| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
National Medical Center, Institute of Hematology and Immunology, Membrane Research Group, and Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary
ABSTRACT I. INTRODUCTION: MULTIDRUG/XENOBIOTIC RESISTANCE AND ABC TRANSPORTERS II. GENERAL STRUCTURE AND MECHANISM OF ACTION OF MDR-ABC TRANSPORTERS A. Basic Features of the ABC Transporters 1. Conserved domains, structural motifs, and catalytic mechanism 2. Composition and membrane topology of human MDR-ABC transporters B. Structural Basis of the Molecular Mechanism of Action in ABC Transporters 1. The ABC fold 2. Communication between the ABC units and the membrane-bound domains III. METHODS FOR FUNCTIONAL INVESTIGATION OF HUMAN MDR-ABC TRANSPORTERS A. Why Bother: Why So Special? B. Cellular Assay Systems 1. Drug resistance studies 2. Transient expression systems 3. Whole cell transport studies: fluorescent dyes and the calcein assay 4. Monolayer transport assays 5. Pharmacogenomic approach to identify ABC substrates C. MDR-ABC Enzymatic or Transport Assays 1. ATPase assay, detection of the catalytic cycle steps 2. Vesicular transport IV. ABCB1 (P-GLYCOPROTEIN, MDR1): THE CLASSICAL HUMAN MDR-ABC TRANSPORTER A. Biochemistry and Cell Biology of MDR1/Pgp B. Transported Substrates of MDR1/Pgp C. MDR1/Pgp in Cancer Multidrug Resistance D. Physiological and Pharmacological Functions of MDR1/Pgp E. Regulation of MDR1/Pgp Expression and Function 1. Transcriptional regulation 2. Posttranscriptional regulation of MDR1/Pgp V. THE ABCG2 (MXR/BCRP) PROTEIN A. Biochemistry and Cell Biology of ABCG2 1. Structure and cellular localization 2. Transport properties 3. Dimer formation B. Transported Substrates of ABCG2 and Its Mutants/Variants 1. Mutant variants of ABCG2 C. ABCG2 in Cancer Multidrug Resistance 1. Anticancer agents and ABCG2 2. ABCG2 expression in tumors 3. Diagnostics of ABCG2 expression and function D. Physiological and Pharmacological Functions of ABCG2: Drugs, Sex, and Survival 1. First line defense and secretion: general xenobiotic resistance 2. Prevention of endogenous toxin accumulation 3. ABCG2 in the placenta 4. ABCG2 in the blood-brain barrier 5. ABCG2 in stem cells 6. Sex differences in ABCG2 expression and function E. Polymorphisms and Regulation of ABCG2 1. SNPs in ABCG2 2. Regulation of ABCG2 F. ABCG2 in Medical Applications: Drug Development and Gene Therapy 1. ABCG2 in drug screening and development 2. ABCG2 in gene therapy VI. ADDITIONAL TRANSPORTERS IN CANCER DRUG/XENOBIOTIC RESISTANCE A. The MDR3 (ABCB4) Protein 1. Tissue distribution and cellular localization of MDR3 2. Transport properties and the physiological function of MDR3 3. Clinical relevance of MDR3 4. MDR3 and drug resistance B. The ABCG5 and ABCG8 Proteins 1. Tissue distribution and cellular localization of ABCG5 and ABCG8 2. Dimer formation 3. Physiological function and clinical relevance of ABCG5/ABCG8 4. Substrates and transport mechanism of ABCG5/ABCG8 VII. ROLE OF ABC TRANSPORTERS IN XENOBIOTIC METABOLISM: THE CONCEPT OF A ''CHEMOIMMUNITY'' DEFENSE SYSTEM A. Chemoimmunity and Toxin Metabolism 1. Toxin extrusion at the gates 2. Extrusion of modified toxins B. Innate Chemoimmunity C. Adaptive Chemoimmunity Response, Memory, and Hypersensitivity D. Chemoimmunity Transporters: Why the MDR-ABC Proteins? GRANTS ACKNOWLEDGMENTS REFERENCES
 |
ABSTRACT |
|---|
 Top NextReferences |
|---|
|
I. INTRODUCTION: MULTIDRUG/XENOBIOTIC RESISTANCE AND ABC TRANSPORTERS |
|---|
|
TopPreviousNextReferences |
|---|
Multidrug resistance transporters belong to the evolutionarily conserved family of the ATP binding cassette (ABC) proteins, present in practically all living organisms from prokaryotes to mammals. ABC transporters are large, membrane-bound proteins, built from a combination of characteristic domains, including membrane-spanning regions and cytoplasmic ATP-binding domains (see sect. II).
In humans, the three major types of multidrug resistance (MDR) proteins include members of the ABCB (ABCB1/MDR1/P-glycoprotein), the ABCC (ABCC1/MRP1, ABCC2/MRP2, probably also ABCC3–6, and ABCC10–11), and the ABCG (ABCG2/MXR/BCRP) subfamily. On the basis of a great deal of clinical and experimental work, it has been established that these pumps recognize a very wide range of drug substrates. Although recognized substrates are mostly hydrophobic compounds, MDR pumps are also capable to extrude a variety of amphipathic anions and cations. As discussed in detail below, ABCB1 preferentially extrudes large hydrophobic molecules, while ABCC1 and ABCG2 can transport both hydrophobic drugs and large anionic compounds, e.g., drug conjugates (Fig. 1). This "promiscuous" character, and the additional overlapping substrate recognition by the three major classes of the MDR-ABC transporters, provide an amazing network of drug resistance capacity in cancer cells.
|
We also describe the general structural and mechanistic features of the MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiological aspects of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins, while a detailed description of the ABCC (MRP) group of transporters is provided by the review of Deeley et al. (73). We also mention here emerging information related to additional ABC transporters with a potential role in drug and xenobiotic resistance and provide a general picture about key aspects of their cellular regulation.
