Drug Metabolism and Disposition Fast Forward
First published on January 19, 2007; DOI: 10.1124/dmd.106.012302
0090-9556/07/3504-623-632$20.00
DMD 35:623-632, 2007
Identification and Functional Assessment of BCRP Polymorphisms in a Korean Population
Sang Seop Lee,
Hye-Eun Jeong,
Joo-Mi Yi,
Hyun-Ju Jung,
Jae-Eun Jang,
Eun-Young Kim,
Su-Jun Lee, and
Jae-Gook Shin
Department of Pharmacology and Pharmacogenomics Research Center (S.S.L., H.-E.J., J.-M.Y., H.-J.J., J.-E.J., S.-J.L., J.-G.S.) and Department of Clinical Pharmacology (E.-Y.K., J.-G.S.), Inje University College of Medicine, Inje University Busan Paik Hospital, Busan, Korea
(Received August 2, 2006;
accepted January 16, 2007)
 |
Abstract
|
|---|
The breast cancer resistance protein (BCRP) is a member of the ATP-binding cassette transporters. The aim of the present study was to identify genetic variants of BCRP in Koreans and to assess the functional consequences of BCRP polymorphisms. Twenty single nucleotide polymorphisms (SNP), including four nonsynonymous SNP, were identified by DNA sequencing of the BCRP gene in 92 Korean subjects. BCRP V12M, Q141K, P269S, and Q126Stop were detected at frequencies of 23, 28, 0.2, and 1.9%, respectively. These four coding variants were also screened in Chinese and Vietnamese subjects; the allelic frequencies among the three populations were compared; and predictions were made as to the potential frequency of each variant. In vitro functional analyses of the P269S protein and the promoter SNP –19031C>T (mutated in the hypoxia-inducible factor-1
binding site) were performed and compared with those of the wild type. P269S exhibited a 35 to 40% decrease in vesicular uptake of [3H]estrone-3-sulfate and [3H]methotrexate compared with the wild type. The promoter SNP –19031C>T did not affect BCRP promoter activity in either the presence or absence of chemical-induced hypoxic stress. Our results suggest that the P269S variant could be a functionally altered variant. Genotyping of this variant in clinical studies is needed to address its phenotypic role. Genetic polymorphisms of BCRP were found to be very common in Koreans, as well as in other ethnic groups. Comparative analyses among three Asian populations revealed different frequencies for the four functional BCRP variants.
The breast cancer resistance protein (BCRP/ABCP/MXR/ABCG2) is a member of the plasma membrane transporter family and is an ATP-binding cassette protein. BCRP was first identified in multidrug-resistant breast cancer cell lines that were selected in the presence of doxorubicin and verapamil (Doyle et al., 1998
). BCRP has also been identified in the human placenta (Allikmets et al., 1998), as well as in cancer cell lines selected in the presence of anticancer agents, such as mitoxantrone (Miyake et al., 1999). The BCRP gene is located on chromosomal region 4q22, consists of 16 exons that span over 66 kb, and encodes a 72-kDa membrane protein that is composed of 655 amino acids (Allikmets et al., 1998; Doyle et al., 1998; Bailey-Dell et al., 2001).
The normal physiological function of BCRP has not been clearly established. High-level expression of ABCG2 has been observed in certain drug-resistant cell lines and tumors, conferring a special multidrug-resistant phenotype on these cancer cells (Miyake et al., 1999; Litman et al., 2001; Volk et al., 2002). In normal human tissues, the highest expression of BCRP is seen in the placenta (Allikmets et al., 1998), whereas BCRP is expressed at lower levels in the brain, prostate, testis, liver, small intestine, colon, ovary, kidney, and heart (Doyle et al., 1998). This expression profile suggests that BCRP plays a role in protecting the fetus from toxins (Allikmets et al., 1998; Maliepaard et al., 1999). BCRP is also known to confer resistance to chemotherapeutic agents, such as mitoxantrone, methotrexate, topotecan, SN38, and flavopiridol (Jonker et al., 2000; Robey et al., 2001).
Several BCRP variants have been reported from direct DNA sequencing of the BCRP gene. Among these, some coding variants of BCRP exhibit increased resistance to anticancer drug treatment (Honjo et al., 2001; Mitomo et al., 2003). It has been recognized that single nucleotide polymorphisms (SNP) of BCRP cause interindividual variations in the pharmacokinetics for BCRP substrates (Sparreboom et al., 2004).
According to the current literature, the most frequent BCRP polymorphisms detected among different ethnic groups are 34G>A, which codes for V12M, and 421C>A, which codes for Q141K (Zamber et al., 2003). The BCRP Q141K SNP has been associated with decreased expression of the BCRP protein in that this genotype produces a reduced transporter activity phenotype (Imai et al., 2002; Kondo et al., 2004). The BCRP V12M SNP has been reported as having similar activity to the wild type in terms of dehydroepiandrosterone sulfate, methotrexate (MTX), p-aminohippuric acid (Kondo et al., 2004), and porphyrin transport (Ai et al., 2006). However, Mizuarai et al. (2004) have shown disrupted membrane localization of the V12M variant, which results in decreased transport activity.
