtained in a typical experiment.

B, effects of 2.5 × 10-6 M P4 in the presence or absence of 2.5 × 10-5 M RU 486 after 72 h

treatment on BCRP protein expression. C, effects of 2.5 × 10-6 M P4 in the presence or

absence of 2.5 × 10-5 M RU 486 after 72 h treatment on BCRP mRNA expression. The

relative BCRP protein or mRNA levels associated with the vehicle controls are set as 1.

Figure 4. Effects of 17β-estradiol (E2) on expression of BCRP protein in BeWo cells.

Relative BCRP protein levels normalized to β-actin were determined as described in

“Materials and Methods”. Data shown are mean ± S.E. from four independent

experiments. The immunoblots shown are the representative results obtained in typical

experiments. The differences in BCRP protein levels are statistically significant: * p <

0.05; ** p < 0.01 as compared with vehicle controls using one-way ANOVA analysis. ∆ p

< 0.05 as compared with E2 treatment alone using Student’s t-test. A, effects of E2 at

various concentrations (10-11 M – 10-7 M) after treatment for 48 h on BCRP protein. The

relative BCRP protein levels associated with vehicle controls are set as 1; B, effects of E2

at 10-7 M on BCRP protein at different treatment times (0 – 72 h). The relative BCRP

protein levels without P4 treatment (time, 0 h) are set as 1; C, effect of 10-6 M ICI 182,

33

780 on E2-mediated down-regulation of BCRP protein after treatment with 10-7 M E2 for

72 h. Relative BCRP protein levels associated with vehicle controls are set as 1.

Figure 5. Combined effects of progesterone (P4) and 17β-estradiol (E2) on expression

of BCRP protein in BeWo cells. Relative BCRP protein levels normalized to β-actin

were determined in “Materials and Methods”. Data shown are mean ± S.E. from three

independent experiments. The immunoblots are the representative results obtained in

typical experiments. The differences in BCRP protein are statistically significant: * p <

0.05; ** p < 0.01 as compared with the vehicle control cells using one-way ANOVA

analysis. ∆ p < 0.05 as compared with P4 treatment alone and # p < 0.05 as compared with

P4 and E2 combination using Student’s t-test. Shown are effects of combinations of 10-6

M P4 (A) and 10-5 M P4 (B) with E2 at various concentrations, and effects of combination

of 10-5 M P4 with 10-8 M E2 in the presence of RU 486 and/or ICI 182,780 (C) on BCRP

protein. Relative BCRP protein levels associated with vehicle controls are set as 1.

Figure 6. Effects of progesterone (P4), 17β-estradiol (E2), RU 486 and ICI 182, 780

on BCRP mRNA in BeWo cells. Cells were treated with P4, E2, RU486 or ICI 182, 780

as in Figures 2 – 5. Total RNA was isolated from cells and relative BCRP mRNA levels

were determined by real-time RT-PCR as described in “Materials and Methods”. Effects

of 10-5 M P4, 10-5 M P4 plus 10-5 M RU 486, 10-5 M P4 plus 10-8 M E2, 10-7 M E2 and 10-7

M E2 plus 10-6 M ICI 182, 780 on BCRP mRNA levels after 24 h (A) and 72 h (B)

treatment are shown. Relative BCRP mRNA levels normalized to β-actin are presented.

The relative BCRP mRNA levels associated with vehicle controls are set as 1. The data

34

shown are mean ± S.E. from four independent experiments. The differences in BCRP

mRNA levels are statistically significant: * p < 0.05; ** p < 0.01 as compared with the

vehicle controls using one-way ANOVA analysis. ∆ p < 0.05 as compared with E2

treatment alone using Student’s t-test.

Figure 7. Accumulation time-dependence of FTC-inhibitable mitoxantrone (MX)

efflux activity of BeWo cells. BeWo cells were treated with 10-5 M P4 (■) or 10-7 M E2

( ) or 0.1 % (v/v) DMSO vehicle (♦) for 72 h. The FTC-inhibitable MX efflux activities

of the BeWo cells were then measured with various accumulation times (30 min, 60 min,

and 90 min) as described in “Materials and Methods”. The data shown are mean ± S.E.

from three independent experiments. The differences in efflux activities are statistically

significant: * p < 0.05; ∆ p < 0.01 as compared with the vehicle controls at the respective

accumulation times using Student’s t-test analysis.

35

36

Table 1. Effects of progesterone (P4) and 17β-estradiol (E2) on FTC-inhibitable MX

efflux activity of BeWo cells.

Hormone treatment FTC-inhibitable MX efflux activity (%) ________________________________________________________________________ DMSO vehicle control 100 P4 at 10-5 M 122 ± 5* P4 at 10-5 M + RU 486 at 10-5 M 121 ± 10* P4 at 10-5 M + E2 at 10-8 M 139 ± 9** # E2 at 10-7 M 70 ± 9* E2 at 10-7 M + ICI 182,780 at 10-6 M 108 ± 10 ∆

The BeWo cells were treated with P4 and/or E2, and the effects of hormone treatment on

the FTC-inhibitable MX efflux activity of the BeWo cells were determined as described

in “Materials and Methods”. The MX efflux activity of the vehicle control cells are set as

100%. The data shown are mean ± S.E. from three independent experiments. The

differences in MX efflux are statistically significant using Student’s t-test: * p < 0.05, **

p < 0.01 as compared with the vehicle controls; # p < 0.05 as compared with P4 treatment

alone; ∆ p < 0.05 as compared with E2 treatment alone.

