MDR1 Synonymous Polymorphisms Alter Transporter …cancerres.aacrjournals.org/content/canres/74/2/598.full.pdfSuresh V. Ambudkar 1, and Michael M. Gottesman Abstract The drug efﬂux

Therapeutics, Targets, and Chemical Biology

MDR1 Synonymous Polymorphisms Alter TransporterSpecificity and Protein Stability in a Stable EpithelialMonolayer

King Leung Fung1, James Pan1, Shinobu Ohnuma1, Paul E. Lund1, Jessica N. Pixley1, Chava Kimchi-Sarfaty2,Suresh V. Ambudkar1, and Michael M. Gottesman1

AbstractThe drug efflux function of P-glycoprotein (P-gp) encoded byMDR1 can be influenced by genetic polymorph-

isms, including two synonymous changes in the coding region ofMDR1. Here we report that the conformation ofP-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayersexpressing P-gp. Several cell lines with similarMDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA(a mutant that carries a different valine codon in position 3435). These cell lines express similar levels ofrecombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However,the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shiftassays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gpshave longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently toP-gp inhibitors that block efflux of rhodamine 123 or mitoxantrone. In addition, cytotoxicity assays show that theLLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with aP-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell's morphology, growth rate,or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPaseactivities are affected in the variant P-gps. Taken together, our findings indicate that "silent" polymorphismssignificantly change P-gp function, which would be expected to affect interindividual drug disposition andresponse. Cancer Res; 74(2); 598–608. �2013 AACR.

IntroductionMDR1 [P-glycoprotein (P-gp), ABCB1] is one of the major

drug transporters found in humans. This gene encodes P-gp, anefflux transporter in the plasma membrane that activelytransports a broad range of drugs in an ATP-dependentmanner (1). It is found in multiple organs (2), and is expressedin the trophoblast layer of the placenta during pregnancy (3).Mice carrying null abcb1a and abcb1b genes are viable, but

have altered pharmacokinetics of many drugs that are P-gpsubstrates (4–6). American collies carrying truncated MDR1genes have lower tolerance to vincristine and the dewormingagent ivermectin, a substrate of P-gp (7, 8). Overexpressionof P-gp is a common cause of acquired drug resistance incultured cancer cells (9–13). In polarized epithelia, P-gp islocated on the apical membrane, facilitating transport in adirectional manner (14, 15).

P-gp contains 2 important functional domains: the sub-strate binding site and the ATPase domain. It is well docu-mented thatmutations in these domains change P-gp function(reviewed in refs. 16 and 17). In humans, the MDR1 gene ishighly polymorphic, with at least 50 coding single-nucleotidepolymorphisms (SNP) in theMDR1 coding region documented.In particular, 3 SNPs at positions 1236C>T, 2677G>T, and3435C>T, which form the most common haplotype, have beenstudied extensively (16, 18–20). Since the first report showingthe alteration of P-gp function with these SNPs (18), manystudies have been done to define the influence of theseSNPs individually, or of the complete haplotype. However, theresults of these population-based studies are indecisive, pos-sibly because of variations in terms of experimental settingsincluding inadequate population sizes to assure statisticalsignificance, incomplete sequence of individuals, differences

Authors' Affiliations: 1Laboratory of Cell Biology, Center for CancerResearch, National Cancer Institute, NIH; and 2Center for Biologics Evalu-ation andResearch, Division of Hematology, Food andDrug Administration,Bethesda, Maryland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for J. Pan: Stanford University Medical School, Stanford,CA; current address for S. Ohnuma: Department of Surgery, TohokuUniversity Hospital, Sendai, Japan; and current address for P.E. Lund:University of Michigan, Ann Arbor, MI.

Corresponding Author: Michael M. Gottesman, Laboratory of Cell Bio-logy, Center for Cancer Research, National Cancer Institute, NIH,Bethesda, MD 20892. Phone: 301-496-1921; Fax: 301-402-0450; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-2064

�2013 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(2) January 15, 2014598

on October 19, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 4, 2013; DOI: 10.1158/0008-5472.CAN-13-2064

http://cancerres.aacrjournals.org/

in tissue-specific P-gp expression, and other unknown envi-ronmental factors (21).The synonymous SNP 3435C>T, usually part of the haplo-

type noted above, plays an influential role in P-gp function,including elevated digoxin, cyclosporin A (CsA), and fexofena-dine bioavailability (22–24). Our previous study using a vac-cinia virus–based transient expression system showed thatwild-type P-gp and its haplotype are different in function (25).We also suggested that differences in protein characteristics of3435C>T, such as those mentioned earlier, might be related tothe introduction of a rare codon that alters the translationalrhythm and folding of P-gp. However, there are technicallimitations in vaccinia virus–based high-level transient expres-sion systems that led us to conduct transport studies andprotein stability experiments in polarized cells. To studyhaplotype P-gp and compare its function with wild-type P-gpunder conditions more physiological than those in the tran-sient expression experiments, we developed stable cell lines inwhich the human MDR1 gene and its variants were translatedfrom recombinant DNA and inserted into genomic DNA in asubclone of LLC-PK1 cells that can form polarizedmonolayers.

Materials and MethodsCell culture and materialsThe LLC-PK1 cell line was obtained from American Type

Culture Collection, and maintained in Medium199 þ 3% (v/v)FBS þ 1% penicillin/streptomycin. The recombinant cell lineswere incubated in the samemediumwith 500mg/mL geneticin.KB-3-1, KB-V1, and KB-8-5 cells were cultured in Dulbecco'sModified Eagle Medium þ 10% FBS and 1% penicillin/strep-tomycin. Cells were cultured at 37�C with 5% CO2 and relativehumidity maintained at 95%.Cell culture media and geneticin were purchased from

Invitrogen. Biotin, paraformaldehyde, verapamil, vinblastine,rodamine 123, calcein-AM, mitoxantrone, trypsin, soybeantrypsin inhibitor, MTT, and valinomycin were obtained fromSigma. Bodipy-FL–vinblastine was obtained from MolecularProbes. Restriction enzymes were obtained from New EnglandBiolabs. The antibodies were purchased from the followingcompanies: DAKO (C219, MRK16), Invitrogen (IgG2a-AlexaFluor 488, CY3-streptavidine), eBiosciences (UIC2-PE, 17F9,IgG2a-HRP; Strepavidin-PE), and Jackson Immuno Research(IgG2a-FITC). ECL reagents were obtained from GE Health-care. 125I-iodoarylazidoprazosin (125I-IAAP; 2,200 Ci/mmol)was obtained from PerkinElmer Life Sciences.

Preparation of pcDNA-MDR1 constructsDetails about the preparation of constructs can be found in

Supplementary Materials and Methods.