Despite several reports on BCRP polymorphisms among various ethnic groups, there have been no reports on BCRP SNP in a Korean population. Therefore, we investigated, for the first time, genetic polymorphisms of the BCRP gene in a Korean population and performed in vitro functional characterization of a novel regulatory SNP and a coding SNP (P269S). The functional capability of the P269S variant to take up [3H]estrone-3-sulfate (ES) and [3H]MTX has been reported as being comparable with that of the wild-type protein (Kondo et al., 2004). However, in the present study, BCRP P269S from human blood samples (not from cell lines) was used for the first time. Functional information on P269S is sparse, as compared with the amount of information that has been collected for other coding variants, such as V12M, Q141K, and the null allele Q126Stop. Because proline is known to be a helix breaker, the three-dimensional structure of the BCRP protein may be affected in the P269S variant. Therefore, we performed a functional study of the P269S variant among the four variants identified in the study (V12M, Q141K, P269S, and Q126Stop). We also compared the allelic frequencies of the BCRP variants in Koreans with those in Chinese and Vietnamese populations, and we discuss possible ethnic differences with respect to BCRP polymorphisms.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 1. LD block structure and haplotype diversity of the BCRP gene. A, genetic organization of BCRP and positions of the identified variations. Eleven common BCRP SNP in HWE and the common haplotype profiles are shown. B, pair-wise LD among the BCRP SNP, as calculated by the Haploview software. The dark black box without a star represents a ID'I value of 1.00, and the dark black boxes that contain a star represent values that are not determinable. The higher ID'I values with strong linkage are indicated by dark coloration. Gray and white colorations indicate weak linkages with low ID'I values.
|
|
|
Materials and Methods
|
|---|
Subjects. In all, 275 Korean subjects were recruited for the BCRP genotyping study. Genomic DNA samples from 159 Vietnamese, 192 Chinese, 100 African-American, and 100 Caucasian subjects were obtained from the DNA Repository of the Pharmacogenomics Research Center, Inje University (Lee et al., 2005). All the participants were healthy according to their medical histories, physical examinations, and routine laboratory tests. All the participants provided written informed consent for the present study, which was approved by the Institutional Review Board of Busan Paik Hospital (Busan, Korea).
DNA Purification and Sequencing. For direct DNA sequencing and pyrosequencing of the BCRP gene, genomic DNA was isolated from whole blood cells using the Qiagen DNA Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Genomic DNA samples from 92 of 275 Korean subjects were directly sequenced for the identification of novel sequence variants, and the DNA samples from the remaining 183 subjects were screened for the identified variants by pyrosequencing. Specific primers were designed using the Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to amplify 16 exons of BCRP, the proximal promoter region, and the 3'-untranslated region. Polymerase chain reaction (PCR) was performed in a reaction volume of 20 µl in the presence of 150 ng of genomic DNA, 0.2 mM deoxynucleoside-5'-triphosphate, 0.2 µM each primer, 1.5 mM MgCl2, and 1 U of Taq polymerase (Roche Pharmaceutical Co., Basel, Switzerland). PCR was performed in the GeneAmp PCR 9700 (Applied Biosystems, Foster City, CA), with an initial denaturation step of 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 54.0 to 64.7°C for 30 s, and extension at 72°C for 30 s. A final termination of elongation step was performed at 72°C for 5 min. The primer sequences and annealing temperatures used are listed in Table 1. The amplified products were directly sequenced using an automated sequencer with the BigDye Terminator Sequencing Kit (Applied Biosystems).
View this table:
[in this window]
[in a new window]
|
TABLE 1 Primer sequences used for the amplification of the BCRP gene fragment and the annealing temperatures used in the PCR
|
|
Genotyping of BCRP Variants by Pyrosequencing. To determine the allelic frequencies of the genetic variants, further genotyping was performed by pyrosequencing the genomic DNA samples from an additional 183 Korean subjects. All the subjects were screened for BCRP V12M, Q126Stop, Q141K, and P269S. To detect the BCRP variants, partial fragments of BCRP gene were amplified using the primer pair, of which one primer was biotinylated at its 5'-end. Sequences of the primers and denaturation temperature for PCR are listed in Table 2. Biotinylated PCR products were immobilized into streptavidin-coated beads (Streptavidin Sepharose High Performance, Amersham Biosciences) following the strand separation for the PSQ 96 Sample Preparation kit (Pyrosequencing Amersham Biosciences). Briefly, Sepharose bead slurry (3 µl) was mixed with binding buffer (37 µl), PCR product (15 µl), and distilled water (25 µl) incubated at room temperature for 10 min at 1400 rpm. The beads were transferred to a filter plate, and liquid was removed by vacuum filtration (Multiscreen Resist Vacuum Manifold, Millipore Inc., Billerica, MA). The DNA strands were separated in denaturation solution (0.5 M NaOH) for 5 s. The immobilized template was washed in 10 mM Tris-acetate, pH 7.6, solution (washing buffer) and transferred to a PSQ 96 plate, resuspended in annealing buffer (20 mM Tris-acetate, pH 7.6) containing sequencing primer as in Table 2. Then, sequencing primer was annealed at 80 to 90°C for 3 min. The sequence was analyzed using a PSQ 96 system with SNP Reagent Kit (Pyrosequencing).
