zinc finger nuclease-mediated gene knockout results in loss of...

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Zinc Finger Nuclease-Mediated Gene Knockout Results in Loss of Transport

Activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Kathleen E Sampson, Amanda Brinker, Jennifer Pratt, Neetu Venkatraman, Yongling Xiao, Jim

Blasberg, Toni Steiner, Maureen Bourner and David C Thompson

Sigma-Aldrich Corporation, St. Louis, MO

Current affiliation: Covance, Madison, WI (K.E.S.); UMKC, Kansas City, MO (A.B.);

Confluence Life Sciences (N.V.)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on November 11, 2014 as DOI: 10.1124/dmd.114.057216

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Running Title: P-gp, BCRP and MRP2 knockout in Caco-2 cells

Corresponding Author:

David C Thompson, Ph.D.

Sigma-Aldrich

2909 Laclede Ave

St. Louis, MO 63103

Phone: 314-236-8997

Email: [email protected]

Manuscript information:

Text pages: 29

Number of tables: 4

Number of figures: 7

Number of references: 51

Number of words:

Abstract: 248

Introduction: 738

Discussion: 1500

Abbreviations: A, apical; B, basolateral; BCRP, breast cancer resistance protein; CDCF, 5-(and-

6)-carboxy-2', 7'-dichlorofluorescein; CDCFDA, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein

diacetate; KO, knockout; MDR1, multidrug resistance 1 gene; MRP2, multidrug resistance-

associated protein 2; P-gp, P-glycoprotein; ZFN, zinc finger nuclease.


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ABSTRACT

Membrane transporters P-glycoprotein (P-gp, MDR1 gene), MRP2, and BCRP impact drug

absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity

concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when

substrates or inhibitors impact more than one actively expressed transporter and when

endogenous or residual transporter activity remains following over-expression or knockdown of

a given transporter. The objective of this study was to selectively knock out three drug efflux

transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a

subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease (ZFN) technology. The wildtype

parent and knockout cell lines were tested for transporter function in Transwell bidirectional

assays using probe substrates at 5 or 10 µM for 2 hr at 37 ºC. P-gp substrates digoxin and

erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate CDCF

each showed a loss of asymmetrical transport in the MDR1, BCRP, and MRP2 knockout cell

lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine,

fexofenadine and colchicine. Compared to the knockout cell lines, standard transporter

inhibitors showed substrate-specific variation in reducing the efflux ratio of the test compounds.

These data confirm the generation of a panel of stable Caco-2 cell lines with single or double

knockout of human efflux transporter genes and a complete loss of specific transport activity.

These cell lines may prove useful in clarifying complex drug-transporter interactions without

some of the limitations of current chemical or genetic knockdown approaches.


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INTRODUCTION

Membrane drug transporters play an important role in the distribution of endogenous molecules

and xenobiotics throughout the body, and are implicated in detoxification mechanisms as well as

multidrug resistance. Members of the ATP-binding cassette (ABC) efflux transporter family

such as P-glycoprotein (P-gp, MDR1, ABCB1), multidrug resistance-associated protein 2

(MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2), actively efflux a wide

variety of small molecule substrates out of the cell to protect cells and organs against harmful

drugs or toxins (Litman et al., 2001; Shitara et al., 2006). ABC transporters have a protective

role in blocking intestinal absorption (Oude Elferink and de Waart, 2007; Takano et al., 2006)

and enhancing excretion of endogenous and xenobiotic compounds from the hepatic canalicular

membrane and the kidney proximal tubules (Köck and Brouwer, 2012; Masereeuw and Russel,

2012). They play a role in clinical drug resistance to multiple chemotherapeutic agents (Szakacs

et al., 2006; Veringa et al., 2013) and in drug-drug interactions (DDI) which may alter systemic

exposure and lead to clinical adverse events (DeGorter et al., 2012; Lin, 2007; Marquez and Van

Bambeke, 2011; Müller and Fromm, 2011).

Guidelines have recently been published by the US Food and Drug Administration (2012) and

European Medicines Agency (2012) on screening new chemical entities for interactions with

clinically relevant transporters. In vitro evaluation of specific transporter interactions can

employ a variety of tools including transporter expressing cell lines, membrane vesicles, and

tissues, along with a panel of substrates and inhibitors as control probes. The standard assay

format for ABC transporter function measures the transcellular permeability of a test article

through a monolayer of cells grown on a permeable filter, and comparison of the absorptive vs.

secretory flux. Caco-2 cells are derived from a human intestinal adenocarcinoma and are widely

used as a model of intestinal absorption and transporter activity (Elsby et al., 2008). These cells

differentiate in culture to an intestinal phenotype with a well defined apical brush border, are

able to form tight junctions, and express the ABC efflux transporters P-gp, BCRP, and MRP2, as

well as other uptake and efflux transporters normally expressed in human intestinal enterocytes

(Hilgendorf et al., 2007). The cells express the transporters in a polarized fashion, enabling the

vectorial transport of substrates, and are considered the gold standard for efflux transporter

screening.


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Transporters recognize and interact with of a broad range of compounds based on their

physicochemical characteristics (Didziapetris et al., 2003; Zhou et al., 2008), with overlapping

substrate recognition between transporters. Transporter “specific” inhibitors are used to help

define interactions, but may also show overlapping interactions between transporters (Matsson et

al., 2009). In addition, substrates may interact with different binding sites per transporter,

necessitating the use of multiple inhibitors with different binding site-specific affinities for each

transporter (Giri et al., 2008). This lack of specificity can cause misinterpretation in biological

systems with multiple transporters or endogenous transporters in transfected cell lines (Goh et

al., 2002; Wang et al., 2008; Mease et al.., 2012). Thus, there exists a need for human testing

systems which allow unambiguous identification of specific substrate interaction without

dependence on chemical inhibition.