37

Table 2. Effects of progesterone (P4) and 17β-estradiol (E2) on mRNA levels of PRA, PRB, ERα and ERβ in BeWo cells. DMSO P4 (10-5) P4 (10-5)+E2 (10-8) E2 (10-7) E2 (10-7)+ICI (10-6) E2 (10-8) E2 (10-8)+ICI (10-6) PRA 1 0.75 ± 0.16 0.71± 0.21 1.03 ± 0.21 0.86 ± 0.14 1.20 ± 0.14 1.02 ± 0.09 PRB 1 0.77 ± 0.16 2.16 ± 0.39* 1.63 ± 0.17* 0.99 ± 0.13 7.49 ± 0.49* 2.0 ± 0.31∆ ERα 1 0.98 ± 0.10 1.03 ± 0.13 1.3 ± 0.3 1.17 ± 0.25 1.02 ± 0.11 1.14 ± 0.15 ERβ 1 1.09 ± 0.15 1.1 ± 0.28 0.58 ± 0.10** 0.89 ± 0.14 0.66 ± 0.10* 0.93 ± 0.15

The BeWo cells were treated with P4 and/or E2 in the absence and presence of ICI 182, 780 and the effects of hormone treatment on

mRNA levels of PRA, PRB, ERα and ERβ were determined as described in “Materials and Methods”. Relative mRNA levels

normalized to β-actin are presented. The relative mRNA levels of the vehicle controls are set as 1. The data shown are mean ± S.E.

from four independent experiments. The differences in mRNA levels are statistically significant: * p < 0.05; ** p < 0.01 as compared

with the vehicle controls using Student’s t-test. ∆ p < 0.05 as compared with respective E2 treatment alone using Student’s t-test. ICI

represents ICI 182, 780. The unit of concentrations: M.

Figure 1

A

B

C

38

Figure 2

BC

RP/β-

actin

Prot

ein

0

1

2

3

0

1

2

3

0 12 24 48 72 Treatment Time (h)

BC

RP/β-

actin

Prot

ein

β-actin

BCRP

BCRP

β-actin

A

*

**

BCRP

β-actin

DMSO 10-9 10-8 10-7 10-6 10-5 P4 (M)

0

1

2

3

DMSO 10-5 10-5 P4 (M)10-5 RU 486 (M)

0

1

2

3

DMSO 10-5 10-5 P4 (M)10-5 RU 486 (M)

B

C

BC

RP/β-

actin

Prot

ein

* *

39

Figure 3

BCRP

β-actin

DMSO 2.5 × 10-6 2.5 × 10-6 P4 (M)2.5 × 10-5 RU 486 (M)

0

0.5

1.0

1.5

2.0

DMSO 2.5 × 10-6 2.5 × 10-6 P4 (M)2.5 × 10-5 RU 486 (M)

BC

RP/β-

actin

mR

NA

A

**

BC

RP/β-

actin

Prot

ein

B2.0

2.5

1.5

1.0

0.5

0

**

C

40

Figure 4

BCRP

β-actin

DMSO 10-11 10-10 10-9 10-8 10-7 E2 (M)

BC

RP/β-

actin

Prot

ein

0

0.5

1.0

***

0 12 24 48 72 Treatment Time (h)

0

0.5

1.0

BCRP

β-actin

0

0.5

1.0

DMSO 10-7 10-7 E2 (M)10-6 ICI 182,780 (M)

** **

∆

**

A

BC

RP/β-

actin

Prot

ein

B

BC

RP/β-

actin

Prot

ein

C BCRP

β-actin

41

Figure 5

0

1

2

3

4

DMSO 10-5 10-5 10-5 10-5 P4 (M)10-9 10-8 10-7 E2 (M)

BC

RP/β-

actin

Prot

ein

**

**

Bβ-actin

BCRP

BCRP

β-actin

DMSO 10-6 10-6 10-6 10-6 P4 (M)10-9 10-8 10-7 E2 (M)

A

BCRP

BC

RP/β-

actin

Prot

ein

β-actinC

DMSO 10-5 10-5 10-5 10-5 10-5 P4 (M)10-8 10-8 10-8 10-8 E2 (M)

ICI 10-6 RU10-5 ICI 10-6 (M)RU 10-5 (M)

#

BC

RP/β-

actin

Prot

ein

***∆

∆

2.52.01.51.00.5

0

0

1

2

3*

**#*

#* #

*

*

42

Figure 6

0

0.5

1.0

1.5

2.0

2.5

3.0

*

*

∆

*

* *

∆

0

0.5

1.0

1.5

2.0

2.5A *

*

*

DMSO

P4 10 -5

(M)

P4 10 -5

(M) + RU 486 10 -5

(M)

P4 10 -5

(M) + E

2 10 -8(M

)

E2 10 -7

(M)

E2 10 -7

(M) + ICI 182, 780 10 -6

(M)

B

E2 10 -7

(M) + ICI 182, 780 10 -6

(M)

DMSO

P4 10 -5

(M)

P4 10 -5

(M) + RU 486 10 -5

(M)

P4 10 -5

(M) + E

2 10 -8(M

)

E2 10 -7

(M)

BC

RP/β-

actin

mR

NA

BC

RP/β-

actin

mR

NA

43

Figure 7

0

0.1

0.2

0.3

0.4

0.5

0 30 60 90 Accumulation time (min)

FTC-

inhi

bita

bleM

X

c

fflu

x A

Etiv

ity

( pm

olpe

r mg

prot

ein) *

*

*

*

*

44