Volume 38, No. 3/2000(March)
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 Int. Journal of Clinical Pharmacology and Therapeutics
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Editorial
Clinical Pharmacology of P-glycoprotein and related transporters
B.G. Woodcock
Review
The prognostic significance of membrane transport-associated multidrug resistance (MDR) proteins in leukemia
M.M. van den Heuvel-Eibrink, P. Sonneveld and R. Pieters
Abstract
M.M. van den Heuvel-Eibrink1,2, P. Sonneveld2 and R. Pieters1
1Department of Pediatric Oncology/Hematology, Sophia Children’s Hospital, and 2Department of Hematology, University Hospital, Rotterdam, The Netherlands
A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents. There are several ways for cancer cells to develop resistance or defense mechanisms against cytotoxic drugs. This review paper will focus on membrane transport-associated multidrug resistance (MDR). The proteins involved, P-glycoprotein (P-gp), MRP1 and LRP/MVP, share the ability to act as drug transport proteins. Following upregulation of the mdr-1 gene, the energy-dependent transmembrane P-gp overexpression results in diminished intracellular concentrations of anthracyclins, vinca-alkaloids and epipodophyllotoxins. The other transmembrane protein, MRP1, also has intracellular epitopes which are involved in intracellular redistribution and sequestration of drugs. The last named mechanism has also been ascribed to LRP, a protein which only occurs intracellularly. In leukemia patients, cellular drug resistance profiles determined in vitro at the time of presentation show a strong correlation with outcome. In AML, mdr-1 overexpression at diagnosis is a strong independent predictor for CR and long-term survival. In ALL, mdr-1 expression is of minor importance for prediction of outcome. In AML, MRP1 expression at diagnosis is not correlated with clinical response and survival in most studies. In ALL, MRP1 expression at diagnosis is not associated with response and long-term survival in the few studies on this aspect which have been published. The studies on LRP in AML emphasize the importance of the correlation between LRP-expression and anthracycline accumulation and suggest that LRP-expression has prognostic value at diagnosis. However, there is an equal number of studies where a predictive value in the case of LRP-expression in de novo AML cannot be shown. The highest levels of LRP have been reported in multiple relapses of ALL. Furthermore, new membrane-associated drug transport proteins have been reported including the transporter associated with antigen processing (TAP), the anthracyclin resistance-associated protein (ARA), five new homologues of MRP (MRP2, or MOAT, MRP3, MRP4, MRP5, and MRP6), the sister of P-glycoprotein (sP-gp) and breast cancer resistance protein (BCRP). Studies on the (clinical) significance of these proteins have not yet been reported.Correspondence to:
Dr. M.M. van den Heuvel- Eibrink; Department of Oncology/Hematology, Sophia Children’s Hospital/University Hospital, Dr. Molewaterplein 60, NL-3015 GJ Rotterdam, The Netherlands
Original
Substrate recognition by P-glycoprotein and the multidrug resistance-associated protein MRP1: a comparison
A. Seelig, X. Li Blatter and F. Wohnsland
Abstract
A. Seelig, X. Li Blatter and F. Wohnsland
Department of Biophysical Chemistry, Biocenter of the University of Basel, Basel, Switzerland
Objectives: It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 ± 0.3 Å (type I units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 ± 0.6 Å (type II units) [Seelig 1998]. Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units. In addition, we searched the putative transmembrane domains (TMD 1 – 12) of P-gp and (TMD 6 – 17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates. Methods: The three-dimensional structures of potential MRP1 substrates were modeled with a force-field approach and were then screened for electron donor units. Helical wheel projections of the 12 putative transmembrane domains of P-gp (1 – 12) and MRP (6 – 17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with p-electron systems. Results: MRP1 recognizes compounds with type I and type II units. At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported. Transport increases with increasing number of electron donor units. Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all. An analysis of the putative transmembrane a-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non-hydrogen-bonding) side chains on the other side. In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with p-electron systems. Conclusions: We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation. Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a p-electron slide guide.Correspondence to:
Dr. A. Seelig; Abtl. Biophysikalische Chemie, Biozentrum, Universität Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland
Original
Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein
R. Wheeler, S.-Y. Neo, J. Chew, S.B. Hladky and M.A. Barrand
Abstract
R. Wheeler, S.-Y. Neo, J. Chew, S.B. Hladky and M.A. Barrand
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK
Background: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells. Material and methods: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means. Conclusions: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.Correspondence to:
Dr. S.B. Hladky; Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK
Original
Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine
D. Ferry, R. Boer, R. Callaghan and W.-R. Ulrich
Abstract
D. Ferry1, R. Boer3, R. Callaghan3 and W.-R. Ulrich2
1CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, UK, 2Byk Gulden-Lomberg GmbH, Konstanz, Germany, and 3Nuffield Department of Clinical Biochemistry, Institute for Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK
Introduction: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype. Methods: In this paper we used the 1,4-dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [3H]-vinblastine transport by P-gp. Results: In cytotoxicity assays, 1 mM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 ± 80 nM to 30 ± 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit with an EC50 of 445 ± 100 nM, again, N-methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyI-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 ± 4.9 nM) and DN (Ki 11.2 ± 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H]-vinblastine transport. Conclusion: These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.Correspondence to:
Dr. D. Ferry; CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, UK
Extended Abstracts
The 13th Symposium of the “Working Group for Pharmacology in Oncology and Hematology”
