PLOS ONE ABCB1 1199G>A Genetic Polymorphism (Rs2229109) Influences the… — Volkswagen 412 Variant

6 Мар 2015 | Author: | Комментарии к записи PLOS ONE ABCB1 1199G>A Genetic Polymorphism (Rs2229109) Influences the… — Volkswagen 412 Variant отключены
Volkswagen 412 Variant

ABCB1 1199GA Genetic (Rs2229109) Influences the Intracellular of Tacrolimus in HEK293 and K562 Cell Lines

Géraldine equal contributor ,

equal Contributed equally to this with: Géraldine Dessilly, Elens

Affiliation: Louvain for Toxicology and Applied Pharmacology, de Recherche Expérimentale et Clinique, Catholique de Louvain, Brussels,

Abstract

Objective

ATP-binding subfamily B, member 1 (ABCB1) or P-glycoprotein, is an efflux protein in the absorption and the distribution of various including tacrolimus and cyclosporine A. In studies suggest an association the ABCB1 1199GA single polymorphism (SNP) and tacrolimus accumulation. The aim of the present experimental was to clarify in vitro the impact of the ABCB1 1199GA SNP on ABCB1 activity towards both drugs.

Method

Two recombinant lines, i.e. Human Kidney (HEK293) and Human Leukemia (K562) cells, ABCB1 carrying either the allele (1199G) or its mutated (1199A), were generated. The of the 1199GA SNP on ABCB1 activity rhodamine (Rh123), doxorubicin, tacrolimus and cyclosporine A was assessed by cytotoxicity and/or kinetic

Results

Tacrolimus accumulation was decreased in cells overexpressing the protein (1199G) compared to cells, confirming the ability of to transport tacrolimus. By contrast, of the variant protein (1199A) had no effect on tacrolimus intracellular whatever the model used and the tested. Unlike tacrolimus, our also indicate that A, Rh123 and doxorubicin are transported in a extent by the wild-type and variant proteins while the variant seems to be more efficient for the of vinblastine.

Conclusion

ABCB1 by the 1199G wild-type allele more efficiently tacrolimus in to the 1199A variant protein. observation indicates that the substitution (Ser400Asn) encoded by the allele drastically decreases the of ABCB1 to drive the efflux of in a substrate-specific manner, in agreement our previously published clinical Our study emphasizes the importance of the 1199GA polymorphism for ABCB1 and its potential to explain differences in response.

Citation: Dessilly G, L, Panin N, Capron A, Decottignies A, et al. ABCB1 1199GA Genetic (Rs2229109) Influences the Intracellular of Tacrolimus in HEK293 and K562 Cell Lines. PLoS ONE e91555. doi:10.1371/journal.pone.0091555

Editor: Zhang, Indiana University of Medicine, United States of

Received: August 6, 2013; February 13, 2014; Published: 12, 2014

Copyright: © 2014 et al. This is an open-access article under the terms of the Creative Attribution License. which unrestricted use, distribution, and in any medium, provided the original and source are credited.

Funding: Dessilly (GD) is a doctoral with the Fonds National de la Scientifique (FNRS)- Télévie, (credit n°7.4539.13). Laure (LE) is a post-doctoral researcher the Fonds National de la Recherche (FNRS, Belgium. The funders had no in study design, data and analysis, decision to publish, or of the manuscript.

Competing interests: The authors declared that no competing exist.

Introduction

P-glycoprotein is a phosphorylated transmembrane glycoprotein of 170 kDa mediates the ATP-dependent efflux of endogenous and exogenous substances biological membranes. P-gp is in humans by the ABCB1 gene spans 28 exons on chromosome 7. The protein (official nomenclature for belongs to the group B of the ATP binding (ABC) superfamily and is composed of amino acids consisting of two and symmetric domains of 6 transmembrane Each domain encloses an nucleotide binding domain which binds ATP [1] –[4]. The of ABCB1 is mainly distributed in organs and in the blood brain [1]. [5]. Consequently, is thought to ensure a protective with respect to the absorption the distribution (blood brain lymphocytes) and the excretion (hepatocytes, of potential harmful substances by their transport from the to the extracellular compartiment [1]. [6] Its substrates spectrum is very and includes important exogenous such as antiretrovirals and immunosuppressive ( e.g. tacrolimus (Tac) and A (CsA) [11] ) and also drugs (e.g. imatinib ). Overall, this protein the pharmacokinetics of various compounds and its in cancer has been suggested as a cause of resistance to treatments [10]. [13] –[16] .

