![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Department of Biopharmaceutical Sciences, University of California, San Francisco, California
Received December 10, 2007; accepted February 19, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
2% were transiently expressed in HEK293T cells, and the transport of calcein acetoxymethyl ester and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-FL)-paclitaxel was measured in the absence or presence of the P-gp inhibitor cyclosporin A. The A893S, A893T, and V1251I variants and the N21D/1236C>T/A893S/3435C>T haplotype altered intracellular accumulation compared with reference P-gp in a substrate-dependent manner. It is interesting that certain variants showed altered sensitivity to cyclosporin A inhibition that was also substrate-specific. These functional data demonstrate that nonsynonymous polymorphisms in ABCB1 may selectively alter P-gp transport and drug-drug interactions in a substrate- and inhibitor-dependent manner.
). The blood-brain barrier expresses P-gp, and brain concentrations of P-gp substrates, such as digoxin and loperamide, are increased in Abcb1a–/– mice (Mayer et al., 1996; Schinkel et al., 1996). P-gp activity in the intestines is a major determinant of oral bioavailability for many therapeutic compounds (Fromm, 2000), and modulators of P-gp, such as rifampicin, can change the bioavailability of P-gp substrates (Niemi et al., 2003). P-gp may also be important in limiting intracellular levels of drugs. The multidrug resistance phenotype seen in the treatment of cancer is often caused by an overexpression of P-gp in solid tumors (DeMario and Ratain, 1998; Litman et al., 2001).
Variability in drug response is widely observed for P-gp substrates. One potential mechanism for this variability is interindividual differences in P-gp activity that result in altered pharmacokinetics. The number of P-gp transporters on the cell membrane and the level of P-gp transport function are the two most important factors that control the apparent activity of P-gp. Genetic variation in ABCB1, which encodes P-gp, is thought to be one of the factors that influence P-gp expression and function. The statistical analysis of ABCB1 genetic variation data indicates that there is considerable nucleotide diversity in this gene (Kroetz et al., 2003; Leabman et al., 2003). A polymorphism that affects P-gp activity at the protein level most probably will be an amino acid changing nonsynonymous variant. Amino acid changes may alter key domains necessary for substrate binding, ATP hydrolysis, or protein folding. It is possible that synonymous or promoter region variants can influence the expression level of P-gp, as suggested by studies on the ABCB1 3435C>T synonymous variation (Wang et al., 2005) and thereby affect P-gp transport function. The majority of pharmacogenetic studies for P-gp have focused on two polymorphisms, the synonymous 3435C>T variation and the nonsynonymous 2677G>T (S893A) variation (Schwab et al., 2003; Marzolini et al., 2004; Pauli-Magnus and Kroetz, 2004; Salama et al., 2006; Schaefer et al., 2006). Unfortunately, there is no clear consensus on how these polymorphisms affect P-gp at the in vivo or cellular level. Single nucleotide polymorphism (SNP) identification studies have discovered numerous other nonsynonymous and synonymous variants of ABCB1 (http://www.hapmap.org). The polymorphisms at positions 2677 and 3435 may simply be markers for other functionally relevant sites that have yet to be investigated. Furthermore, it is unclear what effects certain nonsynonymous P-gp variants have at the cellular level (Kimchi-Sarfaty et al., 2002; Woodahl et al., 2004, 2005). The improved design and analysis of P-gp pharmacogenetic research in vivo is reliant upon functional studies in vitro.
In these studies, we investigated how seven nonsynonymous variants and two common haplotypes of ABCB1 alter P-gp function in an in vitro assay that utilizes transient expression of P-gp in HEK293T cells. The fluorescent substrates, calceinacetoxymethyl ester (calcein-AM) and BODIPY-FL-paclitaxel, were selected to test the function of the P-gp variants. The data from these studies suggest that certain P-gp variants have altered function that is substrate-specific. In addition, some variants have different sensitivities to the P-gp inhibitor cyclosporin A (CsA) that is also substrate-dependent.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture. HEK293T cells were obtained from American Type Culture Collection (Manassas, VA) and maintained according to their instructions. In brief, EMEM containing 10% FBS, 1% nonessential amino acids, 0.11 µg/ml sodium pyruvate, 100 µg/ml streptomycin, and 100 U/m penicillin was used to propagate the cells in 5% CO2 at 37°C. Cells were maintained in T75 flasks and passaged every 2 to 3 days.