Generation of LLC-MDR1 cell linesGeneration of cell lines is detailed in Supplementary Mate-

rials and Methods.

Protein extraction and immunoblot analysisTotal protein extraction from cell culture and protein con-

centration estimation methods were reported previously (26).

For SDS-PAGE, protein samples (50 mg) were loaded onto a 3%to 8% Tris-acetate gel (Invitrogen). Separated proteins weretransferred to a nitrocellulose membrane by iBlot (Invitrogen).The membrane was blocked in PBSþ 20%milk and incubatedovernight with C219 antibody (1:2,000) followed by incubationwith anti-IgG-2a-HRP antibody and ECLþ (GE Healthcare).

Southern blot analysisTotal genomic DNA was extracted using a GenElute Mam-

malian Genomic DNA Purification Kit (Sigma). Total DNA (50mg) and pcDNA-MDR1 (1236C-2677G-3435C; as copy numberstandard) were digested with NdeI for 24 hours and separatedin a 0.7% agarose gel. DNA in the gel was denatured and wastransferred onto a Hybond-XL membrane (GE Healthcare).The membrane was washed briefly with 2 � saline-sodiumcitrate (SSC) and soaked with hybridsol in 42�C for 2 hours. ANdeI-digested DNA [32P]-labeled DNA probe was prepared byReady-To-Go DNA Labeling Kit (GE Healthcare) and wasadded on the membrane for 16-hour incubation at 42�C. Themembrane was washed with 2 � SSC/0.1% SDS at 65�C for30minutes, 2� 0.1� SSC/0.1% SDS at 65�C for 10minutes. Theimage was recorded by a phosphoimager and was analyzed byImageQuant TL (GE Healthcare).

TaqMan qRT-PCRExpression levels of human MDR1 genes were measured

using the TaqMan method. One microgram of total RNA,isolated by the RNeasy Mini Kit (Qiagen), was used tosynthesize cDNA using the high-capacity cDNA ReverseTranscription Kit (Invitrogen). cDNA was mixed with Taq-Man Universal PCR Master Mix (Invitrogen), run on an ABIPrism 7900HT Sequence Detection System (Invitrogen) asper the manufacturer's instructions. Porcine plasma mem-brane calcium ATPase 4 gene (PMCA4) was used as thereference gene.

Confocal microscopyCells were grown on Transwell 3470 membrane inserts

(Corning) until confluent. Tight cell monolayers were fixed bytreatment with 4% paraformaldehyde for 10 minutes, thenincubated with PBSþ 1 mmol/L MgCl2þ 0.1 mmol/L CaCl2þ1% bovine serum albumin for 1 hour. To label P-gp, cellmonolayers were incubated with MRK16 (1:100) for 1 hour,followed by incubation with anti-IgG2a-Alexa Fluor 488 anti-body. To label nuclei, cells were incubated with 4',6-diamidino-2-phenylindole (DAPI; 300 nmol/L). For cell surface labeling,biotin was incubated for 15 minutes before cell monolayerswere fixed. Strepavidin-PE (1:100) was added to the fixed cellmonolayers for 1 hour. Fluorescence images were acquiredwith a Zeiss LSM-510 confocal microscope, and the imageswere analyzed by LSM Image Browser (version 4.2.0; Zeiss).

Cell growth assayCells were seeded on 60-mm culture dishes and cultured at

37�C. Starting from day 3 after cell seeding, 3 dishes of cellsfrom each cell line were trypsinized. Cell numbers wererecorded daily until day 14. Cell counting was measured bythe AutoT4 cellometer (Nexcelom Bioscience).

Effect of Coding Synonymous Polymorphisms on MDR1

www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 599




Cell surface immunolabeling by flow cytometryCell surface P-gp expression was detected by incubating

cells with 1 mg of MRK16, UIC2, and 17F9 antibodies per200,000 cells for 30 minutes. For experiments using CsA(10 mmol/L), cells were preincubated with drug for 10minutes. After primary antibody incubation, cells were incu-bated with anti-mouse IgG2a FITC-conjugated antibody for40 minutes.

Drug influx/efflux assayActively growing cells (2 � 105) were harvested by trypsi-

nization and were incubated in Iscove's modified Dulbecco'smedium (IMDM) þ 5% FBS medium, with rhodamine 123(0.5 mg/mL), mitoxantrone (5 mmol/L), or bodipy-FL–vinblas-tine (0.5 mmol/L) in the presence or absence of CsA (10mmol/L), DCPQ (50 nmol/L), Tariquidar (50 nmol/L), verapa-mil (5 mmol/L), digoxin (125 mmol/L) at 37�C. Cells werewashed and further incubate with IMDM or IMDMwith drugsfor 40 minutes before FACS analysis.

Limited trypsin digestion assayLimited protein digestions by trypsin were performed with

crudemembranes. Threemicrograms of the crudemembraneswere treated with different concentrations of trypsin for 5minutes at 37�C. The reaction was stopped with 5 � excess ofsoybean trypsin inhibitor. Remaining P-gp was detected byWestern blot analysis using C219 antibody.

Cell surface P-gp biotinylationFor cell surface biotinylation, cells werewashedwith ice cold

PBS, and then incubated with 0.2 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) in ice-cold PBS for 30 minutes.Excess biotin was washed with glycine/PBS and PBS. For flowcytometry analysis, cells were trypsinized, washed, and incu-bated with MRK16 þ IgG2a-FITC (0.5 mg per 200,000 cells) þCY3-streptavidine (1:50). Labeling of cells with sulfo-NHS-LC-biotin just before trypsinization was considered as time 0. Thepercentage of biotinylated P-gp remaining at each time pointwas determinedbymeasuring the percentage of biotinylated P-gp cells remaining compared with the number of biotinylatedP-gp cells at time 0. Data calculations were performed byCellQuest software BD Biosciences.

Cytotoxicity assayThe cytotoxicity of drugs on LLC-MDR1 cells was measured

by a colorimetric viability assay using MTT as previouslydescribed (26). Cytotoxicity (IC50) was defined as the drugconcentration that reduced cell viability to 50% of the untreat-ed control.

