Mitochondrial ABC transporters function: the role of ABCB10 (ABC-me) as a novel player in cellular handling of reactive oxygen species

Mitochondria are one of the major sources of reactive oxygen species (ROS) in the cell. When exceeding the capacity of antioxidant mechanisms, ROS production may lead to different pathologies, such as ischemia-reperfusion injury, neurodegeneration, anemia and ageing. As a consequence of the endosymbiotic origin of mitochondria, eukaryotic cells have developed different transport mechanisms that coordinate mitochondrial function with other cellular compartments. Four mitochondrial ATP-binding cassette (ABC) transporters have been described to date in mammals: ABCB6, ABCB8, ABCB7 and ABCB10. ABCB10 is located in the inner mitochondrial membrane forming homodimers, with the ATP binding domain facing the mitochondrial matrix. ABCB10 expression is highly induced during erythroid differentiation and its overexpression increases hemoglobin synthesis in erythroid cells. However, ABCB10 is also expressed in nonerythroid tissues, suggesting a role not directly related to hemoglobin synthesis. Recent evidence points toward ABCB10 as an important player in the protection from oxidative stress in mammals. In this regard, ABCB10 is required for normal erythropoiesis and cardiac recovery after ischemia-reperfusion, processes intimately related to mitochondrial ROS generation. Here, we review the current knowledge on mitochondrial ABC transporters and ABCB10 and discuss the potential mechanisms by which ABCB10 and its transport activity may regulate oxidative stress. We discuss ABCB10 as a potential therapeutic target for diseases in which increased mitochondrial ROS production and oxidative stress play a major role.     

植物ABCB亚家族生物学功能研究进展

ABC转运蛋白超家族结构和功能复杂多样, 包含ABCA-ABCH八个亚家族。ABCB是ABC转运蛋白的一个亚家族, 多数定位于质膜, 少数定位于线粒体膜或叶绿体膜。ABCB与其它生长素转运蛋白(AUX1/LAX、PIN)共同参与调控植物生长素的极性运输, 在植物生长发育的各个阶段发挥作用。此外, ABCB转运蛋白还调控植物的向性运动和重金属抗性等过程。近年来, 随着越来越多植物全基因组测序的完成, ABCB亚家族在禾谷类单子叶植物水稻(Oryza sativa)、玉米(Zea mays)和高粱(Sorghum bicolor)中的生物学功能开始有少量报道, 然而多数ABCB转运蛋白的功能尚未得到阐释。该文对拟南芥(Arabidopsis thaliana)和禾谷类作物ABCB转运蛋白的研究进展进行综述, 以期为全面揭示ABCB亚家族生物学功能提供线索。     

植物ABCB亚家族生物学功能研究进展

ABC转运蛋白超家族结构和功能复杂多样, 包含ABCA-ABCH八个亚家族。ABCB是ABC转运蛋白的一个亚家族, 多数定位于质膜, 少数定位于线粒体膜或叶绿体膜。ABCB与其它生长素转运蛋白(AUX1/LAX、PIN)共同参与调控植物生长素的极性运输, 在植物生长发育的各个阶段发挥作用。此外, ABCB转运蛋白还调控植物的向性运动和重金属抗性等过程。近年来, 随着越来越多植物全基因组测序的完成, ABCB亚家族在禾谷类单子叶植物水稻(Oryza sativa)、玉米(Zea mays)和高粱(Sorghum bicolor)中的生物学功能开始有少量报道, 然而多数ABCB转运蛋白的功能尚未得到阐释。该文对拟南芥(Arabidopsis thaliana)和禾谷类作物ABCB转运蛋白的研究进展进行综述, 以期为全面揭示ABCB亚家族生物学功能提供线索。     

ABCB- and ABCC-type transporters confer multixenobiotic resistance and form an environment-tissue barrier in bivalve gills

Aquatic organisms and, in particular, filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and, presumably, require adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP-binding cassette (ABC) transporters protects mussels against toxicants, but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression, and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiotic transporter types in the gills, where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-glycoprotein and ABCC/MRP-type transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to the ABCB inhibitor PSC-833 and the ABCC inhibitor MK-571. This activity profile is supported by our cloning of the complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity of the amino acid sequences with corresponding homologs from other organisms was 38-50% (ABCB) and 27-44% (ABCC). C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills, providing protection against environmental xenotoxicants.     