Linkage Disequilibrium and Haplotype Analysis. In addition to the unphased SNP analysis, the haplotypes and pair-wise linkages were analyzed using the SNPAlyze software (Dynacom Co., Yokohama, Japan) and Haploview v3.32 (http://www.broad.mit.edu/mpg/haploview/). The linkage disequilibrium (LD) for each pair of segregating sites was estimated by the values of ID'I and r2. From the inferred haplotypes, all the haplotypes with frequencies >1% were regarded as common haplotypes.
Expression of Recombinant Wild-Type and Variant BCRP Proteins. The cDNA that encode the R482 and G482 proteins were amplified by PCR using their plasmid constructs as templates (provided by Dr. Douglas D. Ross, Greenebaum Cancer Center, University of Maryland, Baltimore, MD). For subcloning of the BCRP variant and wild-type cDNA into the baculoviral vector, the BCRP coding region was amplified with the primer pair: 5'-ACGCGTAAGCTTATGTCTTCCAGTAATGTC-3' and 5'-ACGCGTCTCGAGTTAAGAATATTTTTTAAG-3'. The PCR products were ligated into the pFastBac vector (Invitrogen, Carlsbad, CA). The BCRP R482-P269S and G482-P269S variants were generated by an overlap extension procedure using specific primers that introduced amino acid substitutions: 5'-AGGCCTCCTGAGCAGACCCGTGGAACATAAG-3' and 5'-CTTATGTTCCACGGGTCTGCTCAGGAGGCCT-3'. The introduction of mutations was verified by DNA sequencing of all the constructs. The entire open reading frame sequences for the BCRP wild-type and coding variants in the pFastBac plasmid were verified by direct sequencing before expression in insect cells. Sf9 cells were maintained at 27°C in Grace's Insect Cell culture medium that contained 3.3 g/l yeastolate, 3.3 g/l lactalbumin hydrolysate, and 10% heat-inactivated fetal bovine serum. The Sf9 cells were infected with the recombinant baculovirus at a multiplicity of infection of 5 to 6. BCRP expression was under the control of the polyhedron promoter. Cells were harvested 72 h after infection for the preparation of microsomal fractions. Similar amounts of BCRP protein expression were confirmed by immunoblot analysis. The microsomal fraction (baculosome) from the Sf9 cells that expressed BCRP protein was prepared as described previously (Mizuarai et al., 2004). Cell pellet was diluted with hypotonic buffer (0.5 mM Tris-HEPES, pH 7.4, and 0.1 mM EGTA) and homogenated with a Teflon pestle homogenizer. After centrifugation at 2000g, the supernatant was further centrifuged at 100,000g for 30 min. The resulting pellet was suspended in buffer A (0.25 M sucrose, 10 mM Tris-HEPES, pH 7.4), layered over 40% (w/v) sucrose solution, and centrifuged at 100,000g for 30 min. The turbid layer at the interface was collected and centrifuged once more in buffer A. Resulting membrane fraction was resuspended in small volume of buffer A. The membrane fraction was separated on a 10% SDS-polyacrylamide gel. The resolved proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane at 25 V for 30 min using a semidry apparatus (Bio-Rad, Hercules, CA). The membrane was immunoblotted with anti-BCRP antibody (no. 405 clone in Litman et al., 2002). The antibody was provided by Dr. Susan E. Bates (Molecular Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD). The specific expression of BCRP was also confirmed by another anti-BCRP antibody (clone BXP-21 from Alexis Corporation, Lausen, Switzerland). After incubation of the membrane with horseradish peroxidase-conjugated anti-mouse IgG, the BCRP proteins were visualized using the Enhanced Chemiluminescence Western Blotting Detection System (Santa Cruz Biotechnology, Santa Cruz, CA). Gel images were analyzed using the MultiGauge version 2.2 software (Fuji Photo Film Co., Tokyo, Japan) and the LAS-3000 image analyzer (Fuji Photo Film).
Vanadate-Sensitive BCRP ATPase Activity. BCRP baculosome (5 µgof protein per reaction) was incubated in a 40-µl assay mixture that contained 50 mM 3-(N-morpholino)propanesulfonic acid-Tris, pH 7.0, 50 mM KCl, 5 mM sodium azide, 2 mM DTT, 0.1 mM EGTA-Tris, pH 7.0, and 1 mM ouabain for 20 min at 37°C in the presence or absence of 1.0 mM vanadate to eliminate nonspecific phosphatases and ATPases from the vacuolar, mitochondrial, or plasma membrane. To determine the specific ATPase activity of recombinant BCRP, 1 µl of 5 mM prazosin and 0.5 mM Hoechst 33342 were added into the reaction mixture as the representative substrate and inhibitor, respectively. The reactions were stopped, and the amount of inorganic phosphate (Pi) released was determined immediately by measuring the optical density at 630 and 850 nm. Dimethyl sulfoxide used for compound dilution exhibited no effect on BCRP ATPase activity. Specific BCRP ATPase activities were calculated by subtracting the vanadate-sensitive Pi value from the total Pi value in the absence of vanadate.
