DMD #57216
1
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
2
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
3
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
4
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
5
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
6
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
7
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
8
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
9
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
10
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 =
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
11
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
12
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
13
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,
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
14
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
15
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
16
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
17
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
18
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
19
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
20
REFERENCES
Bourdet DL and Thakker DR (2006) Saturable absorptive transport of the hydrophilic organic
cation ranitidine in Caco-2 cells: role of pH-dependent organic cation uptake system and P-
glycoprotein. Pharm Res 23: 1165-1177.
Brouwer KLR, Keppler D, Hoffmaster KA, Bow DAJ, Cheng Y, Lai Y, Palm JE, Stieger B, and
Evers R (2013) In vitro methods to support transporter evaluation in drug discovery and
development. Clin Pharmacol Ther 94: 95-112.
Celius T, Garberg P, and Lundgren B (2004) Stable suppression of MDR1 gene expression and
function by RNAi in Caco-2 cells. Biochem Biophys Res Commun 324: 365-371.
Chu X, Zhang Z, Yabut J, Horwitz S, Levorse J, Li X, Zhu L, Lederman H, Ortiga R, Strauss J,
Li X, Owens KA, Dragovic J, Vogt T, Evers R, and Shin MK (2012) Characterization of
multidrug resistance 1a/P-glycoprotein knockout rats generated by zinc finger nucleases. Mol
Pharmacol 81:220-227.
Collett A, Higgs NB, Sims E, Rowland M, and Warhurst G (1999) Modulation of the
permeability of H2 receptor antagonists cimetidine and ranitidine by P-glycoprotein in rat
intestine and the human colonic cell line Caco-2. J Pharmacol Exp Ther 288: 171-178.
Cvetkovic M, Leake B, Fromm MF, Wilkinson GR, and Kim RB (1999) OATP and P-
glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab
Dispos 27: 866-871.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
21
Dahan A, Sabit H, and Amidon GL (2009) Multiple efflux pumps are involved in the
transepithelial transport of colchicine: combined effect of P-glycoprotein and multidrug-
resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire
small intestine. Drug Metab Dispos 37: 2028-2036.
Darnell M, Karlsson JE, Owen A, Hidalgo IJ, Li J, Zhang W, and Andersson TB (2010)
Investigation of the involvement of P-glycoprotein and multidrug resistance-associated protein 2
in the efflux of ximelagatran and its metabolites by using short hairpin RNA knockdown in
Caco-2 cells. Drug Metab Dispos 38: 491-497.
DeGorter MK, Xia CQ, Yang JJ, and Kim RB (2012) Drug transporters in drug efficacy and
toxicity. Annu Rev Pharmacol Toxicol 52: 249-273.
Didziapetris R, Japertas P, Avdeef A, Petrauskas A (2003) Classification analysis of P-
glycoprotein substrate specificity. J Drug Target 11:391-406.
Drescher S, Schaeffeler E, Hitzl M, Hofmann U, Schwab M, Brinkmann U, Eichelbaum M, and
Fromm MF (2002) MDR1 gene polymorphisms and disposition of the P-glycoprotein substrate
fexofenadine. Br J Clin Pharmacol 53: 526-534.
Elsby R, Surry DD, Smith VN, and Gray AJ (2008) Validation and application of Caco-2 assays
for the in vitro evaluation of development candidate drugs as substrates or inhibitors of P-
glycoprotein to support regulatory submissions. Xenobiotica 38: 1140-1164.
European Medicines Agency, Committee for Human Medicinal Products (2012) Guideline on
the Investigation of Drug Interactions. EMA website (online), http://www.ema.europa.eu/
docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
22
Giacomini K, Huang S-M, Tweedie D, Benet LZ, Brouwer KL, Chu X, Dahlin A, Evers R,
Fischer V, Hillgren KM, Hoffmaster KA, Ishikawa T, Keppler D, Kim RB, Lee CA, Niemi M,
Polli JW, Sugiyama Y, Swaan PW, Ware JA, Wright SH, Yee SW, Zamek-Gliszczynski MJ, and
Zhang L (2010) Membrane transporters in drug development. Nat Rev Drug Discov 9: 215-236.
Giri N, Agarwal S, Shaik N, Pan G, Chen Y, and Elmquist WF (2008) Substrate-dependent
breast cancer resistance protein (Bcrp/Abcg2)-mediated interactions: consideration of multiple
binding sites in in vitro assay design. Drug Metab Dispos 37: 560-570.