Throughout this review, although biased in focusing on selected human ABC proteins, we demonstrate and emphasize the general network characteristics of the multidrug transporters, functioning at cellular and physiological tissue barriers. We try to provide a new framework for the appreciation of their role in physiological defense against chemicals, by suggesting that multidrug transporters are essential parts of an immune-like defense system. This cellular antitoxic network provides a "chemoimmunity," having a number of features reminiscent of innate immunology.
Thus physiology, biochemistry, pharmacology, and toxicology aspects inherently overlap in the present review. We certainly hope that, in addition to a detailed characterization of these transport systems, we will be able to convince the readers about the validity of a general concept that we hope will further our understanding of these multidrug transporter proteins of outstanding medical importance.
|
II. GENERAL STRUCTURE AND MECHANISM OF ACTION OF MDR-ABC TRANSPORTERS |
|---|
|
TopPreviousNextReferences |
|---|
1. Conserved domains, structural motifs, and catalytic mechanism
ABC proteins have been identified in each genome sequenced, and they typically form large families with 30–100 members in various organisms. ABC proteins are named after a conserved, specific ABC domain (140), a 200- to 250-amino acid globular protein unit, which can bind and hydrolyze ATP. The ABC unit (also called nucleotide binding domain or NBD) harbors several conserved sequence motifs. From NH2 to COOH terminal, these are the Walker A (P-loop), a glycine-rich sequence; a conserved glutamine (Q-loop), the family-specific ABC-signature (LSGGQ) motif (also called the C-loop), the Walker B motif, and a conserved His (His-switch). The ABC-signature motif is diagnostic for the family as it is present only in ABC proteins, while Walker A and B motifs are found in many other ATP-utilizing proteins (396).
ABC transporters also contain transmembrane domains (TMD), composed in most cases of six membrane-spanning helices. In archea and in prokaryotes, the ABC and the transmembrane domains are often encoded by separate genes within the same operon, while in some cases a single gene contains the TMD fused to an ABC unit. In bacteria these proteins either function as importers of essential compounds, or they export materials from the cell or lipids into the outer leaflet of the membrane. In eukaryotes, most active ABC transporters export compounds from the cytosol to the outside of the cell, or move molecules into intracellular organelles, like the endoplasmic reticulum, or the peroxisome. The human (mammalian) xenobiotic transporters discussed in this review are all export pumps, predominantly residing in the plasma membrane.
The multidrug/xenobiotic resistance (MDR) ABC proteins are primary active transporters, since they utilize the energy of cellular ATP for the promotion of vectorial, transmembrane movement of drugs or xenobiotics. These ATP hydrolytic enzymes (ATPases) interact with two different types of substrates. The energy donor substrate is the intracellular MgATP complex, and the chemical energy for the active transport of substrates is provided by binding and hydrolysis of ATP within the ABC units. In contrast to P-type ATPases, in MDR-ABC proteins ATP hydrolysis does not involve covalent phosphorylation. The end products of the hydrolysis are intracellular ADP and inorganic phosphate (for recent reviews, see Refs. 13, 34, 48, 102, 118, 123, 156, 210, 213, 300, 313, 322, 331, 389). Based on this molecular mechanism of action, the catalytic and transport properties of MDR-ABC transporters are significantly different from those of the P-type ATPases. Since these data are best provided for the MDR1/P-glycoprotein (Pgp) transporter, we discuss these issues in section IV.
In all MDR-ABC transporters, the sites interacting with the transported substrates are most probably located within the TMDs. It seems likely that a minimum of 12 membrane-spanning helices are required to ensure the complex reaction with the transported substrates. In a phenomenological sense, the transported substrates are bound to intracellular (or in some cases probably intramembrane), high-affinity "on" sites and are unloaded at extracellular, low-affinity "off" sites. However, all recent structural studies indicate a relatively large drug binding pocket within the transmembrane regions of the MDR-ABC proteins (see Refs. 48, 50, 156, 213). The molecular link, transmitting intramolecular signals between the TMDs and the ABCs, that is the substrate binding area and the catalytic machinery, respectively, is still unidentified.
These basic catalytic and active transport features of MDR-ABC transporters have been documented in numerous expression and isolation/reconstitution systems, although the exact binding sites, as well as the energetics and thermodynamics ("uphill" or "downhill" nature) of the transport processes are difficult to estimate. As detailed in this review, many of the transported substrates are hydrophobic molecules, which are concentrated in the membranes, while they have only minimum solubility either in the cytoplasmic or extracellular water phase. Therefore, the classical solute concentration ratios or electrochemical potential gradients cannot be fully appreciated, and even the stoichiometry of the transport and ATP cleavage is difficult to determine. Moreover, it is often questionable if a given molecule is a transported substrate, an inhibitor, or a transport modulator (see sect. IV). In the respective sections we discuss some of the details of the energetics and substrate recognition of individual MDR-ABC transporters.
The main subjects of this review are the human ATP-driven ABC transporter proteins, which can act as xenobiotic exporters. However, there are several membrane-associated human ABC proteins with predominant channel- or even receptor-type functions, which share common structural and regulatory features with the active drug transporters. While active ABC transporters hydrolyze ATP in close coupling with the transmembrane movement of a substrate molecule, channels and receptors use the ATP binding domains mostly for the regulation of opening and/or closing pathways, allowing the passage of ions or conveying information through the membrane. Among the human ABC transporters, well-characterized proteins carrying out such functions are ABCC7/CFTR, ABCC8/SUR1, and ABCC9/SUR2.