the last decade more 50 single-nucleotide polymorphisms (SNPs) been identified in ABCB1 –[19]. The three most SNPs in the protein coding are rs1128503 (1236CT, Gly412Gly), (2677GT/A, Ala893Ser/Thr), and rs1045642 Ile1145Ile) [20]. [21]. three SNPs are in strong disequilibrium (LD) and have extensively investigated. However, the that have been so far are inconsistent and the real functional of these three SNPs controversial [22]. Other frequent SNPs have described and could also explain part of the variability in the expression and/or function of [5]. Of particular interest, the 1199GA coding SNP located in the 11 (rs2229109) is relatively frequent a reported allelic frequency of 6% in the Caucasian population. Therefore, to the Hardy-Weinberg distribution, the expected frequencies are respectively of 0.4% for the mutated AA, 11.2% for the heterozygotes GA and for the homozygotes wild-type GG. This SNP is with a serine to asparagine at position 400 in a cytoplasmic loop of which is involved in substrates [2]. A small number of in studies have assessed the of the ABCB1 1199GA SNP on the ABCB1 activity towards its substrates. One reported that cells the variant protein (ABCB1 cells) were characterized by a efflux of Rhodamine123 (Rh123), a substrate of ABCB1, when to cells overexpressing the wild-type (ABCB1 1199G/wt cells). In the study, cytotoxicity experiments anticancer drugs have that ABCB1 1199G/wt and 1199A/mut cells exhibited resistance to doxorubicin, while 1199A/mut cells were resistant to vinblastine and vincristine in contrast, an increased efflux of the 1199A variant towards anticancer drugs [23]. A recent study suggested an increased efflux activity of the variant towards several inhibitors (PI) used in the of Human Immunodeficiency Virus infection [24]. It can be thus hypothesized that the impact of SNP, may differentially affect the and/or the activity of this in a substrate-dependent manner.

Tacrolimus (Tac) is an immunosuppressive belonging to the calcineurin inhibitor and is widely used in kidney and transplantation to prevent graft [25]. [26]. Tac is characterized by a narrow therapeutic window and a inter-individual variability in its pharmacokinetic and behavior [25]. [27]. Despite the use of therapeutic drug a relatively high rate of failure and toxicity events are encountered. The identification of genetic influencing Tac pharmacokinetics and/or might potentially help the to better adjust Tac therapy after transplantation. In this we have previously linked the variant to an increased accumulation of Tac in biopsies [29] and in peripheral mononuclear cells (PBMCs) include T-lymphocytes, the cellular of Tac [30] and suggesting a decrease in mediated efflux activity Tac in vivo .

Cyclosporin A (CsA) is powerful immunosuppressive drug, in transplantation to prevent graft but to a lesser extent than Tac because the concentrations used in are more nephrotoxic. It was demonstrated in that the 1199GA SNP influences the CsA in PBMCs of patients with transplantation [31]. Carriers of the variant presented lower levels compared to homozygotes patients, arguing for an opposite effect of the ABCB1 1199GA SNP CsA-mediated efflux when to Tac transport.

The aim of the present experimental was to clarify and further confirm in the impact of the ABCB1 1199GA on ABCB1 expression and transport towards both immunosuppressive (Tac and CsA) using two transfected cell lines.

and Methods

Materials

Tac (Prograf IV) and CsA were purchased from Pharma (Brussels, Belgium) and Pharma (Vilvoorde, Belgium), LY335979 (Zosuquidar 3HCl) was from Bio-connect (Huissen, G418 was purchased from Applied Science (Vilvoorde, Rhodamine (Rh123) was purchased Sigma-Aldrich (St-Louis, United Doxorubicin and vinblastine were from Pfizer (Brussels, and Teva (Wilrijk, Belgium),

HEK293 is a cell line from human embryonic cells grown in tissue (ATCC CRL-1573™). K562 is a line derived from the effusion of a 53-year old female chronic myelogenous leukemia in blast crises (ATCC

Cell culture

HEK293 grown in Dulbecco’s Modified medium (DMEM) 1g/l and glutamine (Gibco, Invitrogen) with 10% (v/v) of Fetal Serum (FBS) (Gibco, and 1% (v/v) of Antibiotic-Antimycotic solution (Gibco, Invitrogen) at a temperature of in the presence of 5% of CO 2 . For immunofluorescence staining and accumulation assays, HEK293 were grown in DMEM 4.5 g/l glucose (Gibco, Invitrogen) with 10% (v/v) FBS and 1% (v/v)

K562 were grown in Modified Eagle Dulbecco’s (IMDM) (Gibco, Invitrogen) with 10% (v/v) of FBS (Gibco, and 1% (v/v) A-A (Gibco, Invitrogen) at a of 37°C in the presence of 5% of CO 2 . For intracellular assay, K562 cells grown in IMDM Glutamax Invitrogen), 10% (v/v) FBS and 1% (v/v)