Targeted suppression of gene expression by RNA interference techniques has been explored in

several labs using Caco-2 cells (Celius et al., 2004; Watanabe et al., 2005; Zhang et al.,, 2009;

Darnell et al., 2010; Graber-Maier et al., 2010). Transfection of short hairpin RNA (shRNA)

vectors and the resultant down-regulation of transporters offers an advantage over reliance on

inhibitors to elucidate specific drug-transporter interactions. However, not all RNA oligos are

able to knock down the targeted mRNA efficiently and they may invoke off-target effects on

similar mRNAs. Most importantly, substantial residual activity may remain in a cell line in spite

of reduced mRNA and protein levels (Darnell et al., 2010; Wang et al., 2014).

Zinc finger nuclease (ZFN) technology involves transfection of highly specific gene targeting

reagents linked to DNA cleavage enzymes, allowing exquisite specificity and total gene

knockout in a stable cell line, while minimizing off target effects (Santiago et al., 2008). We

report here the generation and characterization of a panel of knockout (KO) cell lines targeting

MDR1, BCRP and MRP2 transporters in the Caco-2 subclone C2BBe1 cell line using ZFNs. The

resultant panel of single or double KO cells shows disruption of gene sequence as well as

complete loss of transporter function in bidirectional transport assays. These transporter KO cell

lines provide a powerful new tool for elucidating transporter interactions.


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MATERIALS AND METHODS

Materials

Unless otherwise indicated, all cell culture media, biochemical reagents and chemicals were

obtained from Sigma-Aldrich (St Louis, MO). Costar Transwell HTS 24 well plates were

purchased from Sigma-Aldrich. 5-(and-6)-carboxy-2', 7'-dichlorofluorescein (CDCF) and 5-

(and-6)-carboxy-2´,7´-dichlorofluorescein diacetate (CDCFDA) were purchased from Life

Technologies (Carlsbad, CA). Primers specific for MDR1, BCRP, MRP2-4 transporter genes

and the endogenous control human GAPDH gene were purchased as Taqman Gene Expression

Assays from Life Technologies. For Western blotting experiments, a rabbit monoclonal

antibody to MDR1 [ab170904] and a mouse monoclonal antibody to β-actin [ab8226] were

purchased from Abcam Inc. (Cambridge, MA), while rabbit polyclonal antibody to BCRP [cs

4477] and rabbit monoclonal antibody to MRP2 [cs 12559] were obtained from Cell Signaling

Technology, Inc. (Danvers, MA). Secondary antibodies for β-actin (donkey anti-mouse) and

transporters (donkey anti-rabbit) were obtained from Jackson ImmunoResearch Laboratories,

Inc. (West Grove, PA).

Cell culture

The C2BBe1 cell line, a subclone of Caco-2 cells, was obtained from ATCC (Manassas, VA, cat.

no. CRL-2102). The original tissue donor was a 72 year old male with colorectal

adenocarcinoma. The C2BBe1 (“Caco-2 Brush Border expressing”) cell line had been cloned

from the Caco-2 cell line (ATCC HTB-37™) by limiting dilution and was selected on the basis

of morphological homogeneity and exclusive apical villin localization (Peterson and Mooseker,

1992). Cells were maintained in high glucose DMEM with 10% heat inactivated fetal bovine

serum (FBS), 1% (v/v) MEM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium

pyruvate, 100 units/mL penicillin and 100 µg/mL streptomycin. Cells were cultured in

humidified incubators at 37 οC in 5% CO2. Culture medium was refreshed at 2-3 day intervals.

Cells were passaged upon reaching confluence, at least once per week, using 0.25% Trypsin-

EDTA.

ZFN-mediated DNA modification and subclone selection

Knockout cell lines were generated using CompoZr Custom Zinc Finger Nuclease (Sigma-

Aldrich) kit components as previously described (Pratt et al., 2012). Briefly, 2 µg of each ZFN


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forward and reverse mRNA primers for each transporter gene, along with 4 µg gene-specific

mammalian single strand-annealing reporter plasmids containing two complementary portions of

the GFP protein, were nucleofected into C2BBe1 cells using the Amaxa Cell Line Nucleofector

Kit T for Caco-2 cells (Lonza, Basel, Switzerland) as per manufacturer’s directions.

Nucleofected cells were immediately placed in 20% FBS growth medium and cultured in 6 well

plates at 30 ºC for 2 days to increase efficiency of nucleofection, then were moved to 37 ºC.

Medium was refreshed once cells had attached. After growing to >70% confluence (1 -2 weeks),

cells were trypsinized and stained with 1 µg/mL propidium iodide before flow cytometry sorting

based on GFP positive (indicating successful ZFN cutting) and propidium iodide negative

(indicating viable cells) sort gates. Cells were single cell sorted into 96 well plates using the

FACSAria III (BD Biosciences, San Jose, CA) and were cultured for several weeks to form

substantial colonies before testing for mutations. Genomic DNA was obtained using

QuickExtract DNA Extraction Solution (Epicentre Biotechnologies, Madison, WI) and scaled up

using PCR amplification with the ZFN Cel-1 primers specific for each target gene. PCR product

was run on the 96-capillary 3730xl DNA Analyzer using Peak Scanner software v1.0 (Life

Technologies). Clones showing non-wild type and out-of frame mutations were selected for

subcloning. DNA was amplified and subcloned into competent E. coli cells using the TOPO TA

Cloning Kit (Life Technologies), according to manufacturer’s directions. DNA was isolated

from colonies using GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich) and

sequenced to confirm gene disruption through base pair deletion and/or insertion and

identification of specific homozygous KO clones.

mRNA Expression Analysis

Cells at sub-confluent densities were trypsinized from T75 flasks and centrifuged at 800 rpm for

5 min. Medium was aspirated and cell pellets were stored at -80 ºC until use. RNA from each of

the cell pellets was isolated using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA). To

remove genomic DNA, on-column DNase digestion was performed using the On-Column DNase

1 digestion set (Sigma-Aldrich) according to instructions.

RT-PCR reactions were set up using the Taqman RNA-to-Ct 1 Step kit (Life Technologies)

including individual transporter gene primers or endogenous control GAPDH primers (Taqman

Gene Expression Assays, Life Technologies) and 100ng of RNA. PCR cycling conditions


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consisted of 48 ºC for 30 min, 95 ºC for 10 min, followed by 40 cycles of a denaturing step at 95

ºC for 15 sec and an annealing/extension step with fluorescence monitoring at 60 ºC for 1 min.