The ABCC7/CFTR protein forms a chloride ion channel, in which opening and closing is regulated by the binding of ATP, and by a subsequent, relatively slow ATP hydrolysis. Here the driving force of the chloride ion movement is the electrochemical potential gradient, and the ion movement has no stoichiometric relationship with ATP hydrolysis by the CFTR (for recent reviews, see Refs. 99, 142, 286, 391). The sulfonylurea receptors (ABCC8 and -9 or SUR1 and SUR2, respectively) work as regulatory subunits of ATP-dependent potassium channels in the insulin-producing beta cells of the pancreas, and in the heart, respectively. The SUR/KATP channel tetramer, formed by ABCC protein/Kir6.x heterodimer units, is activated by ADP and inhibited by ATP; therefore, the SUR subunit serves as an ADP/ATP sensor that "translates" cellular metabolic changes into alterations of the membrane potential. Again, ATP hydrolysis is very slow in this protein complex (38, 233, 324).
2. Composition and membrane topology of human MDR-ABC transporters
According to a general consensus, all functionally active ABC transporters contain a minimum of two ABC units and two TMDs. These four elements in many cases are present in one single polypeptide chain, called "full transporters," like the MDR1/Pgp/ABCB1 protein. In contrast, "half-transporters," such as the members of the ABCG family, possess only a single ABC and a single TMD. Half-transporters must form homodimers or heterodimers to generate a functional ABC transporter.
The human genome encodes 48 (according to some databases, 49) ABC proteins. Their amino acid sequence alignments revealed that these proteins can be grouped into seven subfamilies, from A to G. The proteins relevant in multidrug transport are depicted in Figure 2A. With the comparison of the individual members, sequence identity/similarity of the ABC units is generally higher than that of the TMDs. However, each subfamily is characterized by typical and somewhat different membrane topology patterns. There are no high-resolution structural data available for any of the eukaryotic ABC transporters; therefore; combination of computer-assisted prediction methods, biochemical experimental data, and model building has been used to establish the position and orientation of the transmembrane segments within the polypeptide chain.
|
As a short summary, members of the ABCA subfamily are "full transporters" with the domain arrangement of TMD1-ABC1-TMD2-ABC2. The TMDs in this family contain very large extracellular loops with numerous glycosylation sites. The ABCB subfamily consists of three full transporters (including MDR1/Pgp/ABCB1) with the domain arrangement of TMD1-ABC1-TMD2-ABC2, and seven TMD-ABC type half-transporters. The membrane topology of the 12 members of the ABCC subfamily, containing an NH2-terminal extension, is discussed in detail in the review by Deeley et al. (73). The ABCD subfamily includes four half-transporters, with a TMD-ABC type arrangement. Members of the ABCE and ABCF family are not involved in membrane transport processes and lack transmembrane domains. The five half-transporters in the ABCG subfamily show a reverse domain arrangement (ABC-TMD).
Of the 48 ABC transporters, MDR1/Pgp, several MRPs, and the ABCG2 protein certainly qualify for the MDR-ABC protein status. MDR3 (ABCB4), a closely related protein to MDR1/Pgp, and the relatives of ABCG2, the heterodimer ABCG5/ABCG8, are also ATP-dependent active transporters, and their involvement in drug and/or xenobiotic transport has also been documented. Figure 2B demonstrates the schematic membrane topology arrangements for the relevant ABCB and ABCG transporters.
Emerging information may suggest a similar, active drug transporter role for ABCG1 and/or ABCG4, as well as for ABCB5 and some ABCA type proteins, but we still have relatively little knowledge about these transporters. There are many other ABC transporters that most probably carry out substrate translocation by using the energy of cellular ATP [ABCB11 (BSEP/sister-Pgp), ABCB2/ABCB3 (TAP1/TAP2), ABCA4, members of the ABCD subfamily], but these have been not implicated in drug or xenobiotic transport and therefore are not discussed in this review.
B. Structural Basis of the Molecular Mechanism of Action in ABC Transporters
As of today, high-resolution structures are only available for bacterial ABC transporters. Therefore, for the discussion of human MDR-ABC proteins we have to rely on these data and models, which indicate a well-conserved structure and suggest a common basic mechanism of action. Unfortunately, the models reveal relatively little information about the substrate recognition or the intramolecular regulation of the individual mammalian homologs. In this section we discuss only general molecular aspects and provide further mechanistic details in the sections dealing with individual transporters.
In several bacterial ABC proteins, the ABC units are expressed as separate proteins, and the first structural information was obtained in such systems. The first high-resolution structures of an ABC unit, that of RbsA (16) and HisP (139), were published in 1998. The HisP structure was solved with a resolution of 1.5 Å and represents an ABC monomer with the typical L-shaped (two lobe) ABC fold. Since then, the structure of several ABC units has been determined (see Fig. 3).