Analysis of Vesicular Uptake of [3H]ES and [3H]MTX. ATP-dependent transport of [3H]ES and [3H]MTX into membrane vesicles was measured by rapid filtration. Transport assays were performed at 37°C for 15 min in a 62.5-µl volume that contained 10 µl of membrane vesicles (5 mg/ml), 1 µlof 0.2 M MgATP or 0.2 M MgAMP, 1 µlof[3H]MTX (1 mCi), and 1 µl of 0.75 mM MTX in transport buffer. In the case of [3H]ES, all the components of reaction were same as with [3H]MTX except [3H]ES. The reaction was stopped on ice, and the vesicles were collected by filtration through a Unifilters GF/B 96-well plate (PerkinElmer, Boston, MA). Radioactivity was measured in a scintillation counter (Beckman Scintillation Counter, Beckman Coulter Inc., Fullerton, CA). ATP-dependent uptake of [3H]ES and [3H]MTX was calculated by subtracting uptake measured in the presence of AMP from uptake measured in the presence of ATP. The data are presented as relative values to the amount of protein in the membrane vesicles.
DNA Constructs and Site-Directed Mutagenesis. The pGL3-BCRP (–1235 to +362) plasmid was used (provided by Dr. Douglas D. Ross). To assay the transcriptional activity of the variant-type promoter (–936C>T, –689A>G, and –114C>T), mutations were introduced into pGL3-BCRP (–1235 to +362) using an oligonucleotide-directed mutagenesis system (Transformer Site-Directed Mutagenesis kit; BD Biosciences, San Jose, CA) according to the manufacturer's instructions. The synthetic oligonucleotides used for mutagenesis were as follows: 5'-CGATAAGGTACCGTCGACCGATGC-3', together with 5'-CCCAAATGTTTGGGTGCATCATGAATAT-3' for –936C>T, 5'-CTAATTTCCTAGGGTGGATGCAGCAGGTAG-3' for –689A>G, and 5'-CAGCGCGGCAGGACATGTGTGCGCTTTCAG-3' for –114C>T. The introduction of each mutation was verified by DNA sequencing.
Transient Transfection and Promoter Activity Analysis. The human embryonic kidney (HEK)-293F cells were plated into a six-well plate in triplicate at 5 x 105 cells/well and were cotransfected with 1 µgof BCRP promoter construct and 0.1 µgof ß-galactosidase-expressing plasmid using the FuGENE 6 transfection reagent according to manufacturer's protocol. Cells were harvested after 48 h and analyzed for luciferase and ß-galactosidase activities using the Luciferase Reporter Gene Assay and Chlorophenolred-ß-D-galactopyranoside (Roche Applied Science), respectively. Luciferase activity was normalized with respect to ß-galactosidase activity. Each set of transfections was performed in triplicate and repeated at least twice. To assess the relative contribution of the –114C>T variation to BCRP transcriptional activity under hypoxic conditions, the cells were cotransfected with 400 ng of hypoxia-inducible factor-1 (HIF-1)-expressing plasmid. The control vectors, ARNT (400 ng) and pCMV-ß-galactosidase (100 ng), were used as internal controls for transfection efficiency. At 24 h post-transfection, the cells were incubated in the presence of 100 µM deferoxamine for 24 h. All the luciferase activities were normalized with respect to ß-galactosidase activity.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 3. In vitro functional characterization of the BCRP P269S variant. A, vesicular uptake of [3H]MTX in membranes obtained from Sf9 cells that express the wild-type BCRP protein or the BCRP P269S variant protein. Isolated Sf9 membrane fractions were incubated with [3H]MTX for 15 min, washed to remove unabsorbed substrate, and the radioactivity was measured in a liquid scintillation counter. BCRP P269S exhibited significantly decreased activity compared with the wild type in a two-tailed Student's t test; *, p < 0.05. B, vesicular uptake of [3H]ES membranes obtained from Sf9 cells that express the wild-type BCRP protein or the BCRP P269S variant protein. All the procedures were same as for [3H]MTX except substrate. C, expression level of wild-type and variant BCRP proteins in Sf9 cell membranes. Empty baculoviral construct was infected to Sf9 cells as a negative control of specific expression of BCRP proteins (Mock). The same membranes in Fig. 3, A and B, were immunoblotted using anti-BCRP antibody as described under Materials and Methods.
|
|
|
Results
|
|---|
Identification of BCRP Genetic Variants in a Korean Population. All 16 exons, intron-exon boundary regions, and the promoter region of the BCRP gene were sequenced to identify BCRP SNP in 92 Korean subjects. Twenty BCRP SNP were identified in this study. Five variants were found in the promoter, 5 in the exons, and 10 in the intron regions. The four coding SNP were 34G>A coding for V12M, 8191C>T coding for Q126Stop, 8825C>A coding for Q141K, and 21850C>T coding for P269S. For more extensive evaluation of the allelic frequencies of the four BCRP variants found in the Korean population, the remaining 183 subjects were screened by pyrosequencing for the presence of V12M, Q126Stop, Q141K, and P269S. The allelic frequencies and locations of the BCRP genetic variants identified in the Korean subjects are listed in Table 2. V12M and Q141K were found in 23 and 28% of Koreans, respectively. Q126Stop and P269S were found in 1.9 and 0.2% of Koreans, respectively. The frequencies for the 10 intronic SNP ranged from 10 to 67%. Recently, Kondo et al. (2004) reported the identification of several BCRP variants, which include A149P, R163K, Q166E, P269S, and S441N, in human cell lines. With the exception of the BCRP P269S variant, these variants were not observed in this study.