The relative expression changes were calculated as described by Livak and Schmittgen (2001).

Immunoblot Protein Expression Analysis

Confluent T75 flasks of Caco-2 cells were lysed with 2 mL of 1X LDS sample buffer (Life

Technologies) containing protease inhibitor cocktail. Cells were scraped from the flask,

homogenized using Qiashredder columns (Qiagen) and stored at -80 °C. Thawed lysates were

denatured by heating for 10 min at 65 °C, then loaded (20 µL per lane) onto NuPage Novex 4-

12% Bis Tris gels (Bio-Rad Laboratories, Hercules, CA) and run at 200v in MOPS SDS buffer.

Gels were transferred to PVDF membranes for 15 min using the Trans-Blot Turbo system (Bio-

Rad). Membranes were blocked with Blotto containing 0.05% Tween (Blotto+T) for 2-3 hr

while shaking at room temperature. Membranes were placed in Blotto+T containing primary

antibodies (diluted 1:250, or 1:1000 for β-actin) and incubated at 4 °C overnight while shaking.

Following multiple 10 min washes in Tris-buffered saline with 0.05% Tween (TBST),

membranes were placed in Blotto+T containing anti-rabbit or anti-mouse secondary antibodies

(diluted 1:10,000) and incubated for 1 hr while shaking. Following multiple 10 min washes in

TBST, proteins were visualized using Super Signal West Dura detection reagent (Thermo

Scientific, Rockford, IL) and imaged on a ChemiDoc imager using Image Lab software v4.0

(Bio-Rad).

Bidirectional Transport Assay

C2BBe1 wild type and KO cell lines were plated at 4 x 104 cells/well onto Costar HTS-

Transwell 24 well permeable support plates (0.4 µM pore size, 0.33 cm2 polyethylene

terephthalate filter). Cells were cultured for 20-22 days to obtain differentiated monolayers with

tight junctions and polarized transporter expression. On day of study, cell monolayers were

rinsed twice and then pre-incubated for 30-60 min with transport buffer (Hank’s Balanced Salt

Solution [HBSS] with 25 mM D-glucose and 10 mM HEPES, pH 7.4) in both chambers at 37 ºC.

Test articles were diluted from 10 mM DMSO stocks to 5 or 10 µM in transport buffer and

placed in either apical (A) or basolateral (B) donor chambers in triplicate, while fresh transport

buffer alone was placed in receiver chambers. Additional transport studies were conducted in

the presence of inhibitors MK-571, Ko143, or verapamil, which were added to both chambers


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during transport assay at indicated concentrations. Plates were incubated at 37 οC for 2 hr, at

which time aliquots were removed from donor and receiver chambers for quantitation. Analyte

concentrations were determined by LC-MS/MS.

For the fluorescent MRP2 substrate CDCF, plates were treated with 10 µM CDCFDA and

incubated at 37 οC for 2 hr as above. Receiver and donor chamber samples were transferred to

black-walled 96 well plates. A triplicate standard curve of 2 fold serial dilutions from 10 µM of

CDCF was generated from a 10 mM DMSO stock. Plates were quantified by fluorescence at

485nm emission, 538nm excitation on a SpectraMax Gemini XS plate reader using SOFTmax

Pro software v 3.1.2 (Molecular Devices, Sunnyvale, CA), both immediately post assay and after

24 hr at room temperature for maximal hydrolysis of dosing and donor solutions to fluorescent

product.

At the conclusion of the transport assay, residual buffer was aspirated from all wells. Fresh

transport buffer was added to basolateral chambers, and Lucifer yellow (dilithium salt) at 0.1

mg/mL in HBSS was added to apical chambers. The plates were incubated at 37 οC for 1 hr.

Samples were transferred from the basolateral chambers to black-walled 96 well plates and

quantified by fluorescence at 485nm emission, 538nm excitation as described above. Lucifer

yellow permeability A to B was calculated; those wells exhibiting permeability >2 x10-6 cm/s

were eliminated from assay results.

Liquid chromatography/tandem mass spectral analysis

Concentration of test articles in samples was analyzed by LC-MS/MS using an API-4000 Q Trap

mass spectrometer with a Turbo V atmospheric pressure electrospray ionization source (AB

SCIEX, Framingham, MA). Samples (40 μl) were injected onto a Fortis C8 column (2.1 × 50

mm, 5 μm) and eluted by a mobile phase gradient optimized for each test article (mobile phase

A: 4 mM ammonium formate; mobile phase B: 4 mM ammonium formate in 90% (v/v)

acetonitrile). Flow rate was 0.5 mL/min. Using positive or negative ionization mode, analytes

were quantitated using multiple reaction monitoring specific for each analyte and internal

standard (tolbutamide) parent-product ion pairs. Peak areas of analyte and internal standard and

resulting ratios were quantified using Analyst 1.5 (AB SCIEX).

Calculations


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The apparent permeability (Papp, cm/sec) was determined for both A to B and B to A directions

by the following calculation:

in which A = area of filter membrane, CD(0) = initial concentration of the test drug, dMr = the

amount of transported drug and dt = time elapsed. The efflux ratio was calculated from:

ER = (Papp, B to A)/(Papp, A to B)

An efflux ratio ≥2 suggests an active transport process, identifying the compound as an apical

efflux transporter substrate.

Statistics

Unless otherwise noted, all transport assays were carried out in triplicate and repeated on at least

3 separate days. The data are presented as mean ± standard deviation. Statistical significance

was determined using one way ANOVA calculations.

dt

dM

CAP r

Dapp *

)0(*

1 =


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RESULTS

ZFN-mediated disruption of genomic DNA sequence.