ABCB1 Plasmids and Transfection. ABCB1 nonsynonymous polymorphisms were identified previously (Kroetz et al., 2003). A full-length ABCB1 cDNA in pCIneo was used as a template to create variant plasmids. Selected variants of ABCB1 were created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Primer sequences are listed in Table 1. Plasmid constructs for 2677T(Ser893) and the 1236T/2677T(893S)/3435T haplotype were made previously (Kroetz et al., 2003). The following variants were created from the reference ABCB1 plasmid in pCIneo: 61A>G (N21D), 1199G>A (S400T), 1596T>G [a nonfunctional nucleotide-binding domain (NBD) mutant], 2005C>T (R669C), 2677G>A (A893T), 3421T>A (S1141T), and 3751G>A (V1251I). The 61G(D21)/1236T/2677T(S893)/3435T construct was made from the 1236T/2677T(S893)/3435T plasmid using the 61A>G primers. All variants and the reference were verified by direct sequencing. Reference ABCB1 was subcloned into the pcDNA5/FRT vector.
|
HEK293T cells were seeded in T25 flasks at 1.2 x 106 cells/flask in EMEM supplemented with 10% FBS, 1% nonessential amino acids, and 0.11 µg/ml sodium pyruvate and allowed to grow for 24 h. The quality of plasmid DNA was confirmed using a 260/280-nm absorbance ratio, and plasmid DNA integrity was verified by gel electrophoresis. Cells were transfected at a confluence of 70 to 85% with 9.2 µg of pcDNA5/FRT, ABCB1 reference plasmid DNA or variant plasmid DNA with 25 µl of Lipofectamine 2000 in a final volume of 5 ml. Fresh medium was replaced 
5 h after the addition of transfection reagents.
Substrate Accumulation Assays. The accumulation assays were repeated a total of three times using 1.5 µM calcein-AM or 100 nM BODIPY-FL-paclitaxel in the absence or presence of 10 µM cyclosporin A. Triplicate samples were used for each P-gp variant in the absence and presence of cyclosporin A. After 24 h, transfected cells were harvested using 0.05% trypsin and counted to achieve a concentration of 5 x 105 cells/sample in PBS. Cells were seeded in 96-well plates at 5 x 105 cells/well in 100 µl of PBS and centrifuged at 150g for 3 min. The PBS was aspirated, and 100 µl of substrate with or without 10 µM cyclosporin A in PBS was added to resuspend the cells. Samples were allowed to incubate for 45 min in the dark at 37°C with a brief agitation period 25 min after substrate addition. All subsequent manipulations were done on ice with cold reagents or at 4°C for the centrifugation steps. Accumulation was stopped by centrifuging the cells at 150g for 3 min, followed by a PBS wash. The cells were resuspended in 75 µl of PBS containing 6.67 µg/ml MRK16 primary antibody and incubated on ice in the dark for 30 min. After two wash steps, 60 µl of PBS containing 2.5 µg/ml secondary antibody with APC was used to resuspend the cells, and the mixture was incubated on ice in the dark for 25 min. Cells were washed twice, resuspended, and transferred into 5-ml tubes in 250 to 300 µl of PBS.