LD and Haplotype Structure. LD was analyzed using D' values across the common SNP, which were found with allelic frequencies of >5%. All the SNP used for the LD analysis exhibited Hardy-Weinberg Equilibrium (HWE). The positions of 11 common BCRP SNP calculated by HWE and their haplotype profiles are shown (Fig. 1A). The LD profile of BCRP showed that all the SNP pairs in the segment of 34G>A-238A>G-7430A>G-8825C>A were in complete LD (D' = 1) (Fig. 1). In the second segment covering six SNP (38485-A>G-40086insA-42288C>T-44072C>T-44997A>G-45235C>T), all the SNP pairs, with the exception of the 40086insA/42288C>T pair, were to a large extent in LD, with D' = 0.9. The BCRP haplotypes for 11 common SNP with frequencies >5% were analyzed using the SNPAlyze program (Table 3). Fourteen haplotypes were predicted to have frequencies >1%. The most frequent allele was the wild type (26%), followed by the Q141K variant. The haplotype analysis suggests that none of the Q141K-containing haplotypes are linked to the V12M variant. To support this strong linkage, the V12M and Q141K variations were assigned to the same haplotype block among two discrete haplotype blocks of the BCRP gene (Fig. 1B).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 4. Prazosin-dependent ATPase activity of the BCRP P269S variant. A, AT-Pase activities of Sf9 cells that express the wild-type BCRP protein or the BCRP P269S variant protein. The ATPase activities of the Sf9 cell membranes were determined by measuring vanadate-sensitive Pi liberation. B, the expression levels of the wild-type and variant BCRP proteins in the Sf9 cells were similar, as confirmed by immunoblotting. Empty baculoviral construct was infected to Sf9 cells as a negative control of specific expression of BCRP proteins (Mock). The data shown are the mean ± S.D. for at least three different experiments.
|
|
Discovery and Functional Characterization of New SNP in the BCRP Promoter. Among the five SNP found in the promoter region of BCRP, three SNP (–19855C>T, –19605A>G, and –19031C>T) were identified as novel variants. The frequencies of these novel SNP were <1.6%, and they appeared on different chromosomes. To evaluate the effects of these variants on transcriptional modulating activity, the BCRP promoter-conjugated luciferase reporter assay was performed. No significant differences were observed between each of the SNP and the wild-type promoter (Fig. 2A). BCRP expression has been reported to be up-regulated through HIF-1 binding to a proximal hypoxia response element (HRE) at the –114 position (Krishnamurthy et al., 2004), and one of the novel BCRP SNP was localized to the 5-base pair consensus element of the HRE sequence. Therefore, we investigated whether the variation in the HRE affected the responsiveness of the BCRP promoter to hypoxic stress. The wild-type promoter and a variant promoter that included –19031C>T were introduced into HEK-293F cells and treated with deferoxamine, a hypoxia-mimetic agent. In the untreated condition, the variant and wild-type promoters showed similar transcriptional activities (Fig. 2B). In the presence of deferoxamine, and with cotransfection of the HIF-1-expressing plasmid, the transcriptional activities of the variant and wild type were increased to similar extents. These results suggest that this novel SNP in the HRE core site does not cause any alteration of HIF-1-mediated transcription. The location of –114C>T relative to HRE core region is indicated in Fig. 2C.
Transporter Activity of the P269S Variant. Two different comparisons of the wild-type and P269S variant proteins were performed. Vesicular uptake of [3H]MTX and [3H]ES into the microsomal fraction was measured for the BCRP wild-type and P269S proteins expressed in Sf9 cells. BCRP P269S-expressing microsomes showed about 35 to 40% decrease in [3H]MTX and [3H]ES uptake compared with microsomes that expressed the wild-type BCRP (Fig. 3, A and B). The expression levels of the wild-type and P269S BCRP proteins were similar, as confirmed by immunoblotting (Fig. 3C). To investigate whether the decreased functionality of BCRP P269S was derived from the modulation of substrate-dependent ATPase activity, prazosin-induced ATPase activity was measured in the wild-type and P269S-expressing microsomes from Sf9 cells. The basal ATPase activity in Sf9 cell membranes enriched with wild-type or variant BCRP proteins in the absence of prazosin showed no difference between wild-type and variant BCRP membranes. The BCRP ATPase activity in the presence of prazosin of the P269S variant was also the same as that of the wild-type protein, which indicates that the decreased activity of P269S is not related to ATPase activity. The expression levels of the recombinant BCRP proteins were similar, as confirmed by immunoblotting (Fig. 4).
Nonsynonymous Variants of the BCRP Gene in Other Asian Populations. From the screening of four nonsynonymous variants in other ethnic groups, the allelic frequencies of V12M, Q126Stop, Q141K, and P269S were obtained (Table 4). The frequency of V12M was 10 to 13% higher in Chinese and Vietnamese subjects than in Koreans, whereas Q141K showed no significant differences among the three Asian ethnic groups. The frequency of the Q126Stop variant in Chinese and Vietnamese was <0.5%, although this null variant exhibited a slightly higher incidence (1.9%) in Koreans than in other ethnic groups. The P269S variant was not observed in 100 Caucasians and 100 African-Americans, which suggests that this variant is a rare variant that is found only in Asians.
|
Discussion
|
|---|
BCRP was first identified in cell lines that exhibited resistance to mitoxantrone or adriamycin, in which the overexpression of BCRP conferred protection from the cytotoxicities associated with these drugs (Brangi et al., 1999; Maliepaard et al., 1999; Ross et al., 1999). Many studies of these drug-resistant cell lines have indicated that BCRP confers resistance to mitoxantrone, anthracyclines, topotecan, irinotecan, and SN-38 by pumping out the drugs from the cells (Litman et al., 2000; Zhou et al., 2001).