Following nucleofection with ZFN pairs and single cell sorting, C2BBe1 clones exhibiting

mutations in all 4 alleles were initially identified by fragment analysis. These clones were

further expanded for genomic DNA sequencing within the ZFN target area. Small insertions

and/or deletions (indels) were confirmed within each allele in the tetraploid cells for each single

and double KO clone. Genotype analysis of the single KO clones is shown in Table 1. Each

MDR1 and BCRP KO clone contained out-of-frame indels resulting in the generation of a

premature stop codon. For the MDR1 KO clone each allele contained a unique modification,

while for BCRP KO only two modified sequences were observed among the four alleles. The

MRP2 KO clone contained two separate in-frame deletions (-9, -36); however, these deletions

overlapped a splice site and thus were still effective at disruption of translation into a functional

MRP2 protein. The MDR1 KO, BCRP KO, and MDR1/BCRP KO clones were all generated

independently; the MRP2 KO was used to generate the MDR1/MRP2 KO and the MRP2/BCRP

KO clones in a second round of ZFN nucleofections.

Protein Expression Analysis

Western blots were run to confirm the absence of the targeted transporter protein in each of the

single KO clones (Figure 1). For each transporter, the protein was detected in the wild type

(parental) cells but was completely absent in the appropriate KO cell line. Equivalent loading of

protein samples per lane was confirmed by measuring β-actin staining.

mRNA Expression Analysis

In order to determine whether substantive changes in expression of related ABC transporters

occurs when single or double transporter genes are knocked out, mRNA expression levels of

MDR1, BCRP, MRP2, MRP3 and MRP4 were measured in each of the single and double KO

cell lines. In Figure 2A, relative mRNA expression levels of these five transporters are shown

for each of the single KO cell lines compared to wild type (normalized to 1). As expected, the

mRNA level of each target gene was reduced in its respective KO line, most likely due to

decreased stability of the mutated transcript. Only small changes in expression levels were

detected for any of the other transporters. Maximum changes detected were a 2-fold increase in


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MRP3 mRNA expression in BCRP KO cells and a 2-fold decrease in MRP3 mRNA expression

in MDR1 KO cells. Similar modest compensatory changes were observed in the double KO

cells (Figure 2B). Here, a 2.5-fold increase in expression of MRP3 mRNA was noted in the

MDR1/MRP2 KO cells.

Cell Line Characterization

Each of the KO cell lines exhibited morphology and growth characteristics that were similar to

the parental C2BBe1 cells, with the exception of a slight lag in growth rate for the MRP2 KO

cell lines. The cellular phenotype for each KO cell line (loss of activity toward a model

substrate) was stable out to at least 40 passages post generation of the master cell bank (data not

shown); KO cells were not tested past P40. All cell lines took a typical 21 day growth period to

fully differentiate and form tight junctions on Transwell plates. Passive permeability data for

two marker compounds, atenolol (low permeability, <1 x 10-6 cm/s) and metoprolol (high

permeability, >15 x 10-6 cm/s) were used to compare the passive permeability of wild type cells

with each KO cell line, and also to serve as a quality control when running test compounds in

these assays. Passive permeability of both atenolol and metoprolol were similar in wild type and

all KO cell lines (data not shown). As an additional control used in all assays, Lucifer yellow A

to B permeability was checked as a paracellular permeability marker post-assay to insure that

tight junctions remained intact.

Bidirectional transport activity using probe substrates

The transport of probe substrates for each targeted transporter was examined in the full panel of

KO cell lines generated, and results were compared to those achieved in the wild type

C2BBe1cells. The A to B and B to A permeability values and the resultant efflux ratios are

shown in Tables 2-4. Efflux ratios for digoxin and erythromycin were reduced to near unity in

the MDR1 single and double KO cell lines (Table 2). This was a reflection of both an increase in

permeability in the A to B (absorptive) direction and a decrease in the B to A (secretory)

direction. The use of transporter-specific inhibitors in the parental C2BBe1 cell line was

compared to results obtained using KO cells. The P-gp inhibitor verapamil (100 μM)

successfully inhibited digoxin and erythromycin efflux in wild type cells to the same extent seen

in the KOs. Surprisingly, digoxin permeability rates in the A to B direction were decreased

somewhat in the BCRP, MRP2 and MRP2/BCRP KO cell lines (from 0.99 to approximately 0.25


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x10-6 cm/s) while the B to A rates remained nearly unchanged (12.5 to 18.2 x10-6 cm/s). This

resulted in higher efflux ratios for digoxin in these cell lines compared to wild type.

Nitrofurantoin and estrone 3-sulfate were used as model substrates to test for loss of function in

the BCRP KO cell lines. The efflux ratios for both compounds were reduced to near unity in the

BCRP single and double KO cell lines (Table 3). As a comparator, the BCRP inhibitor Ko143 (1

μM) reduced the efflux ratios for estrone sulfate and nitrofurantoin to between 2 and 3 in the

wild type cells, suggesting that Ko143 is less effective in blocking BCRP function than the ZFN-

mediated gene knockout.

For the MRP2 KO cell line, the nonfluorescent compound CDCFDA was used as the probe

substrate. CDCFDA passively diffuses into cells where it is hydrolyzed by intracellular esterases

to the fluorescent product CDCF, which is then rapidly excreted by MRP2 (Siissalo et al., 2009).

The CDCF efflux ratio was reduced to approximately 2 in the MRP2 single and double KO cell

lines (Table 4). In contrast, the MRP2 inhibitor MK571 (25 μM) only partially inhibited CDCF

transport with an efflux ratio >8 in wild type cells.

Additional test compounds

In order to further probe the utility of these cell lines several additional compounds (ranitidine,

cimetidine, fexofenadine and colchicine) were tested for transporter interactions. Ranitidine was

identified as a substrate of P-gp only, based on loss of polarized transport in all MDR1 KO lines

(Figure 3). In contrast, the efflux ratio for cimetidine was only partially reduced in both the

MDR1 and BCRP single KO lines (Figure 4). However, in the MDR1/BCRP double KO cells

the efflux ratio was fully reduced to unity, thus identifying cimetidine as a substrate for both P-

gp and BCRP. The P-gp and BCRP inhibitors verapamil and Ko143 gave a similar pattern of

results. Verapamil fully inhibited ranitidine efflux whereas Ko143 had no effect (Figure 3),

confirming the role of P-gp as sole transporter. For cimetidine, a combination of either [KO +

inhibitor] or both inhibitors, resulting in a cumulative loss of both Pgp and BCRP activity, was

required to reduce the efflux to near unity (Figure 4).