|
-sheets. This subdomain contains the Walker A motif (P-loop), the Q-loop (
-phosphate linker), the Walker B motif, and the "His-switch" (see Fig. 3A). The ABC-specific 
-subdomain is built of four -helices and contains the family-specific ABC signature (LSGGQ) motif, while the antiparallel -subdomain contains elements responsible for the ribose/adenine orientation and interaction. The F1-type core and the ABC-specific -subdomain form the larger lobe (the longer arm of the L-shaped structure), while the -subdomain forms the smaller lobe (the shorter arm of the L). In the first publications, the ABC structures were obtained for monomers, and the authors suggested a dimer, in which the two ABC units were positioned in a back-to-back orientation (139) (see Fig. 3B, I). However, this assembly represents an energetically unfavorable interaction of the two monomers and suggests two highly exposed nucleotide binding/catalytic sites, which is difficult to reconcile with the regulated function of the active sites. By now it has been convincingly documented that the two functionally interacting ABC subunits dimerize in a "head-to-tail" orientation. The two ABC domains complement each other's active sites, forming two composite catalytic centers. The Walker A sequence of one subunit and the ABC signature motif of the opposite subunit are involved in the formation of each of the two composite ATP-binding/catalytic sites (Fig. 3B, II). This orientation was first suggested in studies describing the Rad50cd structure (a nontransporter bacterial ABC-ATPase) (130) and in models built using the HisP monomers (160). Later, similar arrangements were found in the ABC domains of various bacterial transporters, including that of MJ0796 (348), HlyB (417), and MalK (54). The orientation of the two ABC units in the dimer and the positions of the conserved sequence motifs are illustrated in Figure 3A, based on the high-resolution structure of the HlyB ABC-ABC dimer. The regulated formation of these composite active sites is in harmony with many mechanistic studies, indicating the direct involvement of both the Walker A and the ABC signature regions in ATP binding and hydrolysis. Further analysis of Rad50cd crystal structures obtained in the presence or absence of nucleotide analogs revealed that during the catalytic cycle, the composite site is formed as a result of a major intramolecular rotation, which brings the contralateral Walker A/B and signature sequences within close proximity.
By now we also have detailed structural information regarding the amino acid residues involved in nucleotide binding and the possible catalytic steps of ATP hydrolysis within the ABC dimers. Figure 4 demonstrates the nucleotide contacts within the composite active site, based on a bacterial ABC structure (MJ0796 ABC; see Ref. 416) and sequence comparisons with human ABC transporters. Based on these structures, a detailed molecular mechanism of ATP hydrolysis by ABC transporters was suggested (see Refs. 171, 416). During ATP binding, the residues of the Walker A segment in ABC unit I coordinate the three phosphate groups of ATP, while the adenine ring is oriented by interactions with a neighboring bulky residue. In the same ABC unit, the Q-loop and the Walker B glutamate alternately interact with a water molecule involved in ATP hydrolysis. From ABC unit II, residues of the signature region are also involved in the coordination of the phosphate groups and the ribose part of ATP, while an additional alanine interacts with the catalytic water molecule. During ATP hydrolysis, that is in a change from a "prehydrolytic" state to a Mg-ADP-bound, "posthydrolytic" structure, together with several minor intramolecular alterations, the Q-loop and the signature regions perform major movements. The movement of the signature (LSGGQ) segment is triggered by a rotation of the ABC-specific -subdomain. Interactions between the signature motif and the -phosphate give additional cooperative stabilization to the nucleotide ("ATP-bound") sandwich dimer. ATP can be considered a "molecular glue" with the -phosphate coupling the Walker A and B motifs and the Q-loop of one of the ABCs with the LSGGQ signature motif of the opposite ABC unit. After hydrolysis, ADP remains bound to the Walker A motif, while the cleaved phosphate anion remains bound to the signature region.
|
The high-resolution structures of various ABC-ABC dimers do not answer several questions concerning the structural basis of the ABC-TMD interactions that ensure the transmission of signals from the substrate binding sites to the catalytic machinery. It is also unknown how ATP binding and hydrolysis serve as the "power stroke" of transmembrane transport. Furthermore, in the separate ABC-ABC dimers, the molecular interactions are not influenced by the presence of the TMDs, and thus the ABC units may enjoy more freedom than in the full transporter complexes. In the case of eukaryote ABC transporters, only low-resolution studies, e.g., electron microscopy (EM) of single particles combined with image analysis (289) and EM analysis of two-dimensional crystals of MDR1/Pgp (203, 291, 293), are available. Typically, the resolution of these structures is 12–25 Å, allowing only the detection of major conformational changes and the relative position of the major structural elements. These data are included in the presentation of the individual MDR-ABC transporters in the following sections.
At the time when this review was compiled, several structures of complete bacterial ABC transporters were already obtained at an atomic resolution. These include the dimeric forms of the homodimer BtuCD (213) and the MsbA "half-transporters," the latter being a close homolog of human MDR1/Pgp, for which crystal structures from various bacterial strains and in various catalytic states were solved with high resolution (49, 50, 285).
Importantly, the composite nature of the active/catalytic centers within the ABC domains, discussed above, is reflected in each of these structures. Another key piece of information provided by these structures seems to be trivial, that is, the transmembrane regions are indeed composed of -helices, spanning the membranes with the predicted numbers. Although fully expected, this evidence settles the issue for a number of proposed alternative transmembrane domain arrangements (158, 159). Another major finding of these structures is that an intracellular domain (ICD) forms a bridge between the TMDs and the ABCs. The ICD is built up from three subdomains, formed from elements of the intracellular loops between transmembrane (TM) helices and between the TMD and ABC.
In the first published full MsbA structure (50), the TM helices form a cone-shaped chamber, with a wide opening from the intracellular side. In contrast, the structure of MsbA in the presence of ADP, vanadate, and a transported ligand (287) indicated a large rotation and translation of the TMDs, resulting in the opening and closure of the chamber to the periplasmic direction and the intracellular face, respectively. However, the question still remains open, are these major molecular movements indeed parts of the molecular mechanism, or do they just represent crystallization artifacts? A space-filling model of the human MDR1/Pgp is shown in Figure 5, constructed on the basis of the atomic resolution information obtained in bacterial transporters and low-resolution structures available for this human transporter. Some details of this model are further discussed in section IV.
|
|
III. METHODS FOR FUNCTIONAL INVESTIGATION OF HUMAN MDR-ABC TRANSPORTERS |
|---|
|
TopPreviousNextReferences |
|---|
In addition to "curiosity-driven" basic research, the two major practical causes for measuring MDR-ABC transporter expression and function are related to cancer drug resistance and predicting/following the general fate and effect of pharmaceutical agents in our body. In both cases, the goal is to determine the actual drug transport capacity, that is, the functional expression level of the relevant transporter(s).
