Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In vitro, overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only very distantly related. Studies with MRP-enriched membrane vesicles have demonstrated that the protein can bind and transport cysteinyl leukotrienes, as well as some other glutathione conjugates, with high affinity. In contrast, there is no direct evidence of the ability of MRP to bind or transport unmodified forms of the drugs to which it confers resistance. To facilitate studies of the physiological function(s) of MRP and its ability to cause multidrug resistance in vivo, we cloned and characterized the mRNA specifying its murine homolog. The murine MRP mRNA encodes a protein of 1528 amino acids that is 88% identical to human MRP. Although detectable by Northern blotting at variable levels in a wide range of tissues, in situ hybridization experiments revealed that MRP mRNA expression in some tissues is cell-type specific. High levels of the mRNA were detected in epithelia lining bronchi and bronchioles, as well as stage-specific expression in the seminiferous epithelium of the testes. Comparison of the predicted hydropathy profiles of human and murine MRP suggests a highly conserved membrane topology, the most distinctive feature of which is an extremely hydrophobic NH2-terminal region containing five or six potential transmembrane sequences. This structural feature is shared with the sulfonylurea receptor and the yeast cadmium factor 1 but is not present in members of the superfamily, such as the cystic fibrosis transmembrane conductance regulator and P-glycoproteins. Finally, we used overlapping cDNAs to construct an episomally replicating murine MRP expression vector that was stably transfected into HeLa cells. MRP-Transfected cell populations expressed markedly elevated levels of a 180-190-kDa protein that cross-reacted with a polyclonal antiserum raised against a peptide that is completely conserved in murine and human MRPs. The MRP transfectants also displayed increased resistance to vincristine (5-6-fold) and doxorubicin (< 2-fold).
Mol Pharmacol 1996 Jun
PMID:Structure and expression of the messenger RNA encoding the murine multidrug resistance protein, an ATP-binding cassette transporter. 864 56

The role of the glutathione (GSH) system in vivo or in drug resistance has received much attention, since GSH is a major component of the cellular detoxification system. We Studied the effect of GSH depletion by buthionine sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, on doxorubicin (DOX) toxicity in mice. The administration of BSO (30 mM in drinking water for 5 days) significantly decreased the tissue GSH. The GSH depletion in various tissues by BSO was associated with a decrease in the detoxification of DOX in mice. A single dose of 20 mg/kg of DOX significantly reduced body weight and rectal temperature in mice 3 days after injection. The combination with BSO and cepharanthine (biscoclaurine alkaloid), a P-glycoprotein (P-gp) inhibitor, significantly potentiated decrease in body and hypothermia induced by DOX. The study demonstrates that BSO markedly increases the toxicological effect of DOX with the alterations in GSH of tissues and Suggests that the intracellular accumulation of DOX is not a factor.
Res Commun Mol Pathol Pharmacol 1995 Sep
PMID:Effect of glutathione depletion by buthionine sulfoximine on doxorubicin toxicity in mice. 868 Aug 8

Expression of the multidrug resistance gene MDR1 is reported to be an important determinant of the response to chemotherapy and survival in some cancers. We compared three methods for determining the intrinsic MDR1 expression in soft tissue sarcomas. We studied MDR1 gene expression in 39 samples from 33 cases of soft tissue sarcomas comprising 11 liposarcomas, nine malignant fibrous histiocytomas, six leiomyosarcomas, four malignant schwannomas, three fibrosarcomas, three synovial sarcomas, and three epithelioid sarcomas, and seven cases of benign soft tissue tumors in adult patients. To detect MDR1 mRNA, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in all samples. Furthermore, RNA dot-blot analysis with digoxigenin-labeled RNA probe and immunohistochemistry with JSB-1 and C-219 antibodies for P-glycoprotein were employed in 34 and 37 samples in soft tissue sarcomas, respectively. We compared these three detection techniques. Of the 39 specimens, 18 (46%) showed MDR1 PCR products. Liposarcomas (six of 11), malignant fibrous histiocytomas (six of nine), leiomyosarcomas (four of six), fibrosarcomas (two of three) revealed high or intermediate MDR1 expression at high frequency. No MDR1 expression was detectable in malignant schwannomas, synovial sarcomas, or epithelioid sarcomas. Of seven benign soft tissue tumors, one ganglioneuroma and one lipomatosis showed low levels of MDR1 expression. By RNA dot-blot analysis, MDR1 transcripts were detectable in 12 of 34 specimens (35%). Four samples were negative by dot blot despite positivity with RT-PCR. Concordance between MDR1 expression by RNA level with RT-PCR and dot blot and at the protein level with immunohistochemistry using C-219 was found in 16 (47%) of the 34 comparable specimens. Eight samples showed positive immunoreactivity for C-219 despite negative results in RT-PCR and dot-blot analysis. The intrinsic MDR1 expression in soft tissue sarcoma seemed to depend on certain tumor types, such as liposarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and fibrosarcoma. For the evaluation of MDR1 expression, RT-PCR is useful because of its relative simplicity and sensitivity. However, the clinical significance of such low levels of MDR1 expression detected only by RT-PCR must be discussed within systematically treated patient groups.
Diagn Mol Pathol 1996 Jun
PMID:Reverse transcriptase-polymerase chain reaction amplification of MDR1 gene expression in adult soft tissue sarcomas. 872 96