Flow Cytometry. Cell samples were run on a dual-laser FACS-calibur machine (BD Biosciences, San Jose, CA) with excitation wavelengths at 488 and 635 nM controlled by CellQuest software (BD Biosciences). Emission filters at 530 nM (FL1) detected calcein-AM and BODIPY-FL-paclitaxel, and >670 nM (FL4) detected APC fluorescence. Spectral overlap controls determined that there was negligible interference of substrate fluorescence with the >670-nm filter. Initial studies with control murine and goat IgG antibodies showed there that was no cross-reactivity. A total of 15,000 events were counted, and FlowJo software (Treestar, Ashland, OR) was used to analyze the flow cytometry data. Forward- and side-scatter analysis established the R1 gate for the viable, single-cell population. Substrate (FL1) and APC (FL4) fluorescence for the empty vector samples was determined from cells in the R1 gate. The P-gp-positive gate was established in the FL4 channel of R1 and was set so that <0.5% of the empty vector population resided in the gate. Substrate and APC measurements for the P-gp-transfected samples were determined from the R1/P-gp-positive gate.
Statistical Analyses. Transfection efficiency was calculated in the FL4 channel (APC fluorescence) from the percentage of cells in the R1 gate that also resided in the P-gp-positive gate. The P-gp expression level was determined from the median APC fluorescence of the cells in the R1/P-gp-positive gate. Median calcein and BODIPY-FL-paclitaxel fluorescence values from the R1/P-gp-positive cells were averaged from the three separate experiments for P-gp reference, variants, and NBD mutant. The averaged fluorescence for the NBD mutant and each variant was converted to a percentage of reference (set as 100), and this represented the substrate accumulation in the absence of cyclosporin A. Inhibitor sensitivity was determined from the median calcein and BODIPY-FL-paclitaxel fluorescence values in the presence of cyclosporin A for P-gp reference, variants, and NBD mutant. The percentage difference in fluorescence with and without inhibitor for P-gp reference, variants, and the NBD mutant was calculated from eq. 1.
 | (1) |
Percentage difference values for the variants and NBD mutant were normalized to reference. After three separate experiments, a one-way analysis of variance followed by Student's t test with Bonferroni's correction was used to calculate the statistical significance of the mean values calculated for substrate accumulation and percentage difference of inhibitor.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Thirteen nonsynonymous sites were identified that span the entire region of the ABCB1 gene, and those chosen for functional analysis are shown in Table 2. The allele frequencies of these variants in Caucasians and African Americans range from 0.5 to 46.4%, and three of the variants are ethnic-specific. Amino acid sequence alignments were performed using orthologous amino acid sequences from six other mammalian species. The degree of conservation is often linked to the allele frequency; that is, the reference amino acid for low frequency (<1%) nonsynonymous SNPs tends to be more conserved across the different species. For residue position 21, neither the reference nor variant amino acid for humans is found in the other mammalian species. Grantham values were determined for the nonsynonymous variants to determine how drastic the amino acid change is in terms of chemical properties (Grantham, 1974). All of the amino acid changing variants studied here are located in the loop domains of P-gp (Kroetz et al., 2003). The amino acid-altering variants that were studied were chosen based on an allele frequency greater than 2% in any one ethnic group, evolutionary conservation of the reference amino acid and/or a Grantham value >150 (Table 2). There are six polymorphic residues that meet these criteria: N21D, S400T, R669C, A893S/T, S1141T, and V1251I. Residue 893 is interesting because it is triallelic and occurs naturally in haplotypes, with the two common synonymous SNPs, 1236C>T and 3435C>T as well as N21D (Kroetz et al., 2003). As a result, two different haplotype constructs were also studied that contain Ser893, 1236T, and 3435T with either Asn21 or Asp21.