Generation of ABCB1 1199G/wt and 1199A/mut plasmids

The expression pcDNA 3.1 with cDNA ABCB1 ( ABCB1 1199G/wt ) was provided by Dr Rodney Ho (University of The mutated plasmid designated 1199A/mut was generated by site-directed using the QuickChange II XL Site-directed Kit (Stratagene) with the mismatched 5′-CAG AAA TGT TCA CTT CAA TTA CCC ATC TCG-3′ (forward) and TTC GAG ATG CGT AAT TGA AGT GAA CAT-3′ (reverse). The plasmids sequenced to confirm the presence of the

Generation of stable recombinant lines

HEK293 cells plated the day before transfection in 6 plates (2 10 5 cells/well) and transfected lipofectamine 2000 (Invitrogen) to the manufacturer’s protocol with 4 μg of DNA. To select stable cells were replated at a low 24h after the transfection and G418 was 24h later at a final concentration of 0.8

K562 cells (10 7 cells) transfected by electroporation (ECM630 from BTX, 200 V, 75 Ω, 1300 μF) 50 μg of plasmid DNA. To select transfectants, cells were at a low density 24h after the transfection and was added 48h later at a final of 1 mg/ml.

Volkswagen 412 Variant

After one week on cell surface ABCB1 expression in transfected cells and K562) was analyzed by flow [FITC Mouse Anti-Pglycoprotein diluted 1:10, (clone BD Pharmingen) or isotypic control 1:10 [(FITC Mouse IgG clone 557002, BD Pharmingen) below)].

Characterization of ABCB1

Flow cytometry.

10 6 cells and K562) were recovered by Cells were washed with 2 ml of ice-cold HAFA [filtrated (0.22 μm) Hank’s with 3% decomplemented FBS and NaN 3 (20 mmol/l)]. were resuspended in HAFA containing the primary FITC Anti-P-glycoprotein antibody diluted (clone17F9 557002, BD Pharmingen) or its isotypic control diluted (FITC Mouse IgG 2bk, 555742, BD Pharmingen) and incubated 45 min on ice in the Cells were further with 2 ml of HAFA solution, and finally fixed in 1:1 HAFA/paraformaldehyde (v/v). Samples were on a Fluorescence-activated cell sorting Calibur (BD Sciences).

HEK 1199G/wt or K562 1199G/wt and HEK or K562 1199A/mut cells sorted with fluorescence gated on the same level of to ensure similar ABCB1 The protocol was identical (see except the using of HAFA without NaN 3 .

HEK293 cells rinsed twice with D-PBS (Invitrogen) and recovered triton lysis buffer TritonX100 0.1%, NaCl 150 mM, pH 7.5 25 mM supplemented with complete inhibitors cocktail (Roche)) and for 15 min on ice. The solution was centrifuged for 10 4°C at 14000 rpm and the supernatant was recovered. The lysate was then sonicated for 5 min and diluted in Laemmli sample (Bio Rad, Hemel UK) containing 10% of β-mercaptoethanol. The cell was passed five to six times 26G needle, and samples were at 60°C during 10 min to denature In order to facilitate the migration, were finally centrifuged 5 14000 rpm at room temperature to cellular debris. 30 μl of each (40 μg of total proteins) and 15 μl of protein (Multicolor High Range Ladder, Fermentas) were on a 7.5% Mini-Protean TGX precasted gel as by the manufacturer (Bio Rad). were then transferred a PVDF membrane using the Semi-dry blotter (VWR) at 80 for 120 min. Blots were with bloking buffer Biosciences) for at least 60 min. several washing steps TBS-T, membranes were overnight at 4°C with a monoclonal antibody anti-P-gp (clone mouse, 1:200 dilution, and with a monoclonal anti-calnexin (ADI-SPA-860, 1:15 000 dilution, Life Sciences) diluted in After three steps of in TBS-T, donkey anti-mouse IgG LI-COR Biosciences) and donkey IgG (926-3223 LI-COR Biosciences) antibodies were applied to the at a 1:15000 and diluted in TBS-T for 60 The transferred proteins were using odyssey IR imaging (LI-COR Biosciences).

Immunofluorescence.