Fexofenadine showed a slightly more complex picture of transporter interactions. In wild type

cells the efflux ratio for fexofenadine was 5.11 (Figure 5). This was largely inhibited by

verapamil (efflux ratio = 2.08) but not at all by MK571. In the KO panel, all cell lines lacking P-

gp (i.e. MDR1, MDR1/BCRP and MDR1/MRP2 KOs) showed a complete reduction in efflux,


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demonstrating that fexofenadine is a substrate for P-gp. In contrast, the BCRP, MRP2, and

MRP2/BCRP KO cell lines did not show any inhibition of fexofenadine efflux except in the

presence of verapamil. Similar to the parental cell line, addition of verapamil only partially

reduced the efflux ratio in the KO cell lines. Notably, with each of the three MDR1 KO cell

lines, the efflux ratio was reduced to less than one (0.51 - 0.72), suggesting a net shift toward

basolateral efflux, or absorptive transport, in the absence of P-gp. The addition of MK571

slightly increased the efflux ratio of fexofenadine in these KO cell lines (0.93 - 1.52), suggesting

inhibition of the basolateral transport.

The colchicine efflux ratio in wild type cells was 12.8; this efflux was completely inhibited in the

KO cell lines lacking P-gp, but not in the other KO cell lines, clearly demonstrating that

colchicine is a substrate for P-gp (Figure 6). Similar to results seen with fexofenadine, the efflux

ratio of colchicine in the MDR1 KO cells was slightly less than one (efflux ratios between 0.88 –

0.96), and was also slightly increased in the presence of MK571 (1.0 – 1.4), again suggesting a

basolateral MRP is involved. In the cell lines expressing P-gp, both verapamil and MK571

partially impacted colchicine transport when used alone, and were able to inhibit colchicine

efflux only when used in combination (efflux ratios between 1.4 – 2.1). However, the MRP or

other target of MK571 is currently unknown.

Effect of MK571 on Caco-2 cell permeability

To further investigate the role of MK571 in colchicine transport, we tested colchicine in the wild

type and single KO cell lines with MK571 at 0, 10, 25 and 100 µM. At 100 µM MK571, the

colchicine efflux ratio in wild type cells was reduced from >15 to approximately 1.2, suggesting

total inhibition of active transport. However, the permeability of colchicine had increased

significantly in both directions (data not shown), and the Lucifer yellow data from the post-assay

integrity control indicated a 12 – 16 fold increased permeability in wells that had been exposed

to 100 µM MK571. Additional assays were run using 10 – 100 µM MK571 under the same

experimental conditions but in the absence of any other compound. We observed that Lucifer

yellow A to B permeability increased significantly in C2BBe1 cells with concentrations of

MK571 ≥ 50 µM, to >10 fold higher at 100 μM (Figure 7); similar results were seen in all KO

cell lines.


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DISCUSSION

Clinically relevant drug-drug interactions have been associated with transporter inhibition,

including efflux transporters (Giacomini et al. 2010). In addition, interaction with efflux

transporters has been linked to poor bioavailability and/or altered rates of clearance (Misaka et

al. 2013). Although several cell-based and membrane model systems exist for studying

transporter interactions, these are typically dependent on the use of transporter-specific substrates

and inhibitors, or target a single transporter over-expressed in a non-human cell system which

contain endogenous transporters and lack the full complement of human transporters. In order to

address some of the limitations of these current systems we generated single and double KO cell

lines for the ABC family efflux transporters P-gp, BCRP and MRP2 in human intestinal C2BBe1

cells using ZFN gene editing technology. The KOs were confirmed by genetic analysis, Western

blotting and functional assays using model substrates. The KO cell lines appeared similar to the

wild type in terms of growth rates and morphology, differentiation, formation of tight junctions,

passive permeability of model compounds, and stability of phenotype.

A key concern in all KO models, whether in vivo or in vitro, is the potential for adaptation or

compensation for the loss of the target gene by changes in the expression of related genes. In

order to address this concern, we compared mRNA expression levels of the three efflux

transporters as well as MRP3 and MRP4 in parental and all KO cell lines. Our data suggest little

if any impact on the expression level of these transporters; however, only a few genes were

examined in the present in vitro study, and the possibility of compensation at the protein

expression level cannot be definitively ruled out. Comparative analyses have been carried out in

rat models in which P-gp, BCRP or MRP2 have been knocked out using ZFN technology (Chu et

al. 2012; Huang et al. 2012; Zamek-Gliszczynski et al. 2013). Zamek-Gliszczynski et al. (2013)

reported that expression analyses of a set of 112 ADME-relevant genes in liver, kidney, intestine

and brain tissues of the three KO rat lines demonstrated only modest compensatory changes and

did not preclude their general application to study transporter-mediated pharmacokinetics.

Bidirectional transport studies were carried out in all KO cell lines using well-characterized

substrates specific for each individual transporter and comparing results with the wild type cells.

Compounds used included digoxin and erythromycin for P-gp, estrone sulfate and nitrofurantoin

for BCRP and CDCF for MRP2. Each KO cell line showed an appropriate reduction of efflux


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ratio with the representative substrate. Inhibitory effects were consistent between the single and

double KO cell lines in which the same transporter was absent.

Several additional compounds, including some with known crossover between multiple

transporters, were tested in the KO cell lines. Cimetidine has been reported to be a substrate for

both P-gp and BCRP (Pavek et al. 2005; Taur et al. 2008) while ranitidine is transported

primarily by P-gp (Collett et al. 1999; Bourdet and Thakker, 2006). Our results confirmed that

cimetidine was a substrate of both P-gp and BCRP, as the efflux ratio was reduced to unity only

in the MDR1/BCRP KO cell line, while ranitidine was identified as a substrate for P-gp alone.