Crude membrane preparations and ATPase assaysThe crude membranes from cells were prepared as de-

scribed previously (27). ATPase activities were measured asreported previously (28). Briefly, crude membrane protein(10 mg protein per 100 mL) was incubated at 37�C for 30minutes in 100 mL of reaction buffer (50 mmol/L MOPS-KOH,pH 7.5, 50 mmol/L KCl, 5 mmol/L sodium azide, 1 mmol/LEGTA, 1 mmol/L ouabain, 10 mmol/L MgCl2) in the presence

and absence of 0.3 mmol/L sodium orthovanadate (Vi).The reaction was initiated by the addition of 5 mmol/L ATPfor 20 minutes at 37�C. SDS solution (2.5%, v/v) was addedto terminate the reaction, and the amount of inorganic phos-phate released was quantified with a colorimetric reaction,as described previously (28). The basal P-gp ATPase activitywas determined by subtracting the ATPase activity in theP-gp–containing membranes with the ATPase activity in thecontrol membranes in the same experiment. Drug-stimulatedP-gp ATPase activity was measured in an ATPase reactionmixture that contained 2.5 mmol/L paclitaxel, 10 mmol/Lvinblastine, 10 mmol/L valinomycin, and 30 mmol/L verapamil,and the activity obtained was corrected for the basal ATPaseactivity.

Photoaffinity labeling with 125I-IAAPPhotoaffinity assays were performed by incubating crude

membranes (1 mg/mL) with drugs for 10 minutes at roomtemperature in 50 mmol/L Tris-HCl, pH 7.5 followed by theaddition of 3-6 nmol/L [125I]-IAAP (2,200 Ci/mmol). The sam-ples were cross-linked and incorporation of [125I]-IAAP wasdetermined as described previously (29).

Statistical analysisStatistical significance of the experimental results was

obtained by the 2-sample t test. Results were consideredstatistically significant at P < 0.05.

ResultsCharacterization of LLC-PK1 cell lines expressing wild-type, haplotype, and mutant P-gps

To characterize the effect of P-gp variants, stable cell linesexpressing human P-gp were developed. The original LLC-PK1cell line, isolated from porcine kidney proximal tubule epithe-lial cells (30), expresses a low level of P-gp (31). Taking thisinto consideration, the LLC-PK1 subclone termed LLC-PK1#7was prepared by clonal selection. LLC-PK1#7 has undetect-able endogenous P-gp and is unable to efflux P-gp substratesincluding rhodamine 123 and calcein-AM (data not shown).

The MDR1 wild-type [pTM1-MDR1(1236C-2677G-3435C)],haplotype [pTM1-MDR1(1236T-2677T-3435T)], and a MDR1haplotypemutant encoding the isoleucine at 1145 using a rarertRNA [pTM1-MDR1(1236T-2677T-3435A)] were used for thisstudy. After transfection, LLC-PK1 clones were isolated andgrown under the same culture conditions. During estab-lishment of stable cells, we did not select P-gp expressionwith P-gp substrates such as colchicine or vinblastine toprevent endogenous P-gp induction and/or alteration ofother endogenous resistance mechanisms (32, 33). We des-ignated the cell lines in this study as follows: LLC-vector(vector-transfected cells), LLC-MDR1-WT [MDR1(1236C-2677G-3435C)-expressing cells], LLC-MDR1-3H [MDR1(1236T-2677T-3435T)-expressing cells], and LLC-MDR1-3HA[MDR1(1236T-2677T-3435A)-expressing cells]. As shown in Fig.1A, all the P-gp–expressing cell clones, numbered 1 to 4, have onecopy of MDR1 cDNA. qRT-PCR analysis determined thatthe transfected DNA expressed a comparable level of MDR1

Fung et al.

Cancer Res; 74(2) January 15, 2014 Cancer Research600




transcripts (Fig. 1B, 2–4). Recombinant P-gp expression wasidentified by Western blotting using C219 antibody. In Fig. 1C,P-gp was not detectable in the LLC-vector cells (lane 1), but wasevident in the P-gp–expressing cells (lanes 2–4). P-gp in all P-gp–expressing LLC-PK1 cell lines was found as a single band with anapparent molecular weight of�160 kDa and no immature bandswere detected (Fig. 1C and Supplementary Fig. S1). The P-gpprotein from LLC-MDR1-WT cells had lowermobility than the P-gp expressed in High-Five insect cell membranes, indicating thatP-gp is likely N-glycosylated in the LLC-PK1 cells (SupplementaryFig. S1). The N-glycosylation pattern of the different P-gps maydiffer, because we observed that the WT-P-gp (Fig. 1C, lane 2)had slightly higher mobility than the 3H-P-gp (Fig. 1C, lane 3) andthe 3HA-P-gp (Fig. 1C, lane 4). P-gp–expressing cell linesexpressed high levels of P-gp and all the clones expressed

comparable levels. P-gp expression in the KB-V1 cells (Fig. 1C,lane 5) was greater than in KB-8-5 cells (Fig. 1C, lane 6), asexpected.

We compared the growth rate of the LLC-PK1 cell lines. Thecells were allowed to grow in culture dishes for 2 weeks untilconfluency. The growth curves indicated that these cell lineshad similar growth rates (Fig. 1D).

To determine whether P-gp polarization could be affectedby P-gp polymorphisms and synonymous mutations, cell sur-face immunolabeling was performed on LLC-PK1 cell mono-layers. Cells were grown in Transwell chambers until cellmonolayers were formed. Light microscopy images showedno significant morphologic differences among the testedcell lines (Fig. 1E, top). Using confocal microscopy, in theP-gp–expressing cells, P-gp signals (green) were detected on

Figure 1. Characterization of LLC-MDR1 cell lines. A, copy numberdetermination by Southern blotanalysis. Genomic DNA sampleswere run with a blank control andcopy number standards. B, qRT-PCR analysis. The mean valuesare the average of threeexperiments and are plotted in thehistogram. C, recombinant P-gpexpression by Western analysis.LLC-vector (lane 1), LLC-MDR1-WT (lane 2), LLC-MDR1-3H (lane3), LLC-MDR1-3HA (lane 4), KB-V1(lane 5), and KB-8-5 (lane 6) areshown. The bar chart showsrelative expression level of P-gp ineach cell line calculated bydensitometry analysis. D, cellularmorphology and localization of P-gp. Top, cells visualized by lightmicroscopy. Bottom, X–Z plane ofthe LLC-MDR1 cells. Cellmonolayers were labeled withMRK16 (green, for P-gp), biotin-strepavdin-PE (red, for plasmamembrane), and DAPI (blue, fornucleus). E, LLC-MDR1 cell lineshave comparable growth rates.LLC-vector (black), LLC-MDR1-WT (green), LLC-MDR1-3H (red),and LLC-MDR1-3HA (blue) cellsare shown. Each point in the chartrepresents an average cell numberfrom three samples.






the apical side, but not in the vector-transfected stable cells(LLC-vector). WT-P-gp, 3H-P-gp, and 3HA-P-gp expressionon the apical membranes were comparable (Fig. 1E, middle).On the basolateral side, as expected, P-gp was undetectable(Fig. 1E, bottom). These results indicate that in the LLC-PK1cells, P-gp polymorphisms do not affect protein expressionor targeting to the apical cell surface.