Goh LB, Spears KJ, Yao D, Ayrton A, Morgan P, Roland Wolf C, and Friedberg T (2002)
Endogenous drug transporters in in vitro and in vivo models for the prediction of drug
disposition in man. Biochem Pharmacol 64: 1569-1578.
Graber-Maier A, Gutmann H, and Drewe J (2010) A new intestinal cell culture model to
discriminate the relative contribution of P-gp and BCRP on transport of substrates such as
imatinib. Mol Pharm 7: 1618 – 1628.
Hilgendorf C, Ahlin G, Seithel A, Artursson P, Ungell AL, and Karlsson J (2007) Expression of
thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines.
Drug Metab Dispos 35: 1333-1340.
Huang L, Be X, Tchaparian EH, Colletti AE, Roberts J, Langley M, Ling Y, Wong BK, and Jin
L (2012) Deletion of Abcg2 has differential effects on excretion and pharmacokinetics of probe
substrates in rats, J Pharmacol Exp Ther 343: 316-324.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
23
Köck K and Brouwer KL (2012) A perspective on efflux transport proteins in the liver. Clin
Pharmacol Ther 92: 599-612.
Kuteykin-Teplyakov K, Luna-Tortós C, Ambroziak K, and Löscher W (2010) Differences in the
expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines
affect P-glycoprotein mediated drug transport. Br J Pharmacol 160: 1453-1463.
Lin JH (2007) Transporter-mediated drug interactions: clinical implications and in vitro
assessment. Expert Opin Drug Metab Toxicol 3: 81-92.
Litman T, Druley TE, Stein WD, and Bates SE (2001) From MDR to MXR: new understanding
of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci 58:
931-959.
Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real time
quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.
Marquez B and Van Bambeke F (2011) ABC multidrug transporters: target for modulation of
drug pharmacokinetics and drug-drug interactions. Curr Drug Targets 12: 600-620.
Masereeuw R and Russel FG (2012) Regulatory pathways for ATP-binding cassette transport
proteins in kidney proximal tubules, AAPS J 14: 883-894.
Matsson P, Pedersen JM, Norinder U, Bergström CA, and Artursson P (2009) Identification of
novel specific and general inhibitors of the three major human ATP-binding cassette transporters
P-gp, BCRP, and MRP2 among registered drugs. Pharm Res 26: 1816-1831.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
24
Matsushima S, Maeda K, Hayashi H, Debori Y, Schinkel AH, Schuetz JD, Kusuhara H, and
Sugiyama Y (2008) Involvement of multiple efflux transporters in hepatic disposition of
fexofenadine. Mol Pharmacol 73: 1474-1483.
Mease K, Sane R, Podila L, and Taub ME (2012) Differential selectivity of efflux transporter
inhibitors in Caco-2 and MDCK-MDR1 monolayers: a strategy to assess the interaction of a new
chemical entity with P-gp, BCRP, and MRP2. J Pharm Sci 101: 1888-1897,
Ming X, Knight BM, and Thakker DR (2011) Vectorial transport of fexofenadine across Caco-2
cells: involvement of apical uptake and basolateral efflux transporters. Mol Pharmacol 8: 1677-
1686.
Misaka S, Müller F and Fromm MF (2013) Clinical relevance of drug efflux pumps in the gut,
Curr Opin Pharmacol doi: 10.1016/j.coph.2013.08.010
Müller F and Fromm MF (2011) Transporter-mediated drug-drug interactions.
Pharmacogenomics 12: 1017-1037.
Oude Elferink RP and de Waart R (2007) Transporters in the intestine limiting drug and toxin
absorption. J Physiol Biochem 63: 75-81.
Pavek P, Merino G, Wagenaar E, Bolscher E, Novotna M, Jonker JW, and Schinkel AH (2005)
Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary
carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine. J
Pharmacol Exp Ther 312: 144-152.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
25
Peterson MD and Mooseker MS (1992) Characterization of the enterocyte-like brush border
cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2. J Cell Sci 102: 581-
600.
Pratt J, Venkatraman N, Brinker A, Xiao Y, Blasberg J, Thompson DC, and Bourner M (2012)
Use of zinc finger nuclease technology to knock out efflux transporters in C2BBe1 cells. Curr
Protoc Toxicol 23: 2.1-2.22.
