Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein (original) (raw)
Related papers
Journal of Neurochemistry, 2002
The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothehum or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-i (PECAM-i) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [ 3H]vincristineaccumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adhereru astrocyte foot processes. In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.
Journal of Biological Chemistry, 1999
Multidrug resistance in tumor cells is often accompanied by overexpression of multidrug resistance protein (MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding cassette superfamily of transport proteins. MRP mediates ATP-dependent transport of a variety of conjugated organic anions and can also transport several unmodified xenobiotics in a glutathione-dependent manner. To facilitate structure-function studies of MRP, we have generated a panel of MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2,-3,-4, and-6, bind intracellular conformationdependent epitopes, and we have shown that they can inhibit the transport of several MRP substrates. Binding competition and immunoprecipitation assays indicated that mAbs QCRL-4 and-6 probably recognize the same detergent-sensitive epitope in MRP, whereas mAbs QCRL-2,-3, and-4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the mAbs with MRP. Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 and-3 are amino acids 617-858 and 617-932, respectively, which encompass the NH 2proximal nucleotide-binding domain (NBD). In contrast, the epitope bound by mAb QCRL-4 localized to amino acids 1294-1531, a region that contains the COOH-proximal NBD. However, none of the mAbs inhibited photolabeling of intact MRP with 8-azido-[␣-32 P]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering with substrate binding or by trapping MRP in a conformation that does not allow transport to occur. Our results also demonstrate for the first time that the NBDs of MRP can be expressed as soluble polypeptides that retain a native conformation.
Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP)
British Journal of Cancer, 1998
Inherent or acquired resistance to multiple natural product drugs in human tumour cells is often associated with increased expression of multidrug resistance protein (MRP), a 190-kDa integral membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. Both clinical and experimental investigations of MRP have been facilitated by several monoclonal antibodies (MAbs) generated against intracellular epitopes of the molecule. Recently, however, several new ABC transporters that are quite closely related to MRP have been identified, raising concems about the specificity of the MRP-reactive MAbs. In the present study, we have mapped the epitopes of MAbs MRPr1 and MRPm6 to the decapeptides 23GSDLWSLNKE247 (located in the intracellular loop between the first and second membrane-spanning domains of MRP) and 1511PSDLLQQRGL'1 (located near the carboxy terminus of MRP) respectively. Alignment of the MRPr1 and MRPm6 epitope sequences with the comparable regions in mammalian ABC proteins most closely related to MRP indicates that, with the exception of munne mrp, the sequences are poorly conserved. We conclude that MAbs MRPm6 and MRPr1, together with MAb QCRL-1, which has previously been mapped to the heptapeptide 918SSYSGDI924, remain highly specific probes for detection of different regions of the MRP molecule.
Role of multidrug transporters in neurotherapeutics
Annals of Indian Academy of Neurology, 2009
Acquired resistance to antibiotics and other chemotherapeutic agents is a major problem in the practice of neurology and other branches of medicine. There are several mechanisms by which drug resistance is acquired. Multidrug transporters are important glycoproteins located in the cell membrane that actively transport small lipophilic molecules from one side of the cell membrane to the other, most often from the inside to the outside of a cell. They have important protective role yet may prove inconvenient in chemotherapy. In epilepsy and other disorders this mechanism augments the elimination of drugs from their target cells and leads to drug resistance. In this review, we have discussed the biochemical characteristics of multidrug transporters and the mechanisms by which these membrane bound proteins transport their target molecules from one side to the other side of the cell membrane. We have also briefly discussed the application of this knowledge in the understanding of drug resistance in various clinical situations with particular reference to neurological disorders. These proteins located in the placenta have important role in preventing the transplacental movement of drugs in to the fetus which may result in congenital malformations or other defects. The molecular genetic mechanisms that govern the expression of these important proteins are discussed briefly. The potential scope to develop targeted chemotherapeutic agents is also discussed.