Molecular players of auxin transport systems: advances in genomic and molecular events*

Phytohormone auxin plays an indispensable role in the plethora of plant developmental process starting from the cell division, and cell elongation to morphogenesis. Auxins are transported to different parts of the plant by different sophisticated transporter molecules known as ‘auxin transporters’.There are four auxin transporter families that have been reported so far in the plant kingdom which includes AUX/LAX (AUXIN-RESISTANT1–LIKES), PIN (PIN-FORMED, auxin efflux carriers), ABCB ((ATP-binding cassette-B (ABCB)/P-glycoprotein (PGP)) and PILS (PIN-Likes). Auxin influx and efflux carriers are distributed in a polar fashion in the plasma membrane whereas ABCB and PILS are present in a non-polar fashion. Other than AUX/LAX, other auxin transporters harbor N-and C-terminal conserved domains along with a variable hydrophilic loop in the transmembrane domain. The AUX/LAX, ABCB and PIN transporters mediate long distance auxin transport whereas PILS and PIN5 protein involved in intracellular auxin homeostasis.     

Molecular evolutionary analysis of ABCB5: the ancestral gene is a full transporter with potentially deleterious single nucleotide polymorphisms

Background

ABCB5 is a member of the ABC protein superfamily, which includes the transporters ABCB1, ABCC1 and ABCG2 responsible for causing drug resistance in cancer patients and also several other transporters that have been linked to human disease. The ABCB5 full transporter (ABCB5.ts) is expressed in human testis and its functional significance is presently unknown. Another variant of this transporter, ABCB5 beta posses a “half-transporter-like” structure and is expressed in melanoma stem cells, normal melanocytes, and other types of pigment cells. ABCB5 beta has important clinical implications, as it may be involved with multidrug resistance in melanoma stem cells, allowing these stem cells to survive chemotherapeutic regimes.

Methodology/Principal Findings

We constructed and examined in detail topological structures of the human ABCB5 protein and determined in-silico the cSNPs (coding single nucleotide polymorphisms) that may affect its function. Evolutionary analysis of ABCB5 indicated that ABCB5, ABCB1, ABCB4, and ABCB11 share a common ancestor, which began duplicating early in the evolutionary history of chordates. This suggests that ABCB5 has evolved as a full transporter throughout its evolutionary history.

Conclusions/Significance

From our in-silco analysis of cSNPs we found that a large number of non-synonymous cSNPs map to important functional regions of the protein suggesting that these SNPs if present in human populations may play a role in diseases associated with ABCB5. From phylogenetic analyses, we have shown that ABCB5 evolved as a full transporter throughout its evolutionary history with an absence of any major shifts in selection between the various lineages suggesting that the function of ABCB5 has been maintained during mammalian evolution. This finding would suggest that ABCB5 beta may have evolved to play a specific role in human pigment cells and/or melanoma cells where it is predominantly expressed.     

ATP-binding cassette transporters as pitfalls in selection of transgenic cells

Puromycin, hygromycin, and geneticin (G418) are antibiotics frequently used to select genetically engineered eukaryotic cells after transfection or transduction. Because intrinsic or acquired high expression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1) and multidrug resistance-associated proteins (MRP/ABCC1), can hamper efficient selection, it is important to know whether these antibiotics are substrates and/or inducers of efflux transporters. Therefore, we investigated the influence of these antibiotics on drug transporter expression by quantitative real-time polymerase chain reaction in the induction model cell line LS180. Moreover, we assessed whether ABC transporters influence the growth inhibitory effects of these antibiotics by proliferation assays using Madin-Darby canine kidney II (MDCKII) cells overexpressing the particular transporter. The results obtained indicate that puromycin and G418 are substrates of several ABC transporters, mainly Pgp/ABCB1. In contrast, hygromycin seems to be no good substrate for any of the ABC transporters investigated. Puromycin induced ABCC1/MRP1, whereas G418 suppressed ABCB1/Pgp, at the messenger RNA (mRNA) level. In contrast, hygromycin had no effect on ABC transporter mRNA expressions. In conclusion, this study emphasizes the significance of ABC transporters for the efficacy of selection processes. Consciousness of the results is supposed to guide the molecular biologist to the right choice of adequate experimental conditions for successful selection of genetically engineered eukaryotic cells.     