|
Transient Expression of P-gp in HEK293T Cells. Fluorescence measurements of intracellular substrate levels in the P-gp reference and variant-transfected HEK293T cells were only based upon the subpopulation of cells staining positively for surface P-gp expression with the MRK16 antibody. P-gp staining for empty vector, reference, NBD, and variant P-gp-expressing cells is shown with a "P-gp-positive" gate superimposed to mark the population of cells overexpressing P-gp (Fig. 1). Two important expression measurements are determined from the P-gp-staining histogram: transfection efficiency and P-gp expression levels. Transfection efficiency is the percentage of healthy cells located in the P-gp-positive gate. Intra- and interexperimental replicates (n = 3) for reference, NBD, and variant P-gp samples have consistently similar levels of transfection efficiency (52–60%). The second measurement, P-gp expression level, is the median APC fluorescence of the cells that are P-gp-positive. P-gp variant expression differs by less than 15% of reference levels. Although the empty vector has detectable APC staining, the endogenous P-gp levels in the HEK293T cells are 100-fold less than the P-gp-transfected cells. Consistent with the flow cytometry analysis, Western blots showed that P-gp-transfected HEK293T cells expressed a single 170-kDa protein (data not shown).
|
|
). Intracellular levels of calcein in the P-gp-positive cells for reference and the variants were dramatically less than for the NBD mutant and empty vector (Fig. 2a). In general, calcein levels for the P-gp variants were similar to those with the reference, and no variant had a complete loss of function. Median calcein fluorescence values for all variant constructs were normalized to the reference sample, which was arbitrarily set at 100. The median calcein fluorescence of all variants ranged from 69 to 97% (Fig. 2c). Variants Thr893 and Ile1251 showed increased function as evident from decreased intracellular calcein levels [69 ± 16 and 75 ± 13% of reference (p < 0.01), respectively]. Intracellular calcein levels were also investigated in the presence of 10 µM cyclosporin A (Fig. 2, b and c), and the data were analyzed to show how sensitive each variant is to inhibition. The percentage difference in intracellular fluorescence between control and cyclosporin A-treated cells was calculated individually for each variant and then normalized to reference, which was arbitrarily set at 100. The NBD mutant has very high levels of calcein accumulation both in the absence and presence of cyclosporin A. As a result, the percentage difference for the NBD mutant is very low (2.1 ± 0.9%), and this corresponds to negligible sensitivity to inhibition. In contrast, the reference and variant P-gp constructs have low calcein levels without cyclosporin A but have high calcein levels with cyclosporin A. Most P-gp variants showed similar inhibition of calcein-AM transport compared to reference. It is interesting that the N21D, R669C, and A893S variants are more sensitive to cyclosporin A (136 ± 28, 139 ± 43, and 130 ± 22%, respectively; p < 0.05) compared to reference (Fig. 2c).
BODIPY-FL-Paclitaxel Transport by P-gp Variants. The P-gp-positive cells expressing reference or any variant plasmid DNA had significantly less intracellular levels of 100 nM BODIPY-FL-paclitaxel than NBD or empty vector-transfected cells (Fig. 3a). In addition, the presence of cyclosporin A dramatically increased BODIPY-FL-paclitaxel levels (Fig. 3b). Median BODIPY-FL-paclitaxel fluorescence values for all variant samples were normalized to reference, which was arbitrarily set at 100. The N21D, S400N, R669C, and A893T variants and the 1236C>T/A893S/3435C>T haplotype were within 5% of the reference (Fig. 3c). In contrast, the A893S and V1251I P-gp variants and the N21D/1236C>T/A893S/3435C>T haplotype showed intracellular paclitaxel levels that were 114 ± 13, 118 ± 12, and 124 ± 13% of reference, respectively (p < 0.01), indicating decreased P-gp function.
|
P-gp transport of 100 nM BODIPY-FL-paclitaxel was also measured in the presence of 10 µM cyclosporin A. Three variants (A893S, A893T, and S1141T) were 30 ± 5, 30 ± 8, and 27 ± 17%, respectively, less sensitive to cyclosporin A inhibition than reference, whereas V1251I was 65 ± 26% less sensitive than reference (p < 0.01). The haplotype containing N21D/1236C>T/A893S/3435C>T was 53 ± 8% less sensitive to cyclosporin A inhibition than reference (p < 0.01).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) were also investigated in this study. These haplotypes contained A893S, 1236C>T, and 3435C>T either with or without N21D. Whereas the synonymous 1236C>T and 3435C>T changes are not expected to directly affect transport function, they could have indirect effects due to changes in mRNA secondary structure and/or translation (Wang et al., 2005; Kimchi-Sarfaty et al., 2007).