As described in the sections dealing with the individual transporters, this task is not easily achieved by the routinely available classical methods. MDR-ABC protein expression is often not correlated with mRNA levels, as translation, protein processing, localization, and degradation are all regulated processes. Moreover, the physiological or cancer cell expression level of an MDR-ABC transporter is often below the detection threshold, as relatively few active transporter molecules may cause major alterations in drug transport. Even if the overall determination of the transporter protein levels is successful, in many cases the variable, regulated localization of the protein may result in misleading conclusions. In addition, these proteins cannot be studied by the classical membrane transport methods, developed for, e.g., ion exchangers, P-type ATPases, or regulated channels. The major differences may lie in the hydrophobic or amphipathic nature of the transported substrates and the relatively loose coupling of transport and ATP hydrolysis in these proteins (see sect. II and below). Based on these problems, the quantitative, functional determination of MDR-ABC transporters gained a special emphasis in the research efforts.
Another major issue is the relevance of in vitro experiments to the in vivo role of MDR-ABC transporters. Although initial indications for the role of membrane transporters in cancer multidrug resistance originated from clinically oriented studies, it is important to emphasize that the role of these transporters in clinical anticancer drug resistance is still unsettled. According to our view, this is mostly due to the still underdeveloped methodological arsenal of the clinical laboratory studies, especially the lack of proper, quantitative assay methods directly applicable for studying the function of all relevant MDR-ABC proteins in human solid cancer tissues.
In the initial in vitro experiments, cellular multidrug resistance was modeled by the in vitro drug selection of the tumor cells. However, since the transported drugs/compounds are mostly hydrophobic, their cellular accumulation strongly depends on the availability of intracellular binding sites, sequestration, as well as the "passive" permeability of the cell membrane. Vesicular transport studies encountered similar problems; if there is no binding or sequestration, the "leakage" of the accumulated drug rapidly counteracts the accumulating transport process. This is the reason why many indirect methods (drug-stimulated ATPase activity, fluorescent dye transport, "nucleotide trapping," drug binding, etc.) were developed to appreciate the drug transport and related drug resistance functions, but a consensus in their application is still lacking.
While this wide array of methods to investigate the function and substrate interaction of MDR-ABC transporters has been developed by various research laboratories, the pharmacological industry became a major consumer and contributor to these studies as well. This is partly due to the development of new anticancer agents, but even more to the interest in defining the role of ABC transporters in drug absorption, distribution, metabolism, excretion, and toxicology (ADME-Tox). As detailed in the relevant sections, MDR-ABC transporters are key determinants of drug permeation into different tissues, as these proteins are located in the absorption, secretion, and sanctuary barriers.
Based on the above-described questions, in section III we discuss the relevant in vitro systems, the various cellular and enzymatic/vesicular models applied for the studies of MDR-ABC transporters. We focus on in vitro functional assays, the analysis of cytotoxicity, translocation of substrates, and ATP hydrolysis and emphasize methods that can be used to quantitatively estimate transporter function. We refer to all in vivo investigations in the sections discussing the individual multidrug transporters.
Typically, in vitro studies use cell lines overexpressing a desired ABC transporter. Such cells may be readily engineered using routine molecular biology techniques. Alternatively, cell lines with pleiotropic drug resistance may be generated through exposure to increasing concentrations of antitumor drugs. Fulfilling their role in detoxification, several ABC transporters (such as ABCA2, ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2) have been found to be overexpressed in cell lines cultured under selective pressure (367). For example, elevated levels of ABCB1 were found in cells selected with Vinca alkaloids, anthracyclines, and colchicine, among others. Similarly, ABCG2 was overexpressed in cells selected for resistance to topotecan and mitoxantrone. To some extent, in vitro selection of cells resembles the in vivo acquisition of the MDR phenotype. However, resistant cells have to be cultured under constant selective pressure to ensure a stable phenotype. Under these conditions, cells usually develop multiple mechanisms of resistance (15), involving the overexpression of further transporters. Discerning signal from noise in selected cells can be achieved by control experiments using parental cells, or cells treated with specific inhibitors. In general, availability of appropriate control cells is a limitation, since cells undergoing selection may have inherent differences that are not readily identified or controlled. Furthermore, inhibitors are rarely specific. Despite these constraints, selected cell lines overexpressing an ABC transporter are extensively used both in research and industrial settings.
Some of the ABC transporters implicated in MDR have never been found overexpressed in drug-selected cells. Still, these transporters (such as ABCB11, ABCC3, ABCC5, ABCC6, ABCC10, and ABCC11) could confer drug resistance when they were transfected into cells. Again, cell lines stably overexpressing these ABC transporters showed characteristic resistance to compounds that are substrates for transport. Thus cytotoxicity assay is a convenient tool that is often used to search for substrates and reversing agents. Cytotoxicity assays can be well quantitated, and "killing curves" provide proper IC50 values for the estimation of changes in cellular drug resistance. However, drug sensitivity may be entirely different in different immortalized or tumor-derived cell lines, and the complex cellular sensitivity and resistance mechanisms greatly modify the effects of such studies.
2. Transient expression systems
Studies using mammalian cells subjected to drug selection with or without the introduction of MDR1 cDNA are subject to contention because of the pleiotropic effects of the drugs (12). To overcome this concern, ABC transporters may be expressed in transient expression systems. Gottesman and colleagues (283) adapted a vaccinia virus-T7 RNA polymerase hybrid transient expression system that does not involve selection for the functional expression of MDR1/Pgp. In this system, high levels of expression can be achieved within 48 h posttransfection, allowing the study of transport and drug-stimulated ATPase.