The evolutionary repertoires of the eukaryotic-type ABC transporters in terms of the phylogeny of ATP-binding domains in eukaryotes and prokaryotes

ABC (ATP-binding cassette) transporters play an important role in the communication of various substrates across cell membranes. They are ubiquitous in prokaryotes and eukaryotes, and eukaryotic types (EK-types) are distinguished from prokaryotic types (PK-types) in terms of their genes and domain organizations. The EK-types and PK-types mainly consist of exporters and importers, respectively. Prokaryotes have both the EK-types and the PK-types. The EK-types in prokaryotes are usually called "bacterial multidrug ABC transporters," but they are not well characterized in comparison with the multidrug ABC transporters in eukaryotes. Thus, an exhaustive search of the EK-types among diverse organisms and detailed sequence classification and analysis would elucidate the evolutionary history of EK-types. It would also help shed some light on the fundamental repertoires of the wide variety of substrates through which multidrug ABC transporters in eukaryotes communicate. In this work, we have identified the EK-type ABC transporters in 126 prokaryotes using the profiles of the ATP-binding domain (NBD) of the EK-type ABC transporters from 12 eukaryotes. As a result, 11 clusters were identified from 1,046 EK-types ABC transporters. In particular, two large novel clusters emerged, corresponding to the bacterial multidrug ABC transporters related to the ABCB and ABCC families in eukaryotes, respectively. In the genomic context, most of these genes are located alone or adjacent to genes from the same clusters. Additionally, to detect functional divergences in the NBDs, the Kullback-Leibler divergence was measured among these bacterial multidrug transporters. As a result, several putative functional regions were identified, some corresponding to the predicted secondary structures. We also analyzed a phylogeny of the EK-type ABC transporters in both prokaryotes and eukaryotes, which revealed that the EK-type ABC transporters in prokaryotes have certain repertoires corresponding to the conventional ABC protein groups in eukaryotes. On the basis of these findings, we propose an updated evolutionary hypothesis in which the EK-type ABC transporters in both eukaryotes and prokaryotes consisted of several kinds of ABC transporters in putative ancestor cells before the divergence of eukaryotic and prokaryotic cells.     

ABC transporters coordinately expressed during lignification of Arabidopsis stems include a set of ABCBs associated with auxin transport

The primary inflorescence stem of Arabidopsis thaliana is rich in lignified cell walls, in both vascular bundles and interfascicular fibres. Previous gene expression studies demonstrated a correlation between expression of phenylpropanoid biosynthetic genes and a subset of genes encoding ATP-binding cassette (ABC) transporters, especially in the ABCB/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) and ABCG/pleiotropic drug resistance (ABCG/PDR) subfamilies. The objective of this study was to characterize these ABC transporters in terms of their gene expression and their function in development of lignified cells. Based on in silico analyses, four ABC transporters were selected for detailed investigation: ABCB11/MDR8, ABCB14/MDR12, ABCB15/MDR13, and ABCG33/PDR5. Promoter::glucuronidase reporter assays for each gene indicated that promoters of ABCB11, ABCB14, ABCB15, and ABCG33 transporters are active in the vascular tissues of primary stem, and in some cases in interfascicular tissues as well. Homozygous T-DNA insertion mutant lines showed no apparent irregular xylem phenotype or alterations in interfascicular fibre lignification or morphology in comparison with wild type. However, in abcb14-1 mutants, stem vascular morphology was slightly disorganized, with decreased phloem area in the vascular bundle and decreased xylem vessel lumen diameter. In addition, abcb14-1 mutants showed both decreased polar auxin transport through whole stems and altered auxin distribution in the procambium. It is proposed that both ABCB14 and ABCB15 promote auxin transport since inflorescence stems in both mutants showed a reduction in polar auxin transport, which was not observed for any of the ABCG subfamily mutants tested. In the case of ABCB14, the reduction in auxin transport is correlated with a mild disruption of vascular development in the inflorescence stem.     