Numerous expression systems have been used to measure the effects of P-gp polymorphisms on transport function. Stable cell lines yield a homogenous population of cells that overexpress P-gp (Kim et al., 2001; Morita et al., 2003; Woodahl et al., 2004; Salama et al., 2006). An obvious drawback is that creating stable cell lines can be very time-consuming, and in the end, many cell lines will have P-gp transport that is similar to the reference transporter. The transient expression system we used has consistent transfection efficiency and P-gp expression levels that allow for a reproducible method to quickly screen P-gp variants against multiple substrates. Furthermore, the endogenous P-gp expression in the HEK293T cells is negated by the substantial overexpression of P-gp in transfected cells.
P-gp function is characterized by the efflux of intracellular substrates. Direct measure of efflux is generally performed with transcellular transport assays that use polarized cells (Rautio et al., 2006). The assay used in the present studies involves incubation of transfected HEK293T cells with substrate and quantitation of intracellular substrate levels. It is assumed that substrate uptake is constant in the transfected cells and that differences in substrate accumulation reflect differences in efflux among the P-gp variants. Calcein-AM is a well documented substrate with a high affinity for P-gp (Holló et al., 1994). The A893T and V1251I variants had lower intracellular levels of calcein, indicating increased efflux of calcein-AM by P-gp (Table 3). To our knowledge, the Ile1251 variant has not been studied previously, but recent data show that Thr893 has increased transport of vincristine (Schaefer et al., 2006). Residues 893 and 1251 are located in the intracellular loops adjacent to the 11th and 12th transmembrane domains (TMD11 and TMD12), respectively, which have been shown to be important for drug binding and ATP hydrolysis (Loo and Clarke, 1997, 1999). It is possible that amino acid changes at these sites may alter important functions of TMD11 and TMD12. Previous work consistent with the current findings has shown that N21D, S400N, and A893S do not change calcein-AM transport (Kimchi-Sarfaty et al., 2002; Kroetz et al., 2003). The transport of digoxin by the A893S variant was originally reported to be increased (Kim et al., 2001), although a subsequent study found similar digoxin transport by the Ser893 and reference P-gp (Morita et al., 2003). Decreased function for Ser893 in accumulation and transepithelial flux assays has been reported for rhodamine 123, vinblastine, and vincristine (Salama et al., 2006). The A893S variant has been the focus of many in vivo and in vitro pharmacogenetic studies, but it is still not clear whether this variant is a causative SNP or merely a marker. In an LLC-PK1 stable cell line expressing Asn400 P-gp, rhodamine 123 transport was reduced, whereas transport of the human immunodeficiency virus protease inhibitors saquinavir and ritonavir was increased (Woodahl et al., 2004, 2005). This suggests that the S400N variation alters P-gp function in a substrate-specific manner. Likewise, we found that the functional effects of A893S, A893T, V1251I, and N21N/1236C>T/A893S/3435C>T were substrate-dependent. It is important in the design of clinical P-gp pharmacogenetic studies to consider these substrate-dependent effects of P-gp variants.
|
Paclitaxel is another well established P-gp substrate (Rautio et al., 2006). The ABCB1 S400N variant has been shown to confer higher drug resistance to paclitaxel, but the stable cell line overexpressing this P-gp was selected for G418 resistance (Crouthamel et al., 2006). It is unknown what other background changes occur during cell line creation that could affect total cellular resistance. BODIPY-labeled paclitaxel was used in the current studies, and although it is possible that the BODIPY modification may alter interactions with P-gp, multiple studies, including ours, show that the compound is transported to a high degree (Kimchi-Sarfaty et al., 2002; Robey et al., 2006).