In the case of several ABC transporters, including MDR1/Pgp and its close relatives, expression of the gene products in insect (e.g., Spodoptera frugiperda, Sf9) cells, using recombinant baculoviruses, proved to be an efficient tool for analyzing various aspects of transporter function. This system allowed studying the protein interactions with substrates and also the substrate-stimulated ATPase activity (307). Although the baculovirus expression system ensures relatively high expression levels (4–5% of the total membrane protein), expression is transient, and the functional analysis of the expressed proteins in most cases has to be performed using microsomes prepared from the infected cells. Despite the lack of full glycosylation in insect cells (proteins are only core-glycosylated), we have successfully used this system for the expression and characterization of several ABC transporters (22, 260, 305, 307, 346). In case a mammalian protein has to be complemented by (an)other protein(s) for function, a heterologous system may not be suitable for functional analysis. Still, functional expression in insect cells can be used to characterize mammalian proteins outside the context of interacting networks. We have used this argument to show that ABCG2, an ABC half-transporter, functions as a homodimer (see sect. V). As mentioned below, transient expression systems are especially useful for direct enzymatic or transport studies of MDR-ABC transporters.
3. Whole cell transport studies: fluorescent dyes and the calcein assay
The multidrug resistance phenotype suggests the overexpression of an MDR-ABC transporter and the decreased cellular accumulation of the toxic compounds. To verify this relation, experiments can be designed to follow the steady-state cellular accumulation of radioactively labeled or fluorescent compounds. In case a reduced accumulation or an increased extrusion is detected, experiments can be performed to define the kinetic parameters, energy dependence, and the specificity of the efflux. However, as mentioned above, intracellular binding, sequestration, and "membrane leakage" of the compounds are major difficulties in quantitating these studies. Therefore, a large number of indirect transport assays, where substrates and inhibitors are identified by following the transport of a reporter substrate, have been developed. Reporter substrates should be generally not toxic, their cellular fate should be well characterized, and an easily measurable fluorescence is a major advantage for such test compounds (Fig. 6).
|
We found calcein-AM to be the dye of choice, because 1) the hydrophobic, nonfluorescent AM form is an excellent substrate for MDR1/Pgp; 2) the fluorescent, hydrophilic free acid is trapped inside the cells, does not bind to intracellular proteins, and is no longer a MDR1/Pgp substrate; and 3) the fluorescence of free calcein is not sensitive to changes in pH and ion concentrations. Due to the enzymatic enhancement of the dye-trapping process, the sensitivity of an assay measuring calcein accumulation highly surpasses that of other functional assays (Fig. 7, Ref. 124). Based on these considerations, we developed a quantitative calcein transport assay, which correlated with functional MDR1/Pgp expression. The assay kit is suitable for flow cytometry-based clinical laboratory applications and was found applicable in predicting multidrug resistance in acute leukemia (170). The calcein assay can be used for the estimation of the transport properties of certain MRPs as well, while ABCG2 does not transport either calcein-AM or free calcein (see sect. V).
|
In vivo, drugs have to cross pharmacological barriers to get absorbed (intestinal epithelial cells), distributed (blood-brain barrier endothelial cells), or excreted (hepatocytes, proximal tubule epithelial cells). This transcellular movement is modeled by cellular monolayer efflux ("vectorial transport") assays. The cell lines used in these assays are polarized epithelial or endothelial cells, such as the human intestinal epithelial line Caco-2. Caco-2 cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarized monolayer, with a well-defined brush border on the apical surface, and intercellular junctions. Measuring the rate of transport across the Caco-2 monolayer provides insight into absorption across the gut wall. Test drugs can be applied to either side of the cell layer, and the rate of transport across the monolayer is measured from the apical to basolateral (A-B) or from the basolateral to apical (B-A) direction. The bidirectional apparent "permeability" of the test compound reflects the complex transport processes across the cell membranes. Measuring transport in both directions across the cell monolayer provides an indicator of active transport, chiefly mediated in Caco-2 cells by MDR1/Pgp (see sect. IV).
To establish specific roles of different MDR-ABC transporters, transfected versions of the canine kidney cell lines MDCKI, MDCKII, or the porcine kidney epithelial cells LLC-PK1 can be used. In these assays, polarized cells are grown on semipermeable filters, and the MDR-ABC transporters are localized to the proper apical or basolateral surfaces of the cells (for reviews, see Ref. 197). Fluorescent compounds can also be used in indirect transport assays in such polarized cells, e.g., the "vectorial calcein assay" can be applied for the estimation of drug interactions with various MDR-ABC proteins (33, 288). Especially in ADME-Tox drug research assays the application of cell lines with well-defined "efflux" and "influx" transporters can be an advantage. For such complex cellular or monolayer transport assays, the stable coexpression of various MDR-ABC transporters and a number of "influx" transporters are already available (70, 189, 206, 308, 309, 421).
5. Pharmacogenomic approach to identify ABC substrates
To determine substrate specificities of ABC transporters and their role in drug resistance of cancer cells, Szakacs et al. (363) have measured the expression profile of the 48 ABC transporters in the National Cancer Institute 60 (NCI-60) cancer cell panel. The NCI-60 cell panel was set up by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI), which has screened the cytotoxicity profiles of more than 100,000 chemical compounds in the 60 cell lines (97). Through the measurement of ABC transporter expression levels, it was possible to link ABC transporter function to a variety of already determined molecular, physiological, and pharmacological features of the cells. Analysis of the correlations between ABC transporter expression and known patterns of drug activity for 1,429 compounds across the 60 cancer cell lines yielded strongly inverse-correlated pairs, where the expression of an ABC transporter was strongly correlated with decreased sensitivity to a drug. As expected, good agreement was found between the mRNA expression of MDR1/Pgp and the reduced cellular sensitivity to anticancer drugs that are known to be substrates of this transporter. Furthermore, the method also allowed the identification of previously unknown MDR1/Pgp substrates (363). Interestingly, the same approach indicated that ABC transporters other than the well-characterized MDR proteins can provide resistance in naive (unselected) cancer cell lines. Follow-up experiments, using cells transfected with ABCC2, ABCC11, or ABCC4 (401), validated the predictions. These results suggested that this pharmacogenomic approach provides an unbiased method for discovering the substrate specificities of known, as well as yet uncharacterized members of the ABC superfamily.