The ER-localized TWD1 immunophilin is necessary for localization of multidrug resistance-like proteins required for polar auxin transport in Arabidopsis roots

Multidrug resistance ABC transporters in plants are required for polar transport of the hormone auxin (indole-3-acetic acid). They are studied in animals primarily because their overexpression confers resistance to anticancer agents. Immunophilins are studied in both plants and animals for their roles in folding and trafficking of proteins, particularly those with signal transducing functions and susceptibility to immunosuppressant drugs. Previous genetic and molecular studies in Arabidopsis thaliana established a physical and functional interaction between some ABCB transporters and the TWISTED DWARF1 (TWD1) immunophilin. In this work, confocal microscopy of fluorescently tagged TWD1 shows it to reside at the endoplasmic reticulum (ER). Mutations in TWD1 caused mislocalization of ABCB1, ABCB4, and ABCB19 to the ER instead of the plasma membrane as shown by confocal microscopy of fluorescently tagged fusion proteins and transmission electron microscopy of immunogold-labeled samples in the case of ABCB19. Localization of the unrelated PIN-FORMED2 auxin transporter or plasma membrane marker proteins was not affected by loss of TWD1. Abnormal spread of auxin signaling into the elongation zone of twd1 roots, attributable to mislocalized ABCB transporters and suppressed by an auxin transport inhibitor, appeared to cause the twisted cell files characteristic of twd1 roots.     

Regulation of progenitor cell fusion by ABCB5 P-glycoprotein,a novel human ATP-binding cassette transporter

Cell fusion involving progenitor cells is a newly recognized phenomenon thought to contribute to tissue differentiation. The molecular mechanisms governing cell fusion are unknown. P-glycoprotein and related ATP-binding cassette transporters are expressed by progenitor cells, but their physiological role in these cell types has not been defined. Here, we have cloned ABCB5, a rhodamine efflux transporter and novel member of the human P-glycoprotein family, which marks CD133-expressing progenitor cells among human epidermal melanocytes and determines as a regulator of membrane potential the propensity of this subpopulation to undergo cell fusion. Our findings show that polyploid ABCB5+ cells are generated by cell fusion and that this process is specifically enhanced by ABCB5 P-glycoprotein blockade. Remarkably, multinucleated cell hybrids gave rise to mononucleated progeny, demonstrating that fusion contributes to culture growth and differentiation. Thus, our findings define a molecular mechanism for cell fusion involving progenitor cells and show that fusion and resultant growth and differentiation are not merely spontaneous events, but phenomena regulated by ABCB5 P-glycoprotein.     

Lipid transport by mammalian ABC proteins

ABC (ATP-binding cassette) proteins actively transport a wide variety of substrates, including peptides, amino acids, sugars, metals, drugs, vitamins and lipids, across extracellular and intracellular membranes. Of the 49 hum an ABC proteins, a significant number are known to mediate the extrusion of lipids from membranes or the flipping of membrane lipids across the bilayer to generate and maintain membrane lipid asymmetry. Typical lipid substrates include phospholipids, sterols, sphingolipids, bile acids and related lipid conjugates. Members of the ABCA subfamily of ABC transporters and other ABC proteins such as ABCB4, ABCG1 and ABCG5/8 implicated in lipid transport play important roles in diverse biological processes such as cell signalling, membrane lipid asymmetry, removal of potentially toxic compounds and metabolites, and apoptosis. The importance of these ABC lipid transporters in cell physiology is evident from the finding that mutations in the genes encoding many of these proteins are responsible for severe inherited diseases. For example, mutations in ABCA1 cause Tangier disease associated with defective efflux of cholesterol and phosphatidylcholine from the plasma membrane to the lipid acceptor protein apoA1 (apolipoprotein AI), mutations in ABCA3 cause neonatal surfactant deficiency associated with a loss in secretion of the lipid pulmonary surfactants from lungs of newborns, mutations in ABCA4 cause Stargardt macular degeneration, a retinal degenerative disease linked to the reduced clearance of retinoid compounds from photoreceptor cells, mutations in ABCA12 cause harlequin and lamellar ichthyosis, skin diseases associated with defective lipid trafficking in keratinocytes, and mutations in ABCB4 and ABCG5/ABCG8 are responsible for progressive intrafamilial hepatic disease and sitosterolaemia associated with defective phospholipid and sterol transport respectively. This chapter highlights the involvement of various mammalian ABC transporters in lipid transport in the context of their role in cell signalling, cellular homoeostasis, apoptosis and inherited disorders.     