MDR1mutations alter P-gp conformation in the LLC-PK1cells

To determine whether polymorphisms of MDR1 couldchange protein conformation, we examined the LLC-MDR1cell lines with the conformation-sensitive antibody UIC2. Asshown in Fig. 2A, the vector cells showed minimal reactivitytoward all antibodies tested (black histogram). The wild-typeand haplotype P-gp–expressing cells showed high and com-parable reactivity with conformation-insensitive MRK16 and

17F9 antibodies. However, these P-gp–expressing cells showeddifferential reactivity with UIC2 and with UIC2 in the presenceof CsA (10 mmol/L).

To further examine the effect of conformation differences,the accessibility to various trypsin concentrations of P-gp wasdetermined. Figure 2B shows Western blots of wild type andpolymorphic forms of P-gp with trypsin. Membrane proteinsfrom each P-gp–expressing cell line were prepared and theywere digested for 5 minutes with increasing amounts of tryp-sin, and the remaining P-gp protein was analyzed using C219antibody. For each protein sample, the concentration of tryp-sin required for a 50% decrease in P-gp was determined andplotted. We observed differences in trypsin sensitivity withLLC-MDR1-WT (most sensitive) < LLC-MDR1-3H < LLC-MDR1-3HA. The fold change in IC50 for LLC-MDR1-WT andLLC-MDR1-3H was 1.2- and 1.4-fold for LLC-MDR1-3HA. Inthe presence of 50 mmol/L verapamil, a P-gp inhibitor, there

Figure 2. Stably expressedhaplotype and mutant P-gps havealtered protein conformations. A,FACS analysis of cells labeled withdifferent anti-Pgp antibodies. Eachhistogram represents at least twoindependent experiments. B,sensitivity of MDR1 wild-type(circle), haplotype (square), andmutant P-gp (triangle) to trypsin.Crude membranes prepared fromthe cell lines were treated with,from left to right, 0.000, 0.625,1.250, 1.875, 2.500, 3.125, 3.750,4.375, and 5.000 trypsin (mg).Charts and Western blots withtrypsin (left) and with trypsin þ 20mmol/L verapamil (right) are shown.The top panel shows quantifieddata from Western blots shownbelow. Each point in the plotrepresents an average cell numberfrom three independentexperiments.

Fung et al.





was no difference in the IC50 values for trypsin digestion.WT-P-gp, 3H-P-gp, and 3HA-P-gp were more resistant totrypsin in the presence of verapamil, as we have previously

observed using transiently expressed wild-type and haplotypeP-gps (25).

P-gp synonymous polymorphisms influence proteinstability

To elucidate the impact of conformation changes of P-gp byits polymorphisms in LLC-PK1 cells, we determined the half-life of plasma membrane P-gp in LLC-PK1 cell lines by cellsurface biotinylation. Cells were labeled with biotin for 30minutes and cultured for 24, 48, and 72 hours. Cells werestained with strepavidin-PE andMRK16 antibody. The remain-ing biotinylated P-gp at each time point was determined byflow cytometry and a plot was drawn to calculate the timerequired for 50% disappearance of biotinylated P-gp (Fig. 2B).The mean half-lives of plasma membrane P-gp were 45 hours(LLC-MDR1-WT), 47 hours (LLC-MDR1-3H), and 53 hours(LLC-MDR1-3HA), respectively (Fig. 3), which are significantlydifferent from each other (P < 0.05), and consistent with thein vitro trypsin sensitivity experiments shown in Fig. 3.

Synonymous mutations affect P-gp functionThe effect of drug transport function of P-gp wild type

and its variants in LLC-PK1 cells was tested using rhoda-mine 123 with various P-gp inhibitors. In Fig. 4, FACS histo-grams show transport of rhodamine 123 by cells transfectedwith the LLC-vector, LLC-MDR1-WT, LLC-MDR1-3H, andLLC-MDR1-3HA cells. In the absence of P-gp inhibitors, effluxof rhodamine 123 by all P-gp–expressing LLC-PK1 cell lines

Figure 3. Mutant and haplotype P-gps remain longer on the cell surface.LLC-MDR1-WT (circles), LLC-MDR1-3H (squares), and LLC-MDR1-3HA(triangles) cells were labeled with biotin and MRK16 for FACS analysis.The percentage of biotin-labeled MRK16 positive cells remaining ateach time point was plotted. Each point in the plot represents an averagecell number from three independent experiments.

Figure 4. Variant P-gps exhibitdifferent drug efflux functions in asubstrate-dependent manner.Cells were incubated withrhodamine 123 for 20 minutes at37�C, followed by 40minutes effluxat 37�C and then FACS analysis.Assays were repeated at least 3times.






was the same. Complete inhibition of rhodamine 123 effluxwasobserved when a high concentration of verapamil (20 mmol/L)was used (Supplementary Fig. S2). Similar observations werealso made for high concentrations of the P-gp inhibitorstariquidar (200 nmol/L) and CsA (20 mmol/L; data not shown).However, we observed differential inhibition of P-gp functionwhen suboptimal inhibitor concentrations were used. Forexample, in the presence of 50 nmol/L tariquidar, efflux ofrhodamine 123 was least affected in LLC-MDR1-3H cells. Also,in the presence of 10 mmol/L CsA or 5 mmol/L verapamil, theLLC-MDR1-3HA cells showed slightly higher efflux of rhoda-mine 123. When we tested the efflux of rhodamine 123 in thepresence of digoxin (125 mmol/L), the LLC-MDR1-3HA cellsshowed the poorest inhibition to digoxin compared withthe LLC-MDR1-3H cells and the LLC-MDR1-WT cells, consis-tent with the original clinical observations that the P-gphaplotype affected digoxin levels in patients (18). These resultssuggest that the variant forms of P-gp have different sensitiv-ities to the inhibitors digoxin, CsA, and tariquidar.

The LLC-vector cells were used as a control. This cell linewas not able to efflux rhodamine 123, and P-gp inhibitors did

not influence its accumulation of rhodamine 123 (Figs. 4and 5, black histogram).

