Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.     

Subcellular localization and activity of multidrug resistance proteins

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.     

Differential expression of ABC transporters (MDR1, MRP1, BCRP) in developing human embryos

Three ABC transporters (MDR1, MRP1, BCRP), belonging to the family of multidrug resistance (MDR) proteins, play a crucial role in the protection mechanisms during embryogenesis and mediate drug resistance in cancer cells. The distribution of these transporters in the series of human embryonal/fetal intestine, liver and kidneys of various stages of intrauterine development (IUD) by indirect two-step immunohistochemical method was investigated. The organ- and age-specific expression patterns of these transporters were depicted and compared with the expression in adult organs. The evaluation of intestine and liver samples demonstrate differences in expression pattern of ABC transporters during IUD. On the contrary, in kidneys the age-specific localization was not observed. However, the increasing positivity from the kidney surface towards deeper, more differentiated parts was found. Hopefully, our study may contribute to elucidation of the role of multidrug resistance (MDR) pathways during IUD in man.     

Human MDR1 and MRP1 recognize berberine as their transport substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Human MDR1 and MRP1 Recognize Berberine as Their Transport Substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Overexpression,purification, and functional characterization of ATP-binding cassette transporters in the yeast,Pichia pastoris

The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.     

ABC multidrug transporters in schistosomes and other parasitic flatworms

Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own.     

Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris

The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.     

Gene expression of ABC proteins in hepatocellular carcinoma,perineoplastic tissue,and liver diseases

BACKGROUND: The development of hepatocellular carcinoma (HCC) is a frequent event during the natural history of cirrhosis. Effective treatment is, however, hampered by drug resistance related to the expression of multidrug resistance (MDR) proteins belonging to the ABC family transporters. Studying expression of genes coding for these proteins may help to explain the potential sensitivity of HCC to chemotherapy. MATERIAL AND METHODS: The expression of MRP1, MRP2, MRP3, MDR1, and MDR3 was investigated by quantitative RT-PCR analyses in paraffin-embedded tissues obtained from 9 cases of HCC, 16 cases of cirrhosis, 10 cases of chronic extrahepatic cholestasis, and 16 cases of normal liver. In HCC cases, gene expression was assessed both in neoplastic and perineoplastic tissue after microscopically assisted microdissection. RESULTS: MRP1 was significantly and similarly overexpressed in HCC and perineoplastic tissue. MRP2 and MDR1 were also increased in HCC, but the level of expression did not correlate with that of perineoplastic tissue. The level of expression was either reduced or normal in cirrhotic liver and during chronic cholestasis. Expression of MDR3 was unchanged in all conditions investigated. CONCLUSIONS: The genetic expression of multi-drug resistance proteins, in particular MRP1, MRP2, and MDR1, is increased during HCC. In the case of MRP1, the extent of expression is similar in neoplastic and perineoplastic tissue, but this is not the case for MRP2 and MDR1. The assessment of ABC protein expression pattern may provide important information for the diagnosis and treatment of HCC.     

ABC转运蛋白结构及在植物病原真菌中的功能研究进展

ABC (ATP-binding cassette)转运蛋白是最大的膜转运蛋白超家族之一,其主要功能是利用ATP水解产生的能量将底物进行逆浓度梯度运输.所有生物体都含有大量ABC蛋白. ABC蛋白位于细胞的不同空间,如细胞膜、液泡、线粒体和过氧化物酶体.通常,ABC转运蛋白由跨膜结构域(TMD)和核苷酸结合结构域(NBD)组成,分别与底物和ATP结合.NBD执行与ATP结合和水解,是ABC转运蛋白的动力引擎,TMD识别特异性配体.大多数ABC转运蛋白最初是通过研究生物体耐药性而被发现的,包括多效耐药(PDR)和多药耐药(MDR).本文对ABC转运蛋白的结构及作用机制,以及植物病原真菌中ABC转运蛋白功能的研究进展进行综述.     