Limited data exist on the effects of ABCB1 genetic variation on BODIPY-FL-paclitaxel transport. In one study, the N21D, F103L, S400N, A893S, and A998T SNPs and three double mutants (N21N/S400N, N21D/A893S, and S400N/A893S) were investigated using a vaccinia virus expression system with BODIPY-FL-paclitaxel, and no differences in function were noted among the variant and reference P-gps (Kimchi-Sarfaty et al., 2002). Our results are similar for N21D and S400N, but we found that A893S and N21D/A893S have decreased transport of BODIPY-FL-paclitaxel (Table 3). The percentage change in function compared to reference for these hypofunctional variants is at most 24% and would be considered moderate. It should be noted that V1251I P-gp displays decreased function with BODIPY-FL-paclitaxel but increased function with calcein-AM. The 1251 residue is evolutionarily conserved across five other mammalian species and is only found in the Mexican-American population (Kroetz et al., 2003). There is no other data on the function of the V1251I variant, and testing additional substrates may elucidate the importance of this amino acid change.
Recently, a Saccharomyces-based assay tested nine nonsynonymous P-gp variants for cellular resistance to the antibiotic valinomycin and the anticancer drugs daunorubicin, doxorubicin, and actinomycin D (Jeong et al., 2007). The R669C variant showed highly increased function for all substrates, whereas our data showed no difference for calcein-AM and BODIPY-FL-paclitaxel. There was increased function for A893S and S1141T depending on the substrate, and in the current study, the A893S variant reduced P-gp transport of BODIPY-FL-paclitaxel. These differences may reflect inherent disparities in the assays used for these measurements. Nonetheless, evidence from the Saccharomyces-based study highlights the importance of considering substrate-specific effects of P-gp variants.
Limited research is available on how an inhibitor mechanistically alters the interactions between P-gp and a substrate. Various investigations have shown that substrates can bind to different P-gp domains (Dey et al., 1997; Rautio et al., 2006), but it is difficult to predict a priori what domains are important for each class of compounds. In addition, predictive three-dimensional modeling of how an amino acid change will affect transport function is still in its infancy. We tested the hypothesis that an amino acid variant can alter sensitivity to an inhibitor, and our results are supportive. Furthermore, the variants that function differently in the presence of cyclosporin A do so in a substrate-dependent manner. The N21D, R669C, and A893S P-gp variants show increased inhibition of calcein-AM transport by cyclosporin A. In contrast, the A893S, A893T, and S1141T variants and the N21D/1236C>T/A893S/3435C>T haplotype are less sensitive to cyclosporin A inhibition of BODIPY-FL-paclitaxel transport. The coadministration of paclitaxel and cyclosporin A for the treatment of various cancers is currently under consideration (Malingré et al., 2001; Kuppens et al., 2005), and the current data may improve the design and interpretation of such studies. Based on studies showing that 1) P-gp substrates bind different domains (Loo and Clarke, 2002; Loo et al., 2003, 2006a,b) and 2) cyclosporin A is a competitive inhibitor that alters P-gp conformation while in the drug-binding pocket (Loo and Clarke, 1997; Loo et al., 2003; Ejendal and Hrycyna, 2005; Suzuyama et al., 2007), there may be a synergistic effect with these variants in how they influence drug binding in the presence of cyclosporin A.
In summary, P-gp variants and haplotypes were investigated for possible functional effects on P-gp expression and function. Intracellular accumulation of calcein-AM and/or BODIPY-FL-paclitaxel was altered by A893S, A893T, V1251I, and N21D/1236C>T/A893S/3435C>T. In addition, certain variants and haplotypes showed different sensitivities to cyclosporin A inhibition. Our in vitro study generated reproducible data regarding how a genetic variant influences transport function, but the consequences at the in vivo level are not yet known. Unless a P-gp variant is characterized against multiple substrates, it is difficult to predict the extent of its clinical significance. However, in vitro data continue to contribute to a better understanding of how P-gp operates at the molecular level and may influence drug design and discovery.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: P-gp, P-glycoprotein; SNP, single nucleotide polymorphism; FBS, fetal bovine serum; calcein-AM, calcein-acetoxymethyl ester; BODIPY-FL, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; APC, allophycocyanin; EMEM, Eagle's minimum essential medium; NBD, nucleotide binding domain; CsA, cyclosporin A; TMD, transmembrane domain; PBS, phosphate-buffered saline; TMD, transmembrane domain; FRT, Flp recombinase target.