Variant P-gps affect drug efflux and cytotoxicityThe stable LLC-PK1 cells expressing human P-gps enabled

us to conduct short-term assays and long-term assays. Weconfirmed that WT-P-gp, 3H-P-gp, and 3HA-P-gp expressionlevels remain unchanged for at least 96 hours after geneticinremoval (results not shown). Growth assays revealed thatall the LLC-PK1 cell lines tested had comparable growthrates (Fig. 1D), indicating that these cells are suitable forcytotoxicity experiments. We tested 2 anticancer drugs thatare P-gp substrates (vinblastine, mitoxantrone) in the pre-sence or absence of P-gp inhibitors (DCPQ, CsA). We com-pared the efflux function of P-gp in different LLC-PK1 celllines using drug transport influx/efflux assays (short-term)and growth-inhibition assays (long-term) by the MTTmethod. In Fig. 5, the P-gp–expressing cells show differentmitoxantrone efflux. The LLC-MDR1-3H cells showed signi-ficantly higher efflux than the LLC-MDR1-3HA and the LLC-MDR1-WT cells. This difference correlated with increased

Figure 5. P-gp–transfected celllines show differential sensitivityto cytotoxic drugs. LLC-vector(black), LLC-MDR1-WT (green),LLC-MDR1-3H (red), and LLC-MDR1-3HA (blue) cells were testedwith cytotoxicity assays (left) anddrug efflux assays (right). Forcytotoxicity assays, cells wereincubated with increasingconcentrations of drugs for72 hours. For drug accumulation-efflux assays, cells were incubatedwith mitoxantrone � DCPQ for30 minutes followed by 45-minuteefflux. For bodipy-vinblastine �CsA, cells were incubated for20 minutes followed by 30-minuteefflux. Assays were repeated atleast three times. Each point in thecytotoxicity assay chartsrepresents an average cell numberfrom three independentexperiments.

Fung et al.





mitoxantrone resistance of the LLC-MDR1-3H and LLC-MDR1-3HA cells in the presence of mitoxantrone. Thecalculated IC50 values showed that the LLC-MDR1-3H cells(14.6 � 1.9 mmol/L) were 10 times more resistant to mit-oxantrone (P < 0.001) than the LLC-MDR1-WT (1.5 � 0.3mmol/L) cells, and the LLC-MDR1-3HA (9.2 � 2.5 mmol/L)cells were 6.3-fold (P < 0.001) more resistant than the wild-type cells (Table 1). Also, the fold differences of the IC50

values of LLC-MDR1-WT versus LLC-MDR1-3H, LLC-MDR1-WT versus LLC-MDR1-3HA, and LLC-MDR1-3H versus LLC-MDR1-3HA cells were also significant. In the presence of100 nmol/L DCPQ, resistance to mitoxantrone was reversedin the LLC-MDR1-WT (0.2 � 0.04 mmol/L) and the LLC-MDR1-3HA 0.8 � 0.2 mmol/L) cells when compared withLLC-vector cells (0.065 � 0.015 mmol/L). In the presence ofDCPQ, the IC50 of the LLC-MDR1-3H cells decreased from14.6 � 1.9 mmol/L to 3.2 � 0.6 mmol/L. However, they arestill significantly (49.8-fold, P < 0.001) resistant to mitoxan-trone compared with the LLC-vector and the LLC-MDR1-WT cells (Fig. 5 and Table 1). These experiments suggest thatthe LLC-MDR1-3H cells are less sensitive to DCPQ.

The effect of drug efflux function by P-gp variants wastested using vinblastine. For drug accumulation/effluxassays, bodipy-vinblastine was selected as a substrate. Thedrug efflux assays showed that all the P-gp–expressing celllines exported bodipy-vinblastine at the same rate. Inthe presence of 5 mmol/L CsA, all the P-gp–expressing cellsshowed reduced efflux of bodipy-vinblastine. Using MTTassays, the P-gp–expressing cells had comparable resistanceto vinblastine and no statistical significance was found inIC50 values between LLC-MDR1-WT versus LLC-MDR1-3Hand LLC-MDR1-3HA cells (Fig. 5 and Table 1). In the pre-sence of CsA, the P-gp–expressing cells showed reduced andcomparable sensitivity to vinblastine.

Effect of substrates on P-gp wild-type and haplotypeATPase activity

The synonymous SNPs 1236C>T and MDR1 3435C>T arein amino acid codons found in the nucleotide bindingdomains (34). To examine whether P-gp conformation couldchange drug-stimulated ATPase activity or photoaffinitylabeling with a transport substrate, we performed ATPaseactivity assays and photoaffinity labeling assays. Using mem-brane protein isolated from LLC-MDR1-WT cells andLLC-MDR1-3H, the basal rate of ATP hydrolysis could beincreased significantly by paclitaxel, vinblastine, valinomy-cin, and verapamil (Fig. 6A). There were no significantdifferences between the cell lines in basal and drug-stimu-lated ATPase activities.

Photoaffinity labeling assays with [125I]-IAAP in the pre-sence of tasigna, verapamil, paclitaxel, vinblastine, valinomy-cin, and tariquidar indicated that all tested compounds exceptverapamil could effectively inhibit [125I]-IAAP binding to P-gp(Fig. 6B). No significant differences were found between the2 cell lines. To further examine the effect of IAAP binding,experiments showed that the IC50 value of the LLC-MDR1-WT cell line was 1.9 mmol/L and that of the LLC-MDR1-3Hcell line was 2.2 mmol/L (P > 0.05).

Tab

le1.

Cytotox

icity

ofse

lected

substratesan

dinhibito

rsof

P-gpin

wild

-typ

ean

dha

plotype-ex

pressingLL

CPK1stab

lece

lllines

Drug

Inhibitor

IC50(mmol/L)

aRes

istanc

eratio

(fold)b

LLC-vec

tor

LLC-M

DR1-WT

LLC-M

DR1-3H

LLC-M

DR1-3H

ALL

C-vec

tor

LLC-M

DR1-WT

LLC-M

DR1-3H

LLC-M

DR1-3H

A

Mito

xantrone

–0.01

�0.00

31.5�

0.3

14.6

�1.9

9.2�

2.5

111

41,13

871

8Mito

xantrone

DCPQ

0.06

5�

0.01

50.2�

0.04

3.2�

0.6

0.8�

0.2

14

5012

Vinblastine

–0.00

2�

0.00

020.3�

0.02

0.2�

0.03

0.3�

0.06

114

611

117

5Vinblastine

CsA

0.00

06�

0.00

010.06

�0.00

40.05

�0.00

30.05

�0.00

31

101

8395

aTh

eIC

50va

lues

wereca

lculated

byno

nlinea

rregres

sion

analysis

inGraphP

adPris

mus

ingdos

e–resp

onse

datafrom

MTT

assa

y.bTh

eresistan

ceratio

isthefold

ofIC

50va

lues

relativ

eto

theLL

C-vec

torce

lls.