For both cimetidine and ranitidine, the inhibitors verapamil and Ko143 were equally as effective

at inhibiting the efflux ratio as were the KO cells.

The H1 antagonist fexofenadine has been described as a substrate for P-gp (Cvetkovic et al.

1999; Drescher et al. 2002), although the possible involvement of multiple efflux transporters in

its hepatic disposition, including MRP2, has been suggested (Matsushima et al. 2008; Tian et al.

2008). In the present studies, fexofenadine was observed as a substrate for P-gp as reduced

efflux was clearly observed in the P-gp KO cell line whereas no reduction of efflux was observed

in either the BCRP or MRP2 single KO cell lines. The KO cell lines provided a clearer

assessment of interactions with transporters than did the use of chemical inhibitors, since

verapamil was only able to reduce the efflux ratio for fexofenadine to 2.08, 4.20 and 2.74 in the

wild type, BCRP KO and MRP2 KO cell lines, respectively.

Interestingly, the efflux ratio for fexofenadine in the MDR1 KO cells was significantly lower

than one (0.51), suggesting absorptive transport. This was also observed in the two double KO

cell lines that lacked functional P-gp. The potential involvement of a basolateral efflux

transporter in the MRP family is supported by the observation that the addition of MK571

increased the efflux ratio in each of the cell lines lacking P-gp (up to 1.52 in the case of MDR1

KO cells). These data support the conclusions drawn by Ming et al. (2011) that fexofenadine

apical efflux in Caco-2 cells is predominantly mediated by P-gp, whereas basolateral efflux is

predominantly mediated by MRP3. Based on data using MK571 and a P-gp/BCRP inhibitor

(GW120918), Ming et al. (2011) further suggested that MRP2 makes a small contribution to the

apical efflux of fexofenadine, although our data using the MRP2 KO cell lines do not support the

involvement of MRP2.


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Similar results were found for the microtubule polymerization inhibitor colchicine. We observed

that colchicine was a substrate for P-gp, based on reduction in efflux ratio in the P-gp KO cell

lines and lack of effect in the other KO cell lines. Similar to fexofenadine, the ERs in cell lines

lacking P-gp were below unity, but were slightly increased by addition of MK571, suggesting a

basolateral MRP may interact with colchicine in the absence of P-gp. Colchicine has been

reported as a substrate for both P-gp and MRP2 in Caco-2 cells and rodent intestine (Dahan et al.

2009); however, our data does not support colchicine interaction with MRP2. Reasons for this

discrepancy in results may include the lack of MK571 specificity within the MRP family as well

as a negative impact on the Caco-2 cell monolayer at higher concentrations, and point to the

challenges in using chemical inhibitors versus gene KO technology.

In the present experiments, inhibitors gave equivalent results in wild type cells compared to the

KO cells (full inhibition of efflux) for ranitidine and cimetidine, but not for fexofenadine or

colchicine, suggesting substrate dependence as another obstacle when using chemical inhibition

in transport interpretations. The KO cell lines represent complete inhibition of the targeted

transporter, and are an efficient alternative to the suggested use of multiple substrates and

inhibitors to ensure coverage of multiple binding sites, substrate specificity, and affinity that may

occur when characterizing transporter interactions in vitro (Brouwer et al., 2013).

The problem of inadequate specificity for inhibitors used in transporter assays has been well

documented. For example, Matsson et al. (2009) reported that each of the inhibitors used in the

current study (verapamil, Ko143 and MK571) have varying degrees of overlap with other efflux

transporters at higher concentrations, but were chosen for these studies due to their common use

and commercial availability. Although non-selectivity can be partially addressed by carefully

choosing the inhibitor concentration, it is difficult to accurately assess the intracellular

concentration of inhibitor at the transporter site. The most promiscuous of the three, MK571, has

IC50s of 10, 26 and 50 µM for MRP2, P-gp and BCRP, respectively (Matsson et al., 2009). In

addition, our results and the work of others suggest that MK571 inhibits not only MRP2 but

other MRPs, although potencies have not been established. Virtual docking experiments have

shown that MK571 binds to the ATP catalytic site, which may contribute to its relatively non-

specific inhibition profile (Matsson et al. 2009). Since MK571 is often used at 50 µM or higher

concentrations in the literature, off-target effects should be anticipated. Furthermore, we found

that MK571 negatively impacts passive permeability within the cell monolayer when used at 50


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µM or higher, further complicating the interpretation of transporter experiments with this

inhibitor.

In comparison with other formats for studying drug-transporter interactions, KO cell lines

provide a new and complementary approach to determine the profile of efflux transporters with

which a given compound may interact. These cell lines were generated from a human parental

line (Caco-2) extensively used for transporter studies for over two decades. This offers the

advantage of the presence of the full complement of other relevant human transporters in the

same cell system, while avoiding potential contributions from non-human transporters

(Kuteykin-Teplyakov et al. 2010). In addition, the double KO cell lines can be used to confirm

effects seen in the single KO cells, or perhaps to study the remaining apical efflux transporter in

relative isolation.

In summary, we have generated stable MDR1, BCRP and MRP2 single and double KO Caco-2

cell lines using ZFN technology. These KO cell lines show complete loss of transporter function

using specific substrates in the bidirectional transport assay format and are useful in identifying

specific drug-transporter interactions by comparison of transport between the wild type and KO

lines. These cell lines represent a valuable tool for application in drug discovery transporter

interaction assessment without dependence on chemical inhibitors with poorly defined

specificities, or RNA knockdown systems with residual activities.


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ACKNOWLEDGMENTS

We thank Kelly Keys and Gene Pegg for technical support, Tim Brayman and Michael Mitchell

for valuable scientific input, and Cole Meyer for assistance with the tables and figures.

AUTHORSHIP CONTRIBUTIONS: Participated in research design: Sampson, Bourner, Thompson

Conducted experiments: Sampson, Brinker, Venkatraman, Pratt, Xiao, Steiner, Blasberg

Contributed new reagents or analytic tools: Brinker, Venkatraman, Pratt

Performed data analysis: Sampson, Xiao

Wrote or contributed to the writing of the manuscript: Sampson, Thompson


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Figure Legends Figure 1. Western blots of P-gp, BCRP and MRP2 protein expression in wild type and KO cell

lines. Whole cell lysates from Caco-2 or KO cells were analyzed for transporter expression

using 1:250 dilutions of primary antibodies followed by chemiluminescent detection. β-actin

expression was used to confirm consistent protein quantities per lane.