A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression

ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.     

Comparison of mechanistic transport cycle models of ABC exporters

ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane domains bind and translocate substrates and are connected to a pair of nucleotide binding domains, which bind and hydrolyze ATP to energize import or export of substrates. Over four decades of investigations into ABC transporters have revealed numerous details from atomic-level structural insights to their functional and physiological roles. Despite all these advances, a comprehensive understanding of the mechanistic principles of ABC transporter function remains elusive. The human multidrug resistance transporter ABCB1, also referred to as P-glycoprotein (P-gp), is one of the most intensively studied ABC exporters. Using ABCB1 as the reference point, we aim to compare the dominating mechanistic models of substrate transport and ATP hydrolysis for ABC exporters and to highlight the experimental and computational evidence in their support. In particular, we point out in silico studies that enhance and complement available biochemical data. “This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.”     

ATP-binding cassette B4, an auxin-efflux transporter, stably associates with the plasma membrane and shows distinctive intracellular trafficking from that of PIN-FORMED proteins

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.     

Drug resistance in epilepsy and the ABCB1 gene: The clinical perspective

Multidrug resistance is one of the most serious problems in the treatment of epilepsy that is likely to have a complex genetic and acquired basis. Various experimental data support the hypothesis that over-expression of antiepileptic drug (AED) transporters may play a pivotal role in drug resistance. Hyyt 6however, key questions concerning their functionality remain unanswered. The idea that P-glycoprotein, encoded by the ABCB1 gene, might mediate at least part of the drug resistance was met with both enthusiasm and skepticism. As in oncology, initial optimism has been clouded subsequently by conflicting results. The first study reporting a positive association between genetic variation in the P-glycoprotein and multidrug-resistant epilepsy was published in 2003. Since then, several other genetic association studies have attempted to verify this result. However, taken overall, the role of P-glycoprotein in drug resistance in epilepsy still remains uncertain. We intend to critically review the inherent problems associated with epilepsy pharmacogenetic studies in general and with ABCB1 polymorphisms studies in particular. The lessons learnt from the ABCB1 studies can help us to guide future association genetics studies to investigate AED resistance, and thereby taking us closer to the cherished dream of personalized AED therapy.     

Purvalanol A,Olomoucine II and Roscovitine Inhibit ABCB1 Transporter and Synergistically Potentiate Cytotoxic Effects of Daunorubicin In Vitro

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors.     

Mice overexpressing hepatic Abcb11 rapidly develop cholesterol gallstones

Cholelithiasis is a polygenic disease, although the genes responsible for gallstone formation have not yet been clearly identified. QTL analysis has identified the Lith 1 loci on mouse Chromosome 2, and the hepatic bile salt transporter Abcb11 maps to the Lith 1 locus. We have used recently developed TTR-Abcb11 transgenic mice that overexpress Abcb11 to determine the effects of Abcb11 overexpression on cholesterol gallstone formation. TTR-Abcb11 and FVB/NJ strain control mice were fed a lithogenic or chow diet and cholesterol crystal and gallstone formation were measured. Biliary lipids in gallbladder bile and gene expression of canalicular lipid transporters were also analyzed. TTR-Abcb11 mice fed a lithogenic diet had an increased rate of cholesterol crystal and gallstone formation. This was associated with an increase in both the hydrophobic bile salt and cholesterol content of gallbladder bile. Expression of Abcb4, Abcg5, and Abcg8 did not change before gallstone formation. These data indicate that hepatic overexpression of Abcb11 increases the rate of cholesterol gallstone formation. This is likely because of increases in bile salt hydrophobicity but not because of alterations of other biliary lipid transporters. These findings strongly support Abcb11 as a Lith 1 gene.     

Olomoucine II,but Not Purvalanol A,Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.