DiscussionIn this study, we developed 4 recombinant P-gp–expres-

sing cell lines to elucidate the effect of synonymous poly-morphisms and haplotypes of MDR1. Until recently, theeffect of synonymous mutations has generally been ignored.Nevertheless, mounting evidence strongly suggests thesepolymorphisms can influence gene function and cellularactivity via multiple pathways (35–38). In fact, Lawrie andcolleagues reported that a significant portion of synonymousmutation sites in Drosophila melanogaster are subject tostrong purifying selection, and the genes harboring thesemutations are related to key developmental pathways (39).

In the MDR1 gene, there are 2 synonymous polymorphismsthat frequently occur in certain human populations(34, 40, 41). Many clinical and in vitro studies have suggestedthat the 3435C>T SNP plays an important role in thefunction of MDR1. However, the functional effects of syn-onymous mutations in MDR1 have not been studied to anysignificant degree except after transient, high-level expres-sion (25). Therefore, we decided to use the LLC-PK1 epithe-lial cell line, which can form a polarized tight monolayer, toevaluate the effect of MDR1 synonymous polymorphisms,and the effects of codon usage.

Our results show that synonymous mutations do influenceprotein stability in intact cells in addition to trypsin sensi-tivity in vitro as previously reported (25). The MDR1 haplo-type causes a change in P-gp conformation, as determinedusing cell surface labeling by UIC2, a P-gp conformation-sensitive antibody (42). We further showed that the 3435C>Asynonymous mutation increases binding of UIC2 MAb, andincreases resistance to trypsin. The surface protein biotiny-lation experiments and FACS assays showed the wild-typeP-gp had the shortest half-life (45 hours), followed by thehaplotype (47 hours) and 3HA mutant (53 hours). Thesedifferences are consistent with the protein stability differ-ences demonstrated earlier using the trypsin-sensitivityassay (25). We hypothesize that the difference in proteinhalf-life may be linked to protein recycling. Kim and col-leagues (43) have reported that P-gp is recycled through aclathrin-dependent pathway and interacts with the AP-2adaptor complex. The P-gp variations we are studying mightcause a change in protein conformation involving a tightturn structure or a di-leucine motif, which interacts withAP-2 adaptor complexes. Also, our functional assays showedthat, in certain cases, the haplotype mutants are moreresistant to inhibitors because the frequency of haplotypeforms of P-gp varies in different human populations (19, 20).These results suggest an evolutionary force driving MDR1to alter its ability to interact with drugs and dietary materi-als through altered protein conformation resulting from thehaplotype studied here.

However, common synonymous mutations of MDR1 didnot influence mRNA expression, total protein expression, ortranslocation to the apical membrane surface. Also, the pres-ence of P-gp on the cell surface did not affect cell growth orformation of a tight polarized monolayer. Our observationdoes not support some clinical reports suggesting that thehaplotype allele often correlates with lower P-gp expression(18, 44, 45).

Our cell lines with stable P-gp expression allowed us toconduct physiologically relevant experiments, including drugefflux assays and cytotoxicity assays. Drug efflux assays sug-gested that the transport function of P-gp variants is depen-dent on the drug of choice. Our results indicated that P-gppolymorphisms do not affect rhodamine 123 transport in thepresence of verapamil or CsA. In contrast, the LLC-MDR1-3Hcell line is less sensitive to tariquidar than LLC-MDR1-WTcells.The LLC-MDR1-3HA cells are more resistant to inhibition bydigoxin than the cells carrying the haplotype and the wild-typeforms of P-gp. Our results with mitoxantrone indicate that the

Figure 6. P-gp variant forms do not differ from wild type in ATPase andphotoaffinity labeling assays. A, substrate-stimulated ATPase activitiesusing crudemembranes fromLLC-MDR1-WT (black) andLLC-MDR1-3H(gray) cells. B, [125I]-IAAP photoaffinity labeling of P-gp wild type andhaplotype in the absence (lane 1) and the presence of 10 mmol/L tasigna(lane 2), 10 mmol/L verapamil (lane 3), 10 mmol/L tariquidar (lane 4),10 mmol/L vinblastine (lane 5), and 5mmol/L paclitaxel (lane 6). The scatterplot shows the incorporation of [125I]-IAAP in LLC-MDR1-WT (circle)and LLC-MDR1-3H (triangle) membrane proteins with verapamil from0 to 100 mmol/L. Autoradiographs from representative experiments areshown. Similar results were obtained in two additional experiments.

Fung et al.





drug transport function of P-gp is significantly influencedby the synonymous mutations in MDR1. Mitoxantrone is ananthracene derivative that is a relatively poor substrate ofP-gp. Expression of P-gp is able to reduce mitoxantroneaccumulation and therefore increase cytotoxic resistance(46). In this study, we found that P-gp polymorphisms directlyaffect mitoxantrone efflux and cytotoxicity. The exact causeof these observations is unclear, but we hypothesize thatthe conformational differences affect the relative efficiency ofsubstrate and inhibitor binding between wild-type and haplo-type-expressing cells.In this study, the MDR1 1236C>T and 3435C>T SNPs

showed a significant impact on the overall folding of P-gp,and not just a localized effect. These "silent" mutationsencode glycine 412 and isoleucine 1145, which are found inthe first and second ATP-binding domains and areunchanged in the wild-type and haplotype P-gp. These SNPsdo not affect basal and drug-stimulated ATPase activity,suggesting changes in conformation in these polymorphicP-gps do not affect ATP binding or hydrolysis (47). These"silent" polymorphisms do not result in mRNA degradation,nor do the resulting P-gp conformational changes resultin obstruction in the protein folding pathway (like CFTR),mis-localization, posttranslational modification defects,protein truncation, and/or misfolding. In fact, P-gp encodedby the MDR1 common haplotype is expressed properly onthe cell surface, targeted to the apical side in polarizedmonolayers, and functions as a drug transporter. Further-more, some of our results indicate that the function ofhaplotype P-gp might be more efficient than the wild-typeP-gp. These results in stably transfected cells stronglysuggest that human P-gp exists in at least 2 conformationalstates of equivalent stability. The haplotype form foldsdifferently than wild-type P-gp (48). It seems that the silentpolymorphisms encode a protein with alternate (or new)energy minima, similar to the model suggested by Tsai andcolleagues (48). However, the results do not demonstrate themechanism of the effect of synonymous mutations onMDR1. More experiments are warranted to examine varioushypotheses.In summary, the role of P-gp synonymous polymorphisms