Figure 2. Relative mRNA expression of efflux transporters in wild type and KO cell lines.

mRNA was isolated from each cell line and subjected to RT-PCR for 40 cycles using

transporter-specific primers. A. Relative mRNA levels in single KO cell lines. B. Relative

mRNA levels in double KO cell lines. Expression was calibrated to WT levels = 1. Data

represents mean ± SD from ≥ 2 RNA isolations per cell line.

Figure 3. Efflux ratios for ranitidine in wild type and KO cell lines. Experiments were carried

out with ranitidine (5 µM) in Transwell plates for 2 hr at 37°C in the presence or absence of the

P-gp inhibitor verapamil (100 µM) or the BCRP inhibitor Ko143 (1 µM). Values represent mean

± SD, n = 3 replicates in ≥3 assays.

Figure 4. Efflux ratios for cimetidine in wild type and KO cell lines. Experiments were carried

out with cimetidine (5 µM) in Transwell plates for 2 hr at 37°C in the presence or absence of the

P-gp inhibitor verapamil (100 µM) and/or the BCRP inhibitor Ko143 (1 µM). Values represent

mean ± SD, n = 3 replicates in ≥3 assays.

Figure 5. Efflux ratios for fexofenadine in wild type and KO cell lines. Experiments were

carried out with fexofenadine (5 µM) in Transwell plates for 2 hr at 37°C in the presence or

absence of the P-gp inhibitor verapamil (100 µM) and/or the MRP inhibitor MK571 (25 µM).

Values represent mean ± SD, n = 3 replicates in ≥3 assays.

Figure 6. Efflux ratios for colchicine in wild type and KO cell lines. Experiments were carried

out with colchicine (5 µM) in Transwell plates for 2 hr at 37°C in the presence or absence of the

P-gp inhibitor verapamil (100 µM) and/or the MRP inhibitor MK571 (25 µM). Values represent

mean ± SD, n = 3 replicates in ≥3 assays.


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Figure 7. Effect of MK571 on Lucifer yellow permeability in C2BBe1 wild type cells.

Experiments were carried out with MK571 in both chambers at concentrations between 10 - 100

μM in Transwell plates for 2 hr at 37 °C, followed by Lucifer yellow assay for 1 hr at 37 °C.

Values represent Lucifer yellow permeability mean ± SD, n=9-10 in each experimental group.

Statistical significance, as determined by one-way ANOVA, is depicted by *** = p<0.001

compared with untreated control.


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Table 1. Genotype analysis of single KOs. Sequences show the ZFN binding sites for each of

the three target genes; lower case letters designate the ZFN cut site. Underlined bases represent

deletions in each allele, while bold highlighted bases represent insertions.

MDR1 gene Indels Allele 1 GTCCTGTTCTTGGACtgtcaGCTGCTGTCTGGGCAAAG -2 Allele 2 GTCCTGTTCTTGGACtgtcaGCTGCTGTCTGGGCAAAG -4 Allele 3 GTCCTGTTCTTGGACtgtcaGCTGCTGTCTGGGCAAAG -5 Allele 4 GTCCTGTTCTTGGACtgtcAaGCTGCTGTCTGGGCAAAG -9, +1 BCRP gene Allele 1&2 TACACCACCTCCTTCTGTcatcaACTCAGATGGGT -4 Allele 3&4 TACACCACCTCCTTCTGTcGTCATatcaACTCAGATGGGT +5 MRP2 gene Allele 1&2 GTCTCCCTAGTCCATGATggcagtGAAGAAGAAGACGATGAC -9 Allele 3&4 GTCTCCCTAGTCCATGATggcagtGAAGAAGAAGACGATGAC -36


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Table 2. Known P-gp substrates demonstrating functional consequence of MDR1 gene KO. The

permeability rates and efflux ratios of digoxin (5 µM) and erythromycin (5 µM) were tested in

the complete panel of wild type (C2BBe1) and KO cell lines. Verapamil (100 µM) was used as a

P-gp inhibitor in the wild type cells. Values represent mean ± SD; n = 3 replicates in ≥3 assays

in each experimental group.

Digoxin Erythromycin

Permeability (x 10-6 cm/s)

Efflux Ratio


Efflux Ratio Cell line A to B B to A A to B B to A

C2BBe1 0.998 ± 0.514 17.7 ± 8.3 17.7 ± 0.9 0.413 ± 0.160 6.95 ± 1.63 16.8 ± 1.4

C2BBe1 + verapamil

3.85 ± 1.89 3.72 ± 0.83 0.965 ± 0.148 0.638 ± 0.168 1.11 ± 0.17 1.73 ± 0.09

MDR1 KO 3.29 ± 1.00 4.59 ± 1.46 1.40 ± 0.37 1.82 ± 0.81 1.89 ± 0.56 1.04 ± 0.41

BCRP KO 0.251 ± 0.087 12.5 ± 2.6 49.6 ± 4.5 0.322 ± 0.112 8.25 ± 2.54 25.6 ± 15.2

MRP2 KO 0.254 ± 0.054 16.9 ± 3.7 66.4 ± 6.5 0.527 ± 0.649 8.92 ± 1.58 16.9 ± 4.2

MDR1/BCRP KO 3.41 ± 1.33 4.66 ± 1.29 1.37 ± 0.16 0.758 ± 0.135 0.951 ± 0.120 1.25 ± 0.05

MDR1/MRP2 KO 3.57 ± 1.32 5.16 ± 1.86 1.44 ± 0.20 0.870 ± 0.344 0.776 ± .0255 0.892 ± 0.032

MRP2/BCRP KO 0.206 ± 0.093 18.2 ± 9.0 88.7 ± 9.3 0.170 ± 0.044 8.55 ± 0.90 50.2 ± 3.4


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Table 3. Known BCRP substrates demonstrating functional consequence of BCRP gene KO.