C. MDR-ABC Enzymatic or Transport Assays
1. ATPase assay, detection of the catalytic cycle steps
As discussed in section II, MDR-ABC transporters exhibit a catalytic activity that is coupled to drug transport. Indeed, crude and purified preparations of various MDR-ABC transporters exhibit substrate-induced, vanadate-sensitive ATPase activities. The rate of ATP hydrolysis is easily determined by measuring the liberation of inorganic phosphate, using membrane vesicles prepared, e.g., from MDR-ABC expressing insect or mammalian cells. Isolated and reconstituted MDR-ABC transporters have also been successfully applied in this regard (10, 14, 81, 114, 219, 307, 329, 336).
The profile of the drug-stimulated ATPase reflects the nature of interaction: compounds may be substrates or inhibitors or may have no effect on the transporter. In the presence of transported substrates, the ATPase activity of the transporter increases (activation protocol). Noncompetitive inhibitors, or compounds transported at a lower rate, inhibit the ATPase activity of the stimulated transporter (inhibition protocol). In general, most of the efficiently transported compounds stimulate the ATPase. In the case of MDR1/Pgp, exceptions were noted, and some substrates were shown to stimulate activity at lower, and inhibit the ATPase at higher, concentrations. Further complicating the issues, the ATPase activity in most cases has a basal rate, probably related to an endogenous stimulation and/or a partial uncoupling (see below) and may also be affected by the lipid environment and the experimental conditions (9, 248, 330, 332). However, because of its simplicity and reproducibility, the ATPase assay is one of the most widely used assays to search for compounds that interact with various ABC transporters.
The application of the vanadate-sensitive membrane ATPase assay for drug interaction studies circumvents the problems of measuring the transport of hydrophobic substrate compounds, if indeed MDR-ABC ATPase is closely coupled to transport activity. While most studies agree that in general this is the case, a certain "slippage" between ATP hydrolysis and drug transport by the MDR-ABC transporters may be a basic feature of the catalytic mechanism (see sect. II). Studies on the wild-type and mutant variants of the major multidrug transporters may answer this basic question.
As described in more detail in the relevant sections, several methods have been developed for the analysis of the catalytic steps of the ATPase/transport reaction of MDR-ABC transporters. These include the determination of the modulation of the vanadate-dependent adenine nucleotide "trapping" (26, 362), and the vanadate-induced cleavage of the transporter protein (134), induced by transported substrates or modulators. Often applied methods are following the interaction of membrane-bound or isolated MDR-ABC proteins with labeled photoaffinity analogs of ATP or the transported substrates. The conformation-sensitive binding of specific monoclonal antibodies both in the case of MDR1/Pgp (267a) and ABCG2 (265) has been successfully applied to study substrate interactions and models of the catalytic cycle. While these methods may yield valuable information about the mechanism and transport properties of the given ABC protein, they are usually expensive with a low throughput; thus they may not be efficiently applied in cancer drug detection or drug research.
A more direct measurement of substrate translocation and its modulation can be achieved by the quantitation of the intravesicularly trapped substrates in vesicular transport assays. However, as noted above, this assay has major limitations when using hydrophobic substrates, due to significant nonspecific binding and rapid leakage of the compounds from the vesicles.
Successful vesicular transport studies using membranes from various sources (insect cells, transformed and selected cell lines, artificial membrane vesicles) have been reported by several laboratories (337, 422). Given the orientation of ABC transporters in cells (where the NBDs are in the intracellular compartment), in inside-out vesicles, the NBDs face the incubation media (accessible to ATP and other chemicals), and substrates are actively transported into the vesicles. Rapid filtration, using glass fiber filters or nitrocellulose membranes, is used to separate the vesicles from the incubation solution, and the test compound, trapped inside the vesicles, is retained on the filter. The quantity of the transported unlabeled molecules can be determined by high-resolution, high-sensitivity analytical methods. Alternatively, the compounds are radiolabeled or a fluorescent tag is attached, and the radioactivity or fluorescence retained on the filter is quantified.
In the case of MDR1/Pgp, which recognizes mostly hydrophobic compounds, a vesicular transport assay for most of the relevant substrates could not be established, due to the high nonspecific binding and passive diffusion of compounds. For some less hydrophobic, relatively low-affinity substrates, such an assay is available. Transport of hydrophilic quaternary drugs (such as N-methylated derivatives of quinidine) was demonstrated into vesicles isolated from MDR1/Pgp overexpressing insect cells (129). In another experiment, using rat hepatocye canalicular membrane preparations, ATP-dependent uptake of radioactively labeled doxorubicin and N-pentylquinidium could be measured by centrifugation of the vesicles through a gel matrix (397). It has to be noted that vesicles prepared from hepatocytes contain additional ABC transporters, such as ABCB11 (BSEP, or "sister-of-Pgp") and ABCC2/MRP2. For these other ABC transporters, including members of the ABCC and the ABCG subfamilies, a number of transported substrates are less hydrophobic and therefore are trapped inside the vesicle compartment. Vesicular transport can also be performed in an "indirect" setup, where interacting test drugs modulate the transport rate of a labeled reporter compound.