The P-glycoprotein multidrug transporter
General Pharmacology: The Vascular System, 1996
1. P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs. Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype.
Characterization of functional assays of multidrug resistance P-glycoprotein transport activity
Leukemia, 1997
P-glycoprotein-mediated multidrug resistance has emerged as different laboratories. 8 Furthermore, there has been lack of one of the most attractive targets to improve anticancer theragreement on the optimal methodology for characterizing Papy. The P-glycoprotein functions as an energy-dependent, glycoprotein expression. The sensitivity of immunological membrane transport pump capable of decreasing the intrareagents appears to be a significant problem with low levels cellular concentration of a broad range of chemotherapeutic of P-glycoprotein expression. Analysis of extracted RNA has agents. Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials. Charac-been hampered by the inclusion of tissue stroma which terization of P-glycoprotein functional activity is critical in includes capillaries and lymphocytes which may express high determining if these multidrug resistance reversal agents levels of endogenous P-glycoprotein. improve therapeutic responses of tumors expressing P-glyco-Functional assays of P-glycoprotein activity provide the protein. In this report, we directly compare and characterize advantage of directly quantifying the potential to transport assays using rhodamine 123, dimethyloxadicarbocyanine iodsubstrates out of cancer cells. They do not rely on a correide (DiOC2), [ 3 H]daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ([ 99m Tc]Sestamibi) as P-glycoprotein lation between the amount of protein or RNA with transport transport probes to quantitate functional activity. The accumuactivity. Functional assays quantify the accumulation of flulation of certain substrates is concentration dependent and the orescent or radioactive substrates in tumor cells as markers of parameters which determine probe accumulation are impacted P-glycoprotein function. The ability of reversal agents to by the level of P-glycoprotein expression. In addition, higher enhance this accumulation has similarly been utilized to concentrations of reversal agents are required to inhibit multidelineate the role of P-glycoprotein in the transport of these drug resistance in cell lines expressing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents probes. required to inhibit completely P-glycoprotein transport activity Functional assays have traditionally been labor intensive is higher than generally recognized. Thus, the level of P-glycorequiring individual analysis of fresh specimens. In addition, protein expression may confound intersample comparisons they have been limited to hematologic malignancies due to unless sensitive probes are used in combination with saturatthe requirement for single cell suspensions to characterize the ing concentrations of potent reversal agents. These results cellular accumulation of transport substrates. Recently, we highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein have utilized the radiopharmaceutical hexakis(2-methoxyisofunction. butyl isonitrile)technetium(I) ([ 99m Tc]Sestamibi) as a functional
The role of multidrug transporters in drug availability, metabolism and toxicity
Toxicology Letters, 2003
Multidrug resistance is frequently observed when treating cancer patients with chemotherapeutic agents. A variety of ATP binding cassette (ABC) transporters, localized in the cell membrane, cause this phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells. However, the major physiological role of the multidrug transporters is the protection of our cells and tissues against xenobiotics, and these transporters play a key role in drug availability, metabolism and toxicity. Three major groups of ABC transporters are involved in multidrug resistance: the classical Pglycoprotein MDR1, the multidrug resistance associated proteins (MRP1, MRP2, and probably MRP3, MRP4 and MRP5), and the ABCG2 protein, an ABC half-transporter. All these proteins were shown to catalyze an ATPdependent active transport of chemically unrelated compounds. MDR1 (P -glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved. Oral bioavailability, entering the blood Á/brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins. Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics. Here we show that by using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents. We focus on the characterisation of the MRP1 and MRP3 proteins, their relevance in chemoresistance of cancer and in drug metabolism and toxicity. #
Cancer research, 2000
Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues. The panel consists of P 3 II-1 and P 3 II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M 2 I-4, M 2 II-12, M 2 III-5 and M 2 III-6 for MRP2; M 3 II-9 and M 3 II-21 for MRP3; and M 5 I-1 and M 5 II-54 for MRP5. All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of crossreactivity with any of the other family members. Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted. These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.
Proceedings of the National Academy of Sciences, 1989
Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.