Address correspondence to: Dr. Deanna L. Kroetz, UCSF Box 2911, 1550 4th St., Rm. RH584E, San Francisco, CA 94158-2911. E-mail: deanna.kroetz{at}ucsf.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Crouthamel MH, Wu D, Yang Z, and Ho RJ (2006) A novel MDR1 G1199T variant alters drug resistance and efflux transport activity of P-glycoprotein in recombinant HEK cells. J Pharm Sci 95: 2767–2777.[CrossRef][Medline]
DeMario MD and Ratain MJ (1998) Oral chemotherapy: rationale and future directions. J Clin Oncol 16: 2557–2567.[Abstract]
Dey S, Ramachandra M, Pastan I, Gottesman MM, and Ambudkar SV (1997) Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc Natl Acad Sci USA 94: 10594–10599.
Ejendal KF and Hrycyna CA (2005) Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A. Mol Pharmacol 67: 902–911.
Fromm MF (2000) P-glycoprotein: a defense mechanism limiting oral bioavailability and CNS accumulation of drugs. Int J Clin Pharmacol Ther 38: 69–74.[Medline]
Fromm MF (2004) Importance of P-glycoprotein at blood-tissue barriers. Trends Pharmacol Sci 25: 423–429.[CrossRef][Medline]
Grantham R (1974) Amino acid difference formula to help explain protein evolution. Science 185: 862–864.
Holló Z, Homolya L, Davis CW, and Sarkadi B (1994) Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta 1191: 384–388.[Medline]
Jeong H, Herskowitz I, Kroetz DL, and Rine J (2007) Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay. PLoS Genet 3: e39.[CrossRef][Medline]
Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI, Taylor A, Xie HG, McKinsey J, Zhou S, et al. (2001) Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther 70: 189–199.[CrossRef][Medline]
Kimchi-Sarfaty C, Gribar JJ, and Gottesman MM (2002) Functional characterization of coding polymorphisms in the human MDR1 gene using a vaccinia virus expression system. Mol Pharmacol 62: 1–6.
Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, and Gottesman MM (2007) A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 315: 525–528.
Kroetz DL, Pauli-Magnus C, Hodges LM, Huang CC, Kawamoto M, Johns SJ, Stryke D, Ferrin TE, DeYoung J, Taylor T, et al. (2003) Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene. Pharmacogenetics 13: 481–494.[CrossRef][Medline]
Kuppens IE, Breedveld P, Beijnen JH, and Schellens JH (2005) Modulation of oral drug bioavailability: from preclinical mechanism to therapeutic application. Cancer Invest 23: 443–464.[Medline]
Leabman MK, Huang CC, DeYoung J, Carlson EJ, Taylor TR, de la Cruz M, Johns SJ, Stryke D, Kawamoto M, Urban TJ, et al. (2003) Natural variation in human membrane transporter genes reveals evolutionary and functional constraints. Proc Natl Acad Sci U S A 100: 5896–5901.
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]
Loo TW, Bartlett MC, and Clarke DM (2003) Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains. J Biol Chem 278: 1575–1578.
Loo TW, Bartlett MC, and Clarke DM (2006a) Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket. Biochem J 396: 537–545.[CrossRef][Medline]
Loo TW, Bartlett MC, and Clarke DM (2006b) Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket. Biochem J 399: 351–359.[CrossRef][Medline]
Loo TW and Clarke DM (1997) Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12. J Biol Chem 272: 20986–20989.