As a general conclusion, a great variety of assays have been developed for functional studies on MDR-ABC transporters, but none of these methods can be singularly applied to answer all questions of functional expression, substrate handling, and regulation. The adaptation and/or the proper combination of these assays for detection of MDR-ABC effects in cancer cells, or predicting their role in the ADME-Tox properties for a given compound, is still more an art than an established procedure. Another key question is the simple, reliable, if possible high-throughput application of the most informative methods in generally available laboratory settings. International method selection and standardization efforts may soon improve this situation both for the clinic and the pharmacological industry.
|
IV. ABCB1 (P-GLYCOPROTEIN, MDR1): THE CLASSICAL HUMAN MDR-ABC TRANSPORTER |
|---|
|
TopPreviousNextReferences |
|---|
Human ABCB1 (MDR1/Pgp) is the archetypal ABC transporter and has earned its reputation by being the first discovered, the most important medically, the most studied, and the one with the broadest substrate specificity (156). Models describing the function of MDR1/Pgp rely on biochemical experiments, mutagenesis studies, low-resolution structures, and the atomic level structures of various other ABC proteins.
ABCB1 is a member of the ABCB subfamily, which in humans has 11 transporters. The ABCB1 (MDR1) gene is located on chromosome 7q21. It consists of 28 exons, which encode a 1,280 amino acid glycoprotein (MDR1/Pgp). Analysis of the primary sequence delineates a tandem repeat of transmembrane domains and ATP-binding cassettes and a linker region connecting the two homologous halves of the protein. The two halves form a single transporter with a pseudo-twofold symmetry, in which the transmembrane helices define a "pore" for substrate translocation, and the nucleotide binding cassettes harvest the energy of ATP binding and hydrolysis.
The membrane topology of MDR1/Pgp has been elucidated by epitope insertion experiments (172, 173), fully supporting the original topology model of six TM helices in both TMDs of the protein (52). The linker region connecting the two halves of the protein plays a critical role in ensuring proper interaction of two subunits. The overall shape of the molecule has been revealed by low-resolution techniques. Cryoelectron microscopy images (at a resolution of 
8 Å) suggest that the transmembrane domains form a funnel-shaped aqueous chamber in the plane of the membrane (290). The chamber opens towards the extracellular compartment and seems to be closed at the intracellular end. The two NBDs are located intracellularly, in close proximity that allows extensive interactions between the two catalytic sites (Fig. 5).
ATP hydrolysis and drug transport are promoted by different segments of the protein, and the collaboration of these modules ensures that 1) ATP is hydrolyzed when a substrate is presented for transport, 2) the substrate is translocated as the energy of ATP is released, and 3) the two ABC units act in a concerted fashion (see sect. II). While a large body of biochemical experiments, coupled with mutagenesis studies summarized below, have elucidated some key features, in the absence of structural evidence the exact mechanism of how conformational signals are transmitted within the protein, resulting in coupling of ATPase and transport cycles, awaits more relevant and/or higher resolution structural data. In particular, there is much controversy regarding the mechanism by which transported substrates promote ATP hydrolysis (substrates may affect ATP binding, ATP hydrolysis, or both), and the details of how the energy of ATP hydrolysis is harnessed for transport.
MDR1/Pgp recognizes substrates that belong to very diverse chemical classes. Several investigators have attempted to catalog the chemical fingerprint of a "model substrate." The consensus that has emerged from these studies is that MDR1/Pgp substrates are amphipatic, with a molecular mass of 300–2,000 Da. Despite the wealth of mutagenesis and photolabeling studies (reviewed in Ref. 13), the structural basis of the transporter's promiscuity remains unknown. Mutations affecting substrate specificity are clustered predominantly in transmembrane domains 5, 6 and 11, 12, but they are also found throughout the rest of the molecule, including the intracellular loops and the ATP binding domains (12). In a series of experiments, Loo and Clarke (217) have used cysteine scanning mutagenesis to assess the relative position of moieties involved in drug binding within the transmembrane regions. Models based on disulfide cross-linking experiments place transmembrane helices 6 and 12 in close proximity. Similarly, TM helices 5 and 8, as well as TM2/TM11, are close to each other, in agreement with the proposed funnel shape of the channel (214). During transport, drugs are translocated from a high-affinity "loading" site located in the intracellular or inner leaflet compartments to a low-affinity, outward-facing "unloading" site. This energy-dependent translocation event involves the repacking of the membrane-spanning -helices (291, 293).
Crude membranes purified from insect cells expressing MDR1/Pgp show ATPase activity that is stimulated by transported substrates (307). Similarly, purified MDR1/Pgp reconstituted in proteoliposomes exhibit substantial drug-stimulated ATPase activity (14). The MDR1/Pgp ATPase has a low affinity and low specificity for nucleotides with a single apparent Km for MgATP of 0.5–1 mM that is not affected by the transported substrates. The turnover of the maximal, drug-stimulated ATPase is 10–20/s, the activity ranges between 5 and 22 µmol · min–1 · mg MDR1/Pgp–1, and the degree of drug stimulus is 2- to 11-fold. It is generally agreed that the stoichiometry of ATP hydrolysis to drug transport is in the range of 1–3 (14).
ATP binding and cleavage occur at the ABC units, and the close interaction of two ABC units results in the formation of a fully competent ATP-hydrolytic site (see sect. II). MDR1/Pgp (as all ABC transporters) differs from P-type ATPases in that it does not show a high-affinity ATP binding and does not utilize a covalently phosphorylated protein intermediate. Theoretically, the ATPase cycle can be described as containing the following basic steps: ATP binding, cleavage of the terminal phosphate bond, and release of the catalytic products (Pi and ADP). Practically, these steps are empirically defined and characterized in experiments described below (for a summary, see Fig. 8).
|