was examined in polarized epithelial cells. We showed that the

synonymous mutations do influence protein folding and func-tion in stable cells expressing recombinant P-gp. P-gp confor-mational alterations subtly changed drug efflux functionand interaction with P-gp inhibitors. By comparing data fromshort-term transport assays and cytotoxicity assays, thesesubtle changes significantly alter cellular cytotoxicity ofdrugs such as mitoxantrone that do not bind tightly to P-gp.The P-gp–expressing cell lines will help to identify substratesand inhibitors that are influenced by the common variantsof P-gp, allowing better predictions about the pharmaco-kinetics of drugs that are substrates for P-gp, and improveddesign of clinical studies that seek to inhibit the function ofP-gp to reverse drug resistance.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.L. Fung, C. Kimchi-Sarfaty, S.V. Ambudkar, M.M.GottesmanDevelopment of methodology: K.L. Fung, C. Kimchi-Sarfaty, S.V. Ambudkar,M.M. GottesmanAcquisition of data (provided animals, acquired and managed pa-tients, provided facilities, etc.): K.L. Fung, J. Pan, S. Ohnuma, P. Lund, J.N.Pixley, S.V. AmbudkarAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): K.L. Fung, J. Pan, S. Ohnuma, J.N.Pixley, S.V. AmbudkarWriting, review, and/or revision of the manuscript: K.L. Fung, J. Pan,C. Kimchi-Sarfaty, S.V. Ambudkar, M.M. GottesmanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.L. Fung, S.V. Ambudkar, M.M.GottesmanStudy supervision: K.L. Fung, S.V. Ambudkar, M.M. Gottesman

AcknowledgmentsThe authors thank G. Leiman for editorial assistance.

Grant SupportThis research was supported by the Intramural Research Program of the NIH,

National Cancer Institute.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received July 23, 2013; revised November 8, 2013; accepted November 8, 2013;published OnlineFirst December 4, 2013.

References1. Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM,

Pastan I. Partial purification and reconstitutionof the humanmultidrug-resistancepump: characterization of thedrug-stimulatableATPhydro-lysis. Proc Natl Acad Sci U S A 1992;89:8472–6.

2. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I.Expression of a multidrug-resistance gene in human tumors andtissues. Proc Natl Acad Sci U S A 1987;84:265–9.

3. Mason CW, Buhimschi IA, Buhimschi CS, Dong Y, Weiner CP, SwaanPW. ATP-binding cassette transporter expression in human placentaas a function of pregnancy condition. Drug Metab Dispos 2011;39:1000–7.

4. Schinkel AH. The physiological function of drug-transportingP-glycoproteins. Semin Cancer Biol 1997;8:161–70.

5. Schinkel AH, Smit JJ, van Tellingen O, Beijnen JH, Wagenaar E, vanDeemter L, et al. Disruption of the mouse mdr1a P-glycoprotein gene

leads to a deficiency in the blood-brain barrier and to increasedsensitivity to drugs. Cell 1994;77:491–502.

6. Smit JW, Schinkel AH, Weert B, Meijer DK. Hepatobiliary andintestinal clearance of amphiphilic cationic drugs in mice in whichboth mdr1a and mdr1b genes have been disrupted. Br J Pharmacol1998;124:416–24.

7. Krugman L, Bryan JN, Mealey KL, Chen A. Vincristine-induced centralneurotoxicity in a collie homozygous for the ABCB1Delta mutation. JSmall Anim Pract 2012;53:185–7.

8. Roulet A, Puel O, Gesta S, Lepage JF, Drag M, Soll M, et al. MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoproteinsubstrate ivermectin. Eur J Pharmacol 2003;460:85–91.

9. Roninson IB, Chin JE, Choi KG, Gros P, Housman DE, Fojo A, et al.Isolation of human mdr DNA sequences amplified in multidrug-resis-tant KB carcinoma cells. Proc Natl Acad Sci U S A 1986;83:4538–42.






10. Shen DW, Fojo A, Chin JE, Roninson IB, Richert N, Pastan I, et al.Human multidrug-resistant cell lines: increased mdr1 expression canprecede gene amplification. Science 1986;232:643–5.

11. AkiyamaS, FojoA,Hanover JA, Pastan I, GottesmanMM. Isolation andgenetic characterization of human KB cell lines resistant to multipledrugs. Somat Cell Mol Genet 1985;11:117–26.

12. AlemanC, Annereau JP, Liang XJ, Cardarelli CO, Taylor B, Yin JJ, et al.P-glycoprotein, expressed in multidrug resistant cells, is not respon-sible for alterations in membrane fluidity or membrane potential.Cancer Res 2003;63:3084–91.

13. Bech-Hansen NT, Till JE, Ling V. Pleiotropic phenotype of colchicine-resistant CHO cells: cross-resistance and collateral sensitivity. J CellPhysiol 1976;88:23–31.

14. Horio M, Chin KV, Currier SJ, Goldenberg S,Williams C, Pastan I, et al.Transepithelial transport of drugs by the multidrug transporter incultured Madin-Darby canine kidney cell epithelia. J Biol Chem 1989;264:14880–4.

15. Thiebaut F, Tsuruo T,HamadaH,GottesmanMM,Pastan I,WillinghamMC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A1987;84:7735–8.

16. Ieiri I. Functional significance of genetic polymorphisms in P-glyco-protein (MDR1, ABCB1) and breast cancer resistance protein (BCRP,ABCG2). Drug Metab Pharmacokinetics 2012;27:85–105.

17. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM. P-glycoprotein: from genomics to mechanism. Oncogene 2003;22:7468–85.

18. Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, JohneA, et al. Functional polymorphisms of the human multidrug-resistancegene:multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci U S A2000;97:3473–8.

19. Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI,et al. Identification of functionally variant MDR1 alleles amongEuropean Americans and African Americans. Clin Pharmacol Ther2001;70:189–99.

20. TangK,Ngoi SM,GweePC,Chua JM, LeeEJ, ChongSS, et al. Distincthaplotype profiles and strong linkage disequilibrium at the MDR1multidrug transporter gene locus in three ethnic Asian populations.Pharmacogenetics 2002;12:437–50.

21. Marzolini C, Paus E, Buclin T, KimRB.Polymorphisms in humanMDR1(P-glycoprotein): recent advances and clinical relevance. Clin Phar-macol Ther 2004;75:13–33.

22. Johne A, Kopke K, Gerloff T, Mai I, Rietbrock S, Meisel C, et al.Modulation of steady-state kinetics of digoxin by haplotypes of theP-glycoprotein MDR1 gene. Clin Pharmacol Ther 2002;72:584–94.

23. Yi SY, Hong KS, Lim HS, Chung JY, Oh DS, Kim JR, et al. A variant2677A allele of the MDR1 gene affects fexofenadine disposition. ClinPharmacol Ther 2004;76:418–27.

24. YatesCR, ZhangW,SongP, Li S,Gaber AO, KotbM, et al. The effect ofCYP3A5 and MDR1 polymorphic expression on cyclosporine oraldisposition in renal transplant patients. J Clin Pharmacol 2003;43:555–64.

25. Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, CalcagnoAM, AmbudkarSV, et al. A "silent" polymorphism in theMDR1gene changes substratespecificity. Science 2007;315:525–8.

26. Brimacombe KR, Hall MD, Auld DS, Inglese J, Austin CP, Gottes-man MM, et al. A dual-fluorescence high-throughput cell line sys-tem for probing multidrug resistance. Assay Drug Dev Technol2009;7:233–49.

27. Sauna ZE, Muller M, Peng XH, Ambudkar SV. Importance of theconserved Walker B glutamate residues, 556 and 1201, for the com-pletion of the catalytic cycle of ATP hydrolysis by human P-glycopro-tein (ABCB1). Biochemistry 2002;41:13989–4000.

28. Ambudkar SV. Drug-stimulatable ATPase activity in crudemembranesof human MDR1-transfected mammalian cells. Methods Enzymol1998;292:504–14.

29. Ohnuma S, Chufan E, Nandigama K, Jenkins LM, Durell SR, Appella E,et al. Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1)function by 50-fluorosulfonylbenzoyl 50-adenosine: evidence for anATP analogue that interacts with both drug-substrate-and nucleo-tide-binding sites. Biochemistry 2011;50:3724–35.

30. Hull RN,CherryWR,WeaverGW.Theorigin andcharacteristics of apigkidney cell strain, LLC-PK. In Vitro 1976;12:670–7.

31. Ito S, Woodland C, Harper PA, Koren G. P-glycoprotein-mediatedrenal tubular secretion of digoxin: the toxicological significance of theurine-blood barrier model. Life Sci 1993;53:PL25–31.

32. Cornwell MM, Pastan I, Gottesman MM. Certain calcium channelblockers bind specifically to multidrug-resistant human KB carcinomamembrane vesicles and inhibit drug binding to P-glycoprotein. J BiolChem 1987;262:2166–70.

33. Kanamaru H, Kakehi Y, Yoshida O, Nakanishi S, Pastan I, GottesmanMM.MDR1RNA levels in human renal cell carcinomas: correlationwithgrade and prediction of reversal of doxorubicin resistance by quinidinein tumor explants. J Natl Cancer Inst 1989;81:844–9.

34. Fung KL, GottesmanMM. A synonymous polymorphism in a commonMDR1 (ABCB1) haplotype shapes protein function. Biochim BiophysActa 2009;1794:860–71.

35. Plotkin JB, Kudla G. Synonymous but not the same: the causes andconsequences of codon bias. Nat Rev Genet 2010;12:32–42.

36. Sauna ZE, Kimchi-Sarfaty C. Understanding the contribution ofsynonymous mutations to human disease. Nat Rev Genet 2011;12:683–91.

37. Brest P, Lapaquette P, Souidi M, Lebrigand K, Cesaro A, Vouret-Craviari V, et al. A synonymous variant in IRGM alters a binding site formiR-196 and causes deregulation of IRGM-dependent xenophagy inCrohn's disease. Nat Genet 2011;43:242–5.

38. Bruun GH, Doktor TK, Andresen BS. A synonymous polymorphicvariation in ACADM exon 11 affects splicing efficiency and may affectfatty acid oxidation. Mol Genet Metab 2013.

39. Lawrie DS, Messer PW, Hershberg R, Petrov DA. Strong purifyingselection at synonymous sites inD.melanogaster. PLoSGenet 2013;9:e1003527.

40. Lai Y, Huang M, Li H, Wang XD, Li JL. Distinct genotype distributionandhaplotypeprofiles inMDR1geneamongChineseHan, Bai,WaandTibetan ethnic groups. Die Pharmazie 2012;67:938–41.

41. Kimchi-SarfatyC,Marple AH,Shinar S, Kimchi AM,ScavoD,RomaMI,et al. Ethnicity-related polymorphisms and haplotypes in the humanABCB1 gene. Pharmacogenomics 2007;8:29–39.

42. Mechetner EB, Roninson IB. Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody. Proc NatlAcad Sci U S A 1992;89:5824–8.

43. Kim H, Barroso M, Samanta R, Greenberger L, Sztul E. Experimentallyinduced changes in the endocytic traffic of P-glycoprotein alter drugresistance of cancer cells. Am J Physiol 1997;273:C687–702.

44. Siegsmund M, Brinkmann U, Schaffeler E, Weirich G, Schwab M,Eichelbaum M, et al. Association of the P-glycoprotein transporterMDR1(C3435T) polymorphismwith the susceptibility to renal epithelialtumors. J Am Soc Nephrol 2002;13:1847–54.

45. HitzlM, Drescher S, van der KuipH, Schaffeler E, Fischer J, SchwabM,et al. TheC3435Tmutation in thehumanMDR1gene is associatedwithaltered efflux of the P-glycoprotein substrate rhodamine 123 fromCD56þ natural killer cells. Pharmacogenetics 2001;11:293–8.

46. Dalton WS, Durie BG, Alberts DS, Gerlach JH, Cress AE. Character-ization of a newdrug-resistant humanmyelomacell line that expressesP-glycoprotein. Cancer Res 1986;46:5125–30.

47. Ambudkar SV, Kim IW, Xia D, Sauna ZE. The A-loop, a novelconserved aromatic acid subdomain upstream of the Walker Amotif in ABC transporters, is critical for ATP binding. FEBS Lett2006;580:1049–55.

48. Tsai CJ, Sauna ZE, Kimchi-Sarfaty C, Ambudkar SV, Gottesman MM,Nussinov R. Synonymous mutations and ribosome stalling can leadto altered folding pathways and distinct minima. J Mol Biol 2008;383:281–91.

Fung et al.





2014;74:598-608. Published OnlineFirst December 4, 2013.Cancer Res King Leung Fung, James Pan, Shinobu Ohnuma, et al. and Protein Stability in a Stable Epithelial Monolayer

Synonymous Polymorphisms Alter Transporter SpecificityMDR1

Updated version

10.1158/0008-5472.CAN-13-2064doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2013/12/04/0008-5472.CAN-13-2064.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/2/598.full#ref-list-1

This article cites 47 articles, 14 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/74/2/598.full#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/74/2/598To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-2064

http://cancerres.aacrjournals.org/content/suppl/2013/12/04/0008-5472.CAN-13-2064.DC1

http://cancerres.aacrjournals.org/content/74/2/598.full#ref-list-1

http://cancerres.aacrjournals.org/content/74/2/598.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/74/2/598


Documents

MDR1 Synonymous Polymorphisms Alter Transporter …cancerres.aacrjournals.org/content/canres/74/2/598.full.pdfSuresh V. Ambudkar 1, and Michael M. Gottesman Abstract The drug efﬂux