Loo TW and Clarke DM (1999) Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane. J Biol Chem 274: 35388–35392.
Loo TW and Clarke DM (2002) Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein. J Biol Chem 277: 44332–44338.
Malingré MM, Ten Bokkel Huinink WW, Duchin K, Schellens JH, and Beijnen JH (2001) Pharmacokinetics of oral cyclosporin A when co-administered to enhance the oral absorption of paclitaxel. Anticancer Drugs 12: 591–593.[CrossRef][Medline]
Marzolini C, Paus E, Buclin T, and Kim RB (2004) Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance. Clin Pharmacol Ther 75: 13–33.[CrossRef][Medline]
Mayer U, Wagenaar E, Beijnen JH, Smit JW, Meijer DK, van Asperen J, Borst P, and Schinkel AH (1996) Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein. Br J Pharmacol 119: 1038–1044.[Medline]
Morita N, Yasumori T, and Nakayama K (2003) Human MDR1 polymorphism: G2677T/A and C3435T have no effect on MDR1 transport activities. Biochem Pharmacol 65: 1843–1852.[Medline]
Niemi M, Backman JT, Fromm MF, Neuvonen PJ, and Kivisto KT (2003) Pharmacokinetic interactions with rifampicin: clinical relevance. Clin Pharmacokinet 42: 819–850.[CrossRef][Medline]
Pauli-Magnus C and Kroetz DL (2004) Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1). Pharm Res 21: 904–913.[CrossRef][Medline]
Rautio J, Humphreys JE, Webster LO, Balakrishnan A, Keogh JP, Kunta JR, Serabjit-Singh CJ, and Polli JW (2006) In vitro p-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates. Drug Metab Dispos 34: 786–792.
Robey RW, Zhan Z, Piekarz RL, Kayastha GL, Fojo T, and Bates SE (2006) Increased MDR1 expression in normal and malignant peripheral blood mononuclear cells obtained from patients receiving depsipeptide (FR901228, FK228, NSC630176). Clin Cancer Res 12: 1547–1555.
Salama NN, Yang Z, Bui T, and Ho RJ (2006) MDR1 haplotypes significantly minimize intracellular uptake and transcellular P-gp substrate transport in recombinant LLC-PK1 cells. J Pharm Sci 95: 2293–2308.[CrossRef][Medline]
Schaefer M, Roots I, and Gerloff T (2006) In-vitro transport characteristics discriminate wild-type ABCB1 (MDR1) from Ala893Ser and Ala893Thr polymorphisms. Pharmacogenet Genomics 16: 855–861.[Medline]
Schinkel AH, Wagenaar E, Mol CA, and van Deemter L (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 97: 2517–2524.[Medline]
Schwab M, Eichelbaum M, and Fromm MF (2003) Genetic polymorphisms of the human MDR1 drug transporter. Annu Rev Pharmacol Toxicol 43: 285–307.[CrossRef][Medline]
Suzuyama N, Katoh M, Takeuchi T, Yoshitomi S, Higuchi T, Asashi S, and Yokoi T (2007) Species differences of inhibitory effects on P-glycoprotein-mediated drug transport. J Pharm Sci 96: 1609–1618.[CrossRef][Medline]
Wang D, Johnson AD, Papp AC, Kroetz DL, and Sadee W (2005) Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability. Pharmacogenet Genomics 15: 693–704.[Medline]
Woodahl EL, Yang Z, Bui T, Shen DD, and Ho RJ (2004) Multidrug resistance gene G1199A polymorphism alters efflux transport activity of P-glycoprotein. J Pharmacol Exp Ther 310: 1199–1207.
Woodahl EL, Yang Z, Bui T, Shen DD, and Ho RJ (2005) MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells. AIDS 19: 1617–1625.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Naesens, D. R. J. Kuypers, and M. Sarwal Calcineurin Inhibitor Nephrotoxicity Clin. J. Am. Soc. Nephrol., February 1, 2009; 4(2): 481 - 508. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
