Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein. 2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay. 3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity. 4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833. 5. The binding of 8-azido-[alpha-32P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein. 6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.
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PMID:Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein. 931 31

Multidrug resistance (MDR) mediated by the drug efflux pump P-glycoprotein (Pgp), may cause remission failure and relapse in patients with acute myeloid leukaemia (AML) by extruding cytotoxic agents such as anthracyclines from leukaemic cells thus allowing them to survive. Cell line data suggest that reversal of MDR is possible using modifying drugs such as cyclosporin A (CSA) and its analogue PSC 833. We have investigated the effects on cell kill of the addition of CSA and PSC 833 to daunorubicin, idarubicin, mitozantrone, etoposide and cytarabine in 52 fresh cell samples from AML patients using an MTT assay. Pgp status was determined by using monoclonal antibodies JSB-1 and MRK-16 and by assessment of rhodamine efflux. Although overall each cytotoxic-modifier combination produced significant improvements in cell kill compared to cytotoxic alone (P values ranged from P < 0.001 to P = 0.017), modifiers also produced significant cytotoxicity in their own right, and no consistent difference was seen between responses in Pgp-positive and negative groups. Up to one in three Pgp-positive samples failed to show any improvement in cell kill with the addition of CSA or PSC 833, possibly owing to co-expression of alternative resistance mechanisms not affected by the MDR modifiers. The best responses were seen when PSC 833 was added to idarubicin, with 7 out of 22 Pgp-positive cases (32%) showing five-fold improvements in cell kill or better compared to idarubicin alone. Comparison of equimolar concentrations of the two modifiers in the Pgp positive group failed to show a significant difference in cell kill, though PSC 833 was markedly superior to CSA in a minority of highly responsive samples which demonstrated clear evidence of MDR reversal. Our in vitro data suggest that MDR modifiers such as CSA and PSC 833 could play an important role in the therapy of AML and indicate the need for prospective randomised trials to assess their clinical efficacy.
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PMID:Effect on cell kill of addition of multidrug resistance modifiers cyclosporin A and PSC 833 to cytotoxic agents in acute myeloid leukaemia. 939 2

The multidrug resistance phenotype is found to be frequently associated with the overexpression of proteins which lead to a decrease of drug accumulation within human tumor cells. A 170 kDa membrane glycoprotein which is related to the overexpression of the mdr1 gene is inserted in the plasma membrane and pumps the cytotoxic drugs out of the cells. The aim of this work was to study the morphological modifications of resistant CEM/VLB 100 cells relative to their parental drug-sensitive ones and the detection of the ultrastructural localization of P-glycoprotein at the cytoplasmic level. Using a scanning electron microscope, CEM resistant cells showed wide smooth protrusions while CEM sensitive cells showed microvilli and fine folds. With transmission electron microscopy, an enhanced secretory system was observed in CEM resistant cells: both electron transparent and electron opaque vesicles were associated with the Golgi system, revealed by wheat germ agglutinin-colloidal gold labelling. These vesicles were the binding site of C 219 and MRK 16 antimembrane glycoprotein antibodies, and some of them were determined to belong to the lysosomal system after PTA staining. These vesicles may be an additional way to decrease the cellular uptake of drugs in multidrug resistant cells. Moreover, some nuclear and nucleolar modifications were also observed. These observations show that MDR has wide morphological features which concern several organelles.
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PMID:Ultrastructural changes related to multidrug resistance in CEM cells: role of cytoplasmic vesicles in drug exclusion. 941 88

In order to better understand acquired resistance to antitumor agents in acute myelogenous leukemia (AML), we investigated various drug resistance mechanisms; namely, topoisomerase II (topo II), glutathione system and P-glycoprotein (P-gp). Blast cells of 31 patients with AML, 21 before treatment (BT) and 10 at relapse (AR) were studied. Topo II was evaluated by Western blot analysis. Glutathione-S-transferase activity (GST) and glutathione content (GSH) were investigated by spectrophotometric assays. GST isoenzymes (-alpha, -mu and -pi) were tested by Western blot and by immunocytochemical staining. P-gp was evaluated by an immunocytochemical method using MRK 16 antibody. Our results showed that GST, GSH and GST-pi were similar in patients BT and AR GST-mu was detected in 13/21 AML BT and in 5/10 AML AR. GST-alpha expression was higher (p < 0.05) in AML AR (60 +/- 105 AU/mg) compared to AML BT (10 +/- 10 AU/mg). A relationship was found between GST-pi quantitation evaluated by Western blot and immunocytochemical staining, whereas no correlation was observed for the other isoenzymes. Topo II was detected in only 4 AML BT and 3 AML AR. Eleven out of 21 AML BT and 3/10 AML AR expressed P-gp with immunohistochemical study. These results indicate that only the "glutathione system", especially the GST-alpha could be involved in drug resistance in AML.
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PMID:Glutathione system, topoisomerase II level and multidrug resistance phenotype in acute myelogenous leukemia before treatment and at relapse. 949 83

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.
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PMID:Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. 950 34

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.
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PMID:P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels. 960 55

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.
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PMID:The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis. 961 32

Monoclonal antibody MRK-16 recognizes a discontinuous extracellular epitope on the multidrug resistance-associated ATP-binding cassette transporter, P-glycoprotein. The atomic basis for specificity of this antibody is of interest because of its potential as a modulator of P-glycoprotein activity. The crystal structure of Fab MRK-16 is reported to a resolution of 2.8 A. A structure for a portion of the epitope was derived by comparison to regions of solved structures with similar primary sequence. This has permitted a proposal for the mode of binding of the peptide epitope to the antibody, in which the peptide makes specific contacts with complementarity-determining regions H1, H2, and H3 from the heavy chain and L3 from the light chain. These interactions are consistent with epitope mapping studies and with the observation that MRK-16 is specific for human class I P-glycoprotein. This result identifies side chains in MRK-16 that would be amenable to alteration in antibody engineering experiments to derive improved multidrug resistance inhibitors for clinical use during chemotherapy. In particular, Arg-H97 contacts both Glu-746 and Asp-744 of the peptide, Arg-L96 contacts Asp-743, and Thr-H33 interacts with Thr-747. All of these epitope residues were implicated in mediating specificity by epitope mapping studies.
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PMID:Mode of binding of anti-P-glycoprotein antibody MRK-16 to its antigen. A crystallographic and molecular modeling study. 973 9

The discordance between P-glycoprotein (P-gp) expression and functionality [as measured by the efflux of doxorubicin (DOX)] was analyzed in a DOX-sensitive human breast cancer cell line (HTB-123) with high reactivity against four P-gp specific monoclonal antibodies (C219, MRK-16, UIC2, and 4E3). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analyses confirmed the overexpression of MDR1 mRNA and P-gp in this cell line. However, incubation of cells with efflux blockers, verapamil (VPL) or dipyridamole (DPD), did not enhance cellular (DOX) accumulation or cytotoxicity. Upon incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA), HTB-123 cells retained less DOX than control cells and were sensitive to the efflux blockers verapamil or dipyridamole. These observations suggest that 12-O-tetradecanoylphorbol-13-acetate-induced P-gp phosphorylation may be associated with induction of P-gp-mediated drug efflux in the HTB-123 cell line.
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PMID:Phorbol ester-induced P-glycoprotein phosphorylation and functionality in the HTB-123 human breast cancer cell line. 975 Oct 75

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.
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PMID:Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector. 979 10


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