Sulforaphane and erucin increase MRP1 and MRP2 in human carcinoma cell lines

Multidrug resistance (MDR) transporters have been termed the Phase III detoxification system because they not only export endogenous metabolites but provide protection from xenobiotic insult by actively secreting foreign compounds and their metabolites from tissues. However, MDR overexpression in tumors can lead to drug resistance, a major obstacle in the treatment of many cancers, including lung cancer. Isothiocyanates from cruciferous vegetables, such as sulforaphane (SF) and erucin (ER), are known to enhance the expression of Phase II detoxification enzymes. Here we evaluated the ability of SF and ER to modulate MDR mRNA and protein expressions, as well as transporter activity. The expression of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and multidrug resistance protein 2 (MRP2) in liver (HepG2), colon (Caco-2) and lung (A549) cancer cells treated with ER or SF was analyzed by Western blotting. Neither SF nor ER affected P-gp expression in any of the cell lines tested. Both SF and ER increased the protein levels of MRP1 and MRP2 in HepG2 cells and of MRP2 in Caco-2 cells in a dose-dependent manner. In A549 lung cancer cells, SF increased MRP1 and MRP2 mRNA and protein levels; ER caused a similar yet smaller increase in MRP1 and MRP2 mRNA. In addition, SF and ER increased MRP1-dependent efflux of 5-carboxyfluorescein diacetate in A549 cells, although again the effect of SF was substantially greater than that of ER. The implication of these findings is that dietary components that modulate detoxification systems should be studied carefully before being recommended for use during chemotherapy, as these compounds may have additional influences on the disposition of chemotherapeutic drugs.     

Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

Pharmacology and potential physiological significance of schistosome multidrug resistance transporters

Schistosomes are the causative agents of schistosomiasis, a neglected tropical disease affecting hundreds of millions worldwide and a major global health burden. Current control of schistosomiasis depends largely on a single drug, praziquantel (PZQ). One potential physiological target for new antischistosomal drugs is the parasite's excretory system, which removes wastes and xenobiotics. Multidrug resistance (MDR) transporters that are members of the ATP-binding cassette (ABC) superfamily of proteins are ATP-dependent efflux pumps involved in removal of toxins and xenobiotics from cells. They mediate the phenomenon of multidrug resistance, in which cells resistant to one drug show cross-resistance to a broad range of other agents, and are also associated with reduced drug susceptibility in parasitic helminths. In this review, we survey the different types of ABC transporter genes present within the schistosome genome, and examine recent evidence indicating that at least some of these transporters may play a role in fine-tuning susceptibility of schistosomes to PZQ. Disruption of their function may therefore provide a strategy for enhancing drug action or overcoming or attenuating drug resistance. Furthermore, dissection of the roles these transporters may play in normal schistosome physiology could potentially lead to identification of highly "druggable" targets for new antischistosomals.     

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.     

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.     

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.     

Genetic knockdown and pharmacological inhibition of parasite multidrug resistance transporters disrupts egg production in Schistosoma mansoni

P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) are ATP-dependent transporters involved in efflux of toxins and xenobiotics from cells. When overexpressed, these transporters can mediate multidrug resistance (MDR) in mammalian cells, and changes in Pgp expression and sequence are associated with drug resistance in helminths. In addition to the role they play in drug efflux, MDR transporters are essential components of normal cellular physiology, and targeting them may prove a useful strategy for development of new therapeutics or of compounds that enhance the efficacy of current anthelmintics. We previously showed that expression of Schistosoma mansoni MDR transporters increases in response to praziquantel (PZQ), the current drug of choice against schistosomiasis, and that reduced PZQ sensitivity correlates with higher levels of these parasite transporters. We have also shown that PZQ inhibits transport by SMDR2, a Pgp orthologue from S. mansoni, and that PZQ is a likely substrate of SMDR2. Here, we examine the physiological roles of SMDR2 and SmMRP1 (the S. mansoni orthologue of MRP1) in S. mansoni adults, using RNAi to knock down expression, and pharmacological agents to inhibit transporter function. We find that both types of treatments disrupt parasite egg deposition by worms in culture. Furthermore, administration of different MDR inhibitors to S. mansoni-infected mice results in a reduction in egg burden in host liver. These schistosome MDR transporters therefore appear to play essential roles in parasite egg production, and can be targeted genetically and pharmacologically. Since eggs are responsible for the major pathophysiological consequences of schistosomiasis, and since they are also the agents for transmission of the disease, these results suggest a potential strategy for reducing disease pathology and spread.     

Regulation of function by dimerization through the amino-terminal membrane-spanning domain of human ABCC1/MRP1

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.     

Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione,glucuronate or sulfate

Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters. A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs). The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors. The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate. MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH. MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH. MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level. Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting. The human MRP4, MRP5 and MRP6 have only partially been characterized. However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs. MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes. Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown. In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates and GSSG in the cytoplasm, and play, therefore, an essential role in detoxification and defense against oxidative stress.