Recent clinical studies have suggested that the large interindividual variability in drug responses is a consequence of genetic alterations in drug transporter genes. For example, the BCRP Q141K variant has been implicated in the altered pharmacokinetics of diflomotecan (Sparreboom et al., 2004). This BCRP polymorphism has also been associated with the susceptibility of renal carcinoma cells to drug treatments (Korenaga et al., 2005) and with the low expression of BCRP in the human placenta (Kobayashi et al., 2005). Because BCRP plays an important role in clinical treatment, genetic polymorphisms of BCRP have been widely studied in different ethnic groups (Honjo et al., 2002; Backstrom et al., 2003; Wang et al., 2004).
In the present study, we performed a comprehensive evaluation of BCRP polymorphisms in a Korean population. Twenty SNP were identified in 92 Korean subjects. Among the BCRP coding variants, V12M and Q14lK were the most common in Koreans, with allelic frequencies of 23 and 28%, respectively. These variants are also found frequently in other ethnic groups, such as Caucasians, Japanese, and Chinese (Zamber et al., 2003; Kobayashi et al., 2005). The Q141K variant was the most prevalent coding variant in Caucasians (14%), Japanese (35%), and Chinese (35%), as well as in Koreans (28%), whereas it was not detected in African-Americans or in subjects from Africa north of the Sahara (Zamber et al., 2003). The allelic frequency of V12M in Koreans (23%) is much lower than that reported for Southeast Asians (45%), Pacific Islander (64%), Mexican-Indian (90%), and Hispanic (40%) subjects (Zamber et al., 2003). Another nonsynonymous variant, Q126Stop, was detected as a heterozygous genotype in Koreans (1.9%) and Japanese subjects (1%) (Kobayashi et al., 2005). P269S was observed exclusively in Koreans and Vietnamese and not in the Caucasian, African-American, and Chinese populations. Taken together with previously reported studies, our results support the notion that BCRP polymorphisms show ethnic differences.
View this table:
[in this window]
[in a new window]
|
TABLE 5 Expected allelic frequencies of BCRP V12M, Q126Stop, Q141K, and P269S variants in different Asian populations
|
|
Recently, it has been shown that many genes exhibit a block-like pattern of LD and linkage to each other. The LD pattern observed in the present study shows that there are two discrete blocks of BCRP genes in the Korean population (Fig. 1B). These two-block structures of LD have also been reported in a Chinese population (Wang et al., 2004). Most of the SNP in the individual blocks reveal a high frequency of haplotype structure for the BCRP gene in Koreans (Table 3). The wild type showed the highest frequency at 42%. Haplotypes 4, 8, and 11 indicate that a single promoter SNP (20296A>G) and the 238A>G change accompany the V12M nonsynonymous change in Koreans (Table 3). In haplotypes 2, 7, and 13, Q141K (8825C>A) variation is accompanied by 42288C>T and 44997A>G. These strong linkages also show the block-like haplotype structure of BCRP gene. For the association studies between haplotypes and phenotypes such as drug resistance or disease susceptibility, the haplotype-based approach has been known to be more informative than the SNP-based approach. Our results on BCRP haplotypes in the Korean population would provide information on genetic markers for association studies relating to BCRP polymorphism in this population.
The identification of genetic polymorphisms is recognized as an important topic because some SNP can be used as simple genetic markers, as well as for the prediction of in vivo functional phenotypes. In the case of BCRP, the 421C>A variation (encodes Q141K) has been reported to play important roles in determining the expression level of the protein (Imai et al., 2002; Kobayashi et al., 2005), drug resistance, and ATPase activity (Mizuarai et al., 2004). The V12M SNP has been reported to be associated with membrane localization (Mizuarai et al., 2004). Increased BCRP expression has been linked to reduced efficacy of chemotherapy in acute myeloid leukemia patients, which implies that BCRP expression is associated with the development of multidrug resistance (Steinbach et al., 2002). In addition, BCRP expression has been suggested as a marker for human hematopoietic stem cell protection (Kim et al., 2002). Recently, BCRP expression has been shown to be up-regulated by hypoxia through the HRE, which is required for HIF-1 binding during BCRP transcription (Blancher and Harris, 1998; Semenza, 2002; Krishnamurthy et al., 2004). Interestingly, of our three novel SNP in the promoter region of BCRP, we localized –19031C>T SNP (–114 position in relation to transcription start site) to the core HRE region of the BCRP promoter. Krishnamurthy et al. (2004) revealed that there are two functional HRE (centered at –194, –115) in proximal promoter and one distal nonfunctional HRE at –1094 in BCRP gene. They revealed that HRE at –115 is most important in hypoxic induction of BCRP expression. Because the –19031C>T transition variation is localized at the second position in five-nucleotide core region (AC/TGTG) of HRE centered at –115 and the core consensus region is highly conserved, it is speculated that this –19031C>T SNP may affect BCRP transcriptional activation. However, it was found that this variation did not significantly alter BCRP transcription under the chemically induced hypoxic condition and under normal condition. This suggests that the second base variation (C/T) in the consensus HRE may not be essential for the binding of HIF-1 to the HRE of BCRP promoter. The other two promoter SNP found in Koreans also showed normal transactivation activity, which suggests that the regulatory SNP in the BCRP promoter may not contribute to the interindividual variation of BCRP expression.