Santiago Y, Chan E, Liu P-Q, Orlando S, Xhang L, Urnov FD, Holmes MC, Guschin D, Waite
A, Miller JC, Rebar EJ, Gregory PD, Klug A, and Collingwood TN (2008) Targeted gene
knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci
105: 5809-5814.
Shitara Y, Horie T, and Sugiyama Y (2006) Transporters as a determinant of drug clearance and
tissue distribution. Eur J Pharm Sci 27: 425-446.
Siissalo S, Hannukainen J, Kolehmainen J, Hirvonen J, and Kaukonen AM (2009) A Caco-2 cell
based screening method for compounds interacting with MRP2 efflux protein. Eur J Pharm
Biopharm 7: 332-338.
Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM (2006) Targeting
multidrug resistance in cancer. Nat Rev Drug Discov 5:219-234.
Takano M, Yumoto R, and Murakami T (2006) Expression and function of efflux drug
transporters in the intestine. Pharmacol Ther 109: 137-161.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
26
Taur JS and Rodriguez-Proteau R (2008) Effects of dietary flavonoids on the transport of
cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models.
Xenobiotica 38: 1536-1550.
Tian X, Zamek-Gliszczynski MJ, Li J, Bridges AS, Nezasa K, Patel NJ, Raub TJ, and Brouwer
KL (2008) Multidrug resistance-associated protein 2 (Mrp2) is primarily responsible for the
biliary excretion of fexofenadine in mice. Drug Metab Dispos 36: 61-64.
US Department of Health and Human Services, Food and Drug Administration, Center for Drug
Evaluation and Research (2012) Guidance for industry. Drug interaction studies – study design,
data analysis, and implications for dosing and labeling recommendations. US FDA website
(online), http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM292362.pdf.
Veringa SJ , Biesmans D, van Vuurden DG, Jansen MH, Wedekind LE, Horsman I, Wesseling P,
Vandertop WP, Noske DP, Kaspers GJ, and Hulleman E (2013) In vitro drug response and efflux
transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic
pontine glioma. PLoS One 8(4):e61512.
Watanabe T, Onuki R, Yamashita S, Taira K, and Sugiyama Y (2005) Construction of a
functional transporter analysis system using MDR1 knockdown Caco-2 cells. Pharm Res 22:
1287-1293.
Wang Q, Strab R, Kardos P, Ferguson C, Li J, Owen A, and Hidalgo IJ (2008) Application and
limitation of inhibitors in drug-transporter interactions studies. Int J Pharmaceu 356: 12-18.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
27
Wang L, Zhao Z, Meyer MB, Saha S, Yu M, Guo A, Wisinski KB, Huang W, Cai W, Pike JW,
Yuan M, Ahlquist P, and Xu W (2014) CARM1 methylates chromatin remodeling factoer
BAF155 to enhance tumor progression and metastasis. Cancer Cell 25:21-36.
Zamek-Gliszczynski MJ, Goldstein KM, Paulman A, Baker TK, and Ryan TP (2013) Minor
compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract
from their utility in the study of transporter-mediated pharmacokinetics. Drug Metab Dispos 41:
1174-1178.
Zhang W, Li J, Allen SM, Weiskircher EA, Huang Y, George RA, Fong RG, Owen A, and
Hidalgo IJ (2009) Silencing the breast cancer resistance protein expression and function in Caco-
2 cells using lentiviral vector-based short hairpin RNA. Drug Metab Dispos 37: 737-744.
Zhou SF, Wang LL, Di YM, Xue CC Duan W, Li CG, and Li Y (2008) Substrates and inhibitors
of human multidrug resistance associated proteins and the implications in drug development.
Curr Med Chem 15: 1981-2039.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
28
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
29
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.
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
30
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
31
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
Permeability (x 10-6 cm/s)
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
DMD #57216
32
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
Permeability (x 10-6 cm/s)
Efflux Ratio
Permeability (x 10-6 cm/s)
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
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
Permeability (x 10-6 cm/s)
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
0
1
2
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
0
2
4
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
0
2
4
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
0
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
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
012345678
0 10 25 50 75 100
Effect of MK571 on Lucifer Yellow Permeability
MK571 Concentration (µM)
Papp
(× 1
0-6cm
/s)
***
******
Figure 7
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
at ASPE
T Journals on A
ugust 24, 2020dm
d.aspetjournals.orgD
ownloaded from
Top Related