The permeability rates and efflux ratios of estrone 3-sulfate (5 µM) and nitrofurantoin (5 µM)

were tested in the complete panel of wild type (C2BBe1) and KO cell lines. Ko143 (1 µM) was

used as a BCRP inhibitor in the wild type cells. Values represent mean ± SD; n = 3 replicates in

≥3 assays in each experimental group.

Estrone 3-sulfate Nitrofurantoin


Efflux Ratio


Efflux Ratio Cell line A to B B to A A to B B to A

C2BBe1 0.550 ± 0.227 12.5 ± 2.3 22.7 ± 10.9 1.17 ± 0.25 15.4 ± 1.1 13.2 ± 1.6

C2BBe1 + Ko143 1.62 ± 0.47 3.96 ± 1.22 2.44 ± 0.20 3.53 ± 0.80 10.4 ± 1.29 2.93 ± 0.23

MDR1 KO 0.738 ± 0.415 8.56 ± 0.74 11.6 ± 2.4 2.34 ± 0.21 18.9 ± 4.8 8.09 ± 0.53

BCRP KO 1.35 ± 0.19 2.38 ± 0.81 1.76 ± 0.28 4.14 ± 1.04 6.98 ± 2.37 1.68 ± 0.12

MRP2 KO 0.280 ± 0.202 21.4 ± 10.6 76.2 ± 10.4 0.874 ± 0.018 18.1 ± 2.6 20.7 ± 1.3

MDR1/BCRP KO 1.85 ± 0.23 1.70 ± 0.37 0.920 ± 0.48 5.49 ± 1.06 11.8 ± 1.2 2.15 ± 0.13

MDR1/MRP2 KO 0.345 ± 0.048 11.6 ± 7.9 33.5 ± 9.9 1.98 ± 0.64 18.0 ± 2.0 9.06 ± 0.98

MRP2/BCRP KO 1.42 ± 0.19 2.46 ± 0.37 1.73 ± 0.03 8.06 ± 0.92 9.23 ± 1.75 1.15 ± 0.07


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DMD #57216

33

Table 4. Known MRP2 substrate demonstrating functional consequence of MRP2 gene KO. The

permeability rates and efflux ratios of CDCF (10 µM, added as CDCFDA) were assessed in the

complete panel of wild type (C2BBe1) and KO cell lines. MK571 (25 µM) was used as a MRP2

inhibitor in the wild type cells. Values represent mean ± SD; n = 3 replicates in ≥3 assays in

each experimental group.

CDCF


Efflux Ratio Cell line A to B B to A

C2BBe1 0.357 ± 0.117 11.5 ± 2.77 32.3 ± 4.47

C2BBe1 + MK571 0.428 ± 0.136 3.59 ± 1.50 8.38 ± 1.26

MDR1 KO 0.573 ± 0.121 13.9 ± 2.04 24.3 ± 2.12

BCRP KO 0.545 ± 0.051 14.0 ± 1.80 25.7 ± 0.48

MRP2 KO 2.04 ± 0.63 4.12 ± 1.52 2.03 ± 0.37

MDR1/BCRP KO 0.778 ± 0.486 11.3 ± 1.34 14.5 ± 1.51

MDR1/MRP2 KO 2.76 ± 0.36 6.46 ± 0.49 2.34 ± 0.07

MRP2/BCRP KO 2.46 ± 0.83 4.01 ± 1.75 1.63 ± 0.41


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β-Actin β-Actin

WT KOWT KOWT KO

MRP2BCRPMDR1A B C

β-Actin

WT KOWT KO WT KO

174 kDA72

kDA

141 kDA

Figure 1


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0

0.5

1

1.5

2

2.5

3

MDR1 BCRP MRP2 MRP3 MRP4

Rel

ativ

e E

xpre

ssio

n

WTBCRP KO

MDR1 KOMRP2 KO

0

0.5

1

1.5

2

2.5

3

MDR1 BCRP MRP2 MRP3 MRP4

Rel

ativ

e Ex

pres

sion

WTBCRP/MDR1 KO

BCRP/MRP2 KOMDR1/MRP2 KO

Single KO Cell Lines

Double KO Cell LinesB

A

Figure 2


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1

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3

4

5

C2BBe1

no inhibitor100 µM Verapamil1 µM Ko143

Ranitidine Efflux Ratios

MDR1 KO

BCRP KO

MRP2 KO

MDR1/BCRP KO

MDR1/MRP2 KO

MRP2/BCRP KO

Efflu

x R

atio

(B to

A/A

to B

)

Figure 3


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2

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6

8

10

no inhibitor100 µM Verapamil1 µM Ko143100 µM Verapamil + 1 µM Ko143

C2BBe1 MDR1 KO

BCRP KO

MRP2 KO

MDR1/BCRP KO

MDR1/MRP2 KO

MRP2/BCRP KO

Efflu

x R

atio

(B to

A/A

to B

)

Cimetidine Efflux RatiosFigure 4


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2

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6

8

10

12

14

16

C2BBe1 MDR1 KO

BCRP KO

MRP2 KO

MDR1/BCRP KO

MDR1/MRP2 KO

MRP2/BCRP KO

Efflu

x R

atio

(B to

A/A

to B

)

no inhibitor100 µM Verapamil25 µM MK571100 µM Verapamil + 25 µM MK571

Fexofenadine Efflux Ratios

Figure 5


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5

10

15

20

25

30

35

40

C2BBe1 MDR1 KO

BCRP KO

MRP2 KO

MDR1/BCRP KO

MDR1/MRP2 KO

MRP2/BCRP KO

Efflu

x R

atio

(B to

A/A

to B

)

no inhibitor100 µM Verapamil25 µM MK571100 µM Verapamil + 25 µM MK571

Colchicine Efflux RatiosFigure 6


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012345678

0 10 25 50 75 100

Effect of MK571 on Lucifer Yellow Permeability

MK571 Concentration (µM)

Papp

(× 1

0-6cm

/s)

***

******

Figure 7


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