In the present study, we have described the allele frequencies for BCRP variants in a Korean population and analyzed the function of the P269S coding variant. Kondo et al. (2004) have reported that the P269S variant has activity similar to that of the wild-type protein. However, in at least three repeated experiments, we showed that P269S shows altered ES and MTX uptake as compared with the wild type. Both the previous study and our current study show similar protein levels of P269S variant and wild-type protein expression in the systems used. This discrepancy may be partly explained by the different assay systems used, i.e., baculovirus versus adenovirus systems. For example, in Sf9 cells, the cholesterol/phospholipid ratio is much lower than in mammalian cells (Marheineke et al., 1998). Many studies have shown that the lipid composition of membrane alters ATP-binding cassette transporters including P-glycoprotein (Troost et al., 2004). Therefore, the possibility that P269S variation may not change the BCRP activity in mammalian cells cannot be ruled out. Additional experiments in other assay systems will reveal the function of this variant. Similarly, BCRP V12M has been reported to exhibit the same transport function as the wild type (Kondo et al., 2004), whereas another study has shown decreased activity caused by disrupted membrane localization (Mizuarai et al., 2004). Genotyping in clinical studies is needed to address the relationships between these genotypes and phenotypes.
The present study is the first report on the identification of allelic variants of the BCRP gene in a Korean population. In the 92 Korean subjects, 20 SNP, including four functional variants, were identified in the present study. We also evaluated the frequency of the four functional variants in two representative Asian populations, Chinese and Vietnamese, to find ethnic difference of BCRP polymorphism. Future clinical studies will elucidate the genotype/phenotype relationships for these BCRP polymorphisms and the ethnic variation of drug disposition of BCRP substrates.
|
Footnotes
|
|---|
This study was supported by the Ministry of Science and Technology (National Research Laboratory Program) and Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (03-PJ10-PG13-GD01-0002).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.012302.
ABBREVIATIONS: BCRP, breast cancer resistance protein; SNP, single nucleotide polymorphism(s); MTX, methotrexate; ES, estrone-3-sulfate; PCR, polymerase chain reaction; LD, linkage disequilibrium; Pi, inorganic phosphate; HEK, human embryonic kidney; HIF-1, hypoxia-inducible factor-1; HWE, Hardy-Weinberg Equilibrium; HRE, hypoxia response element.
Address correspondence to: Jae-Gook Shin, Department of Pharmacology, Pharmacogenomics Research Center, College of Medicine, Inje University, 633-165 Gaegum-Dong, Jin-Gu, Busan 614-735, Korea. E-mail: phshinjg{at}inje.ac.kr
|
References
|
|---|
Ai T, Watanabe M, Saito H, Nakagawa H, Kamachi T, Okura I, and Ishikawa T (2006) Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport. Mol Pharm 70: 287–296.[Abstract/Free Full Text]
Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V, and Dean M (1998) A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug-resistance. Cancer Res 58: 5337–5379.[Abstract/Free Full Text]
Backstrom G, Taipalensuu J, Melhus H, Brandstrom H, Svensson A-C, Artursson P, and Kindmark A (2003) Genetic variation in the ATP-binding cassette transporter gene ABCG2(BCRP) in a Swedish population. Eur J Pharm Sci 18: 359–364.[CrossRef][Medline]
Bailey-Dell KJ, Hassel B, Doyle LA, and Ross DD (2001) Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene. Biochim Biophys Acta 1520: 234–241.[Medline]
Blancher C and Harris AL (1998) The molecular basis of the hypoxia response pathway: tumour hypoxia as a therapy target. Cancer Metastasis Rev 17: 187–194.[CrossRef][Medline]
Brangi M, Litman T, Ciotti M, Nishiyama K, Kohlhagen G, Takimoto C, Robey R, Pommier Y, Fojo T, and Bates SE (1999) Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR), and potential for glucuronidation in MXR-expressing cells. Cancer Res 59: 5938–5946.[Abstract/Free Full Text]
Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, and Ross DD (1998) A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA 95: 15665–15670.[Abstract/Free Full Text]
Honjo Y, Hrycyna CA, Yan QW, Medina-Perez WY, Robey RW, van de Laar A, Litman T, Dean M, and Bates SE (2001) Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res 61: 6635–6639.[Abstract/Free Full Text]
Honjo Y, Morisaki K, Huff LM, Robey RW, Hung J, Dean M, and Bates SE (2002) Single-nucleotide polymorphism (SNP) analysis in the ABC half-transporter ABCG2 (MXR/BCRP/ABCP1). Cancer Biol Ther 1: 696–702.[Medline]
Imai Y, Nakane M, Kage K, Tsukahara S, Ishikawa E, Tsuruo T, Miki Y, and Sugimoto Y (2002) C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance. Mol Cancer Ther 1: 611–616.[Abstract/Free Full Text]
Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH, and Schinkel AH (2000) Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan. J Natl Cancer Inst 92: 1651–1656.[Abstract/Free Full Text]
Kim M, Turnquist H, Jackson J, Sgagias M, Yan Y, Gong M, Dean M, Sharp JG, and Cowan K (2002) The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 8: 22–28.[Abstract/Free Full Text]
Kobayashi D, Ieiri I, Hirota T, Takane H, Maegawa S, Kigawa J, Suzuki H, Nanba E, Oshimura M, Terakawa N, et al. (2005) Functional assessment of abcg2 (bcrp) gene polymorphisms to protein expression in human placenta. Drug Metab Dispos 33: 94–101.[Abstract/Free Full Text]
Kondo C, Suzuki H, Itoda M, Ozawa S, Sawada J, Kobayashi D, Ieiri I, Mine K, Ohtsubo K, and Sugiyama Y (2004) Functional analysis of SNPs variants of BCRP/ABCG2. Pharm Res (NY) 21: 1895–1903.