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with 35S-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.
Plant Mol Biol 1996 Jun
PMID:A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein. 879 Mar

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.
Mol Pharmacol 1996 Sep
PMID:BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells. 879 85

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp 1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp 1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.
Mol Cell Biochem 1996 Jun 07
PMID:Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences. 881 6

The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl- channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl- currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotonic-induced chloride conductance in neuroblastoma cells.
Cell Mol Neurobiol 1996 Jun
PMID:Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene. 881 5

The mechanism by which chlorpromazine (2-chloro-10-(3-dimethylaminopropyl)-phenothiazine) reverses P-glycoprotein (P-gp2) mediated multidrug resistance was investigated using membrane vesicles prepared from human CCRF-CEM leukaemia cells. Chlorpromazine was transported in an ATP-dependent manner into membrane vesicles prepared from vinblastine resistant (VBL1000) cells but not from drug-sensitive cells. The chlorpromazine uptake was sensitive to osmotic pressure, indicating true transport into the vesicle lumen. The ATP-dependent chlorpromazine uptake was inhibited about 30% by the addition of ammonium chloride, indicating that a pH or electrical gradient could not account for the majority of ATP-dependent chlorpromazine uptake. The results of this study show that chlorpromazine is actively transported my P-glycoprotein and that chemosensitization by phenothiazines may occur by competition of these agents for active transport of anticancer agents by P-glycoprotein.
Biochem Mol Biol Int 1996 Jul
PMID:Chlorpromazine transport in membrane vesicles from multidrug resistant CCRF-CEM cells. 884 36

Multiple drug resistance (MDR) as a result of overexpression of the P-glycoprotein drug transporter, a product of the MDR1 gene, is a significant problem in cancer therapeutics. We demonstrate that phosphorothioate antisense oligonucleotides can reduce levels of MDR1 message, inhibit expression of P-glyco protein, and affect drug uptake in MDR mouse 3T3 fibroblasts. An obligonucleotide (5995) directed against a sequence overlapping the AUG start codon was effective in reduction MDR1 transcript and protein levels when used at submicromolar concentrations in conjunction with cationic liposomes, whereas a scrambled control oligonucleotide (10221) was ineffective. Substantial and specific antisense effects could also be attained with a 5' cholesterol conjugate of the 5995 sequence. In this case, use of cationic liposomes was unnecessary. The 5' cholesterol 5995, but the not 5' cholesterol 10221, reduced MDR1 message and P-glycoprotein levels by 50-60% when used at low micromolar concentrations. In parallel, treatment with 5' cholesterol 5995 also enhanced cellular accumulation of rhodamine 123, a well-known substrate of the P-glycoprotein transporter. The effectiveness of the cholesterol-conjugated 5995 may be due to its rapid and extensive cell uptake, as indicated in flow cytometry and confocal microscopy studies. These observations suggest that cholesterol-conjugated anti-sense oligonucleotides may offer a novel approach to inhibition of P-glycoprotein-mediated MDR and to the modulation of other tumor cell genes whose overexpression contributes to the neoplastic state or to resistance to therapy.
Mol Pharmacol 1996 Oct
PMID:Inhibition of expression of the multidrug resistance-associated P-glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate antisense oligonucleotides. 886 25

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.
Mol Biol Cell 1996 Oct
PMID:Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. 889 56


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