One day the experiment, HEK293 cells plated at a density of 10 5 cells/well in medium. The next day, were rinsed with BSA, fixed with 4% of during 15 min and rinsed with BSA. Cell membranes subsequently permeabilized with Triton X100 during 5 After a washing step PBS/0.1% BSA, cells incubated with the primary antibody Ab4E3 (ab10333, 5 μg/ml, diluted with BSA) or with its isotopic (Mouse Ig2a kappa ab10353, Abcam, 25 μg/ml) for 90 min in the After two washing steps PBS and PBS/0.1% BSA, respectively, were incubated for 60 min with anti-mouse IgG coupled to FITC Abcam, 1 μg/ml) and with (Hoechts 33258 pentahydrate H3569, Invitrogen). Finally, the were fixed once with 4% of paraformaldehyde during 5 Fluorescence was analyzed in fluorescent medium (Dako) with a microscope Evos fluorescence

Rh123 functional test

One day the experiment, 10 5 cells (HEK293 or were plated in poly-L-lysine-coated 96-well plates in complete ABCB1 related transport was investigated by loading the cells for 60 min at with 5 μM of Rh123 in the dark. indicated, ABCB1 inhibition was by pre-incubating the cells for 15 min in the presence of (0.1 and 0.2 μM). After with Rh123, the supernatants discarded. The cells were two times with Dulbecco’s-PBS at 4°C and lysis was performed with X100 1% in DMEM/or IMDM at room temperature for 30 min for HEK293 or respectively. Finally, the intracellular of Rh123 was analyzed by a fluorimeter Gemini Xs (Molecular Devices); wavelength was set at 485 nm and emission at 530 nm.

Thymidine assay

K562 cells plated at 10 4 cells/well in 96-well in complete medium. Cells incubated for 24h at 37°C with concentrations of doxorubicin (from 10 to nM) or vinblastine (from 1.11 to 270 One 1 μCi of [ 3 H] thymidine was then added to well and further incubated for The radioactivity was measured with a NXT liquid scintillation counter

Graphics are represented by the relative (%); CPM samples/CPM control drugs exposure) *100.

Tac and CsA

Tac or CsA accumulation experiments were according to a previously described [32]. [33]. with modifications. One day before the experiment, 3.5 10 5 (HEK293 or K562) were in 24-wells plates in 500 μl of complete Tac or CsA were added at six different (from 0.0015 to 0.5 μM and 0.015 to 5 μM, and cells were incubated for 120 min at 5% of CO 2 . After incubation with Tac or the supernatant was discarded by splashing the and the cells were washed two in cold PBS and detached with PBS supplemented with 0.2% After centrifugation at 4°C, the was discarded and cells pellets conserved at –80°C until analysis.

Tac intracellular kinetics

One day the experiment, 3.5 10 5 cells (HEK293 or were seeded in 24-wells in 500 μl of complete medium. Tac was added at a concentration of 0.05 μM and cells incubated for 120 min at 37°C, 5% of CO 2 . After with Tac, the supernatant was by splashing the plate and the cells allowed to efflux in serum-free for 15 sec, 30 sec, 1 min, 5 10 min, 30 min and 1 h. At each time (including the point measured in experiments, corresponding to 120 min of accumulation), were washed two times in PBS and detached with ice-cold PBS with 0.2% EDTA. centrifugation at 4°C, the supernatant was and cells pellets were at –80°C until LC-MS/MS

Tac was quantified in cell pellets to a previously published LC-MS/MS [34]. with minor The chromatography system used was an (Waters) coupled with a mass spectrometer Quattro (Micromass). The analytic column was a Phenyl 3.5 μm×50 mm (Waters). after alkalinization of cells ammonium hydroxide 5% (v/v), Tac was with chlorobutane containing the standard, consisting of ascomycin at a of 100 ng/ml. Blank cell were processed in parallel for the curve (from 1.25 nM to μM). Then, the organic was decanted, and evaporated to dryness. The dry was then reconstituted with and transferred to a high-performance liquid vial and 10 μl was injected into the The absolute amount of drug in cell extracts was normalized to the of protein present as assessed the BCA kit (Thermoscientific).

We used the chromatography and the analytic column described for Tac quantification. CsA was extracted by precipitation ZnSO 4 0.1 M in aqueous solution and a solution of acetonitrile containing the standard (ascomycin 100 ng/ml). cell pellets were in parallel for the calibration curve 1.25 nM to 1 μM). Then, the was recovered and transferred to a high-performance chromatography vial and 10 μl was injected the LC-MS/MS. The absolute amount of present in extracted cells was to the amount of protein present as using the BCA kit (Thermoscientific).

All experiments repeated minimum twice. analyses were performed by GraphPad InStat (Version Analyses of variance were under the null hypothesis the means of the compared groups equal. Student-Newman-Keuls tests carried out when the differences means were significant. P less than 0.05 considered as statistically significant.


Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant
Volkswagen 412 Variant

Articles

Other articles of the category "Variant":

Twitter-news
Our partners
Follow us
Contact us
Our contacts

dima911@gmail.com

Born in the USSR

About this site

For all questions about advertising, please contact listed on the site.


Volkswagen all cars catalog with specifications, pictures, ratings, reviews and discusssions about cars Volkswagen.