Korenaga Y, Naito K, Okayama N, Hirata H, Suehiro Y, Hamanaka Y, Matsuyama H, and Hinoda Y (2005) Association of the BCRP C421A polymorphism with nonpapillary renal cell carcinoma. Int J Cancer 117: 431–434.[CrossRef][Medline]
Krishnamurthy P, Ross DD, Nakanishi T, Bailey-Dell K, Zhou S, Mercer KE, Sarkadi B, Sorrentino BP, and Schuetz JD (2004) The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J Biol Chem 279: 24218–24225.[Abstract/Free Full Text]
Lee SS, Jeong HE, Liu KH, Ryu JY, Moon T, Yoon CN, Oh SJ, Yun CH, and Shin JG (2005) Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity. Pharmacogenet Genomics 15: 105–113.[Medline]
Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, and Bates SE (2000) The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113: 2011–2021.[Abstract]
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.[CrossRef][Medline]
Litman T, Jensen U, Hansen A, Covitz KM, Zhan Z, Fetsch P, Abati A, Hansen PR, Horn T, Skovsgaard T, et al. (2002) Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2. Biochim Biophys Acta 1565: 6–16.[Medline]
Maliepaard M, van Gastelen MA, de Jong LA, Pluim D, van Waardenburg RC, Ruevekamp-Helmers MC, Floot BG, and Schellens JH (1999) Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer Res 59: 4559–4563.[Abstract/Free Full Text]
Marheineke K, Grünewald S, Christie W, and Reiländer H (1998) Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection. FEBS Lett 441: 49–52.[CrossRef][Medline]
Mitomo H, Kato R, Ito A, Kasamatsu S, Ikegami Y, Kii I, Kudo A, Kobatake E, Sumino Y, and Ishikawa T (2003) A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport. Biochem J 373: 767–774.[CrossRef][Medline]
Miyake K, Mickley L, Litman T, Zhan Z, Robey R, Cristensen B, Brangi M, Greenberger L, Dean M, Fojo T, et al. (1999) Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res 59: 8–13.[Abstract/Free Full Text]
Mizuarai S, Aozasa N, and Kotani H (2004) Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2. Int J Cancer 109: 238–246.[CrossRef][Medline]
Robey RW, Medina-Perez WY, Nishiyama K, Lahusen T, Miyake K, Litman T, Senderowicz AM, Ross DD, and Bates SE (2001) Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells. Clin Cancer Res 7: 145–152.[Abstract/Free Full Text]
Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Greenberger L, Cole SP, and Doyle LA (1999) Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 91: 429–433.[Abstract/Free Full Text]
Semenza GL (2002) HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 8: S62–S67.[CrossRef][Medline]
Sparreboom A, Gelderblom H, Marsh S, Ahluwalia R, Obach R, Principe P, Twelves C, Verweij J, and McLeod HL (2004) Diflomotecan pharmacokinetics in relation to ABCG2 421C>A genotype. Clin Pharmacol Ther 76: 38–44.[CrossRef][Medline]
Steinbach D, Sell W, Voigt A, Hermann J, Zintl F, and Sauerbrey A (2002) BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia 16: 1443–1447.[CrossRef][Medline]
Troost J, Lindenmaier H, Haefeli WE, and Weiss J (2004) Modulation of cellular cholesterol alters p-glycoprotein activity in multidrug-resistant cells. Mol Pharmacol 66: 1332–1339.[Abstract/Free Full Text]
Volk EL, Farley KM, Wu Y, Li F, Robey RW, and Schneider E (2002) Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance. Cancer Res 62: 5035–5040.[Abstract/Free Full Text]
Wang H, Hao B, Zhou K, Chen X, Wu S, Zhou G, Zhu Y, and He F (2004) Linkage disequilibrium and haplotype architecture for two ABC transporter genes (ABCC1 and ABCG2) in Chinese population: implications for pharmacogenomic association studies. Ann Hum Genet 68: 563–573.[CrossRef][Medline]
Zamber CP, Lamba JK, Yasuda K, Farnum J, Thummel K, Schuetz JD, and Schuetz EG (2003) Natural allelic variants of breast cancer resistance protein (BCRP) and their relationship to BCRP expression in human intestine. Pharmacogenetics 13: 19–28.[CrossRef][Medline]
Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, et al. (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7: 1028–1034.[CrossRef][Medline]
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
