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)

Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-3H]benzoyl)aminomethyl verapamil and N-(p-azido[3-125I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM. Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.
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PMID:Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil. 290 25

For the characterization of membrane changes related to Adriamycin resistance in tumor cells, we have developed monoclonal antibodies against Adriamycin-resistant human myelogenous leukemia K562 (K562/ADM). In addition to the monoclonal antibodies which recognize P-glycoprotein, we have obtained two monoclonal antibodies (designated MRK4 and MRK20) which recognize an Mr 85,000 membrane protein. Using MRK20 as a probe, we have studied the expression of the Mr 85,000 protein in various human multidrug-resistant and -sensitive cell lines. The Mr 85,000 protein was overexpressed in K562/ADM and in a human ovarian cancer cell line resistant to Adriamycin, 2780AD. The protein, if any, was not detected in other drug-resistant human cell lines such as colchicine-resistant KB cells (KB-C4), vinblastine-resistant CEM cells (CEM/VLB100), and vincristine-resistant K562 cells (K562/VCR). We have isolated subclones of K562/ADM cells which express different amounts of the Mr 85,000 protein. The expression of the Mr 85,000 protein diminished when the cells were not kept in Adriamycin, and increased when the clones were kept in the presence of Adriamycin. In contrast, the expression of P-glycoprotein remained constant whether in the presence or absence of Adriamycin during these experiments. These findings suggest that the Mr 85,000 membrane protein is closely related to the resistant mechanism specific to Adriamycin resistance, which is different from that of the pleiotropic drug resistance.
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PMID:Mr 85,000 membrane protein specifically expressed in adriamycin-resistant human tumor cells. 290 93

HL60 cells resistant to Adriamycin contain a 32P-labeled, Mr 150,000 surface membrane protein (p150) which is not detected in cells sensitive to drug. The levels of phosphorylation of this protein increase with increasing levels of resistance. Analysis of plasma membranes prepared from cells labeled with [14C]glucosamine shows, however, that both sensitive cells and those exhibiting an 80-fold increase in drug resistance contain essentially identical levels of a highly glycosylated Mr 150,000 protein. Identical results are obtained when cells are labeled with [14C]galactose or [14C]mannose. Limited proteolytic digestion of [14C]glucosamine-labeled p150 from sensitive and resistant cells shows that the glycopeptides formed are identical. Additional studies involving binding of proteins to insolubilized lectin indicate that 32P-labeled p150 is glycosylated. Polyacrylamide gel electrophoresis of p150 followed by silver staining shows no difference in the levels of this protein in sensitive and 80-fold drug-resistant cells. Further studies show that two-dimensional tryptic peptide maps of 125I-labeled p150 of sensitive and resistant cells are essentially the same. It has also been found that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate followed by [14C]glucosamine labeling results in a selective decrease in the glycosylation of p150 of sensitive and resistant cells. TPA has an identical effect on the phosphorylation of p150 in cells resistant to drug. HL60 cells have also been examined for the presence of the Mr 170,000 to 180,000 P-glycoprotein. Using immunoblot analysis with a monoclonal antibody directed against the P-glycoprotein we did not detect the presence of this protein in membranes of drug-sensitive or -resistant HL60 cells. The results of this study suggest that Adriamycin resistance in HL60 cells may be related to a modified form of a protein contained in cells sensitive to drug. Proteins active in drug resistance in this system may be distinct from those described for other cell lines.
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PMID:Adriamycin resistance in HL60 cells and accompanying modification of a surface membrane protein contained in drug-sensitive cells. 362 Nov 92

A major form of multidrug resistance results from the overexpression of P-glycoprotein, a 170 kDa membrane protein. Multidrug resistant (MDR) Chinese hamster ovary (CHO) cells and mdrl transfectants displayed cross-resistance to the channel-forming peptide ionophore gramicidin D, which was reversed by various chemosensitizers, thus directly implicating P-glycoprotein as the mediator of resistance. However, gramicidin D was not able to inhibit [3H]azidopine photolabelling of P-glycoprotein. MDR cells were not resistant to other pore-forming ionophores, but showed a modest level of cross-resistance to the mobile ionophore valinomycin. There was no difference in 125I-gramicidin D uptake by resistant and sensitive cells. Resistant cells showed lower 86Rb+ uptake, relative to the drug-sensitive parent. Addition of GmD increased both the rate and the level of 86Rb+ uptake in sensitive cells, but had no effect on MDR cells. MDR cells also showed much lower rates of gramicidin D-dependent 86Rb+ efflux than sensitive cells, and this was greatly increased by verapamil. These results suggest that P-glycoprotein interferes with the formation of ion-conducting gramicidin D channels. In contrast, valinomycin had the same effect on gramicidin D-dependent cation efflux in MDR and sensitive cells. Gramicidin D is thus unique among the ionophores is being a substrate for P-glycoprotein, which appears to greatly reduce the formation of active dimeric channels in the plasma membrane of MDR cells.
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PMID:Interaction of multidrug-resistant Chinese hamster ovary cells with the peptide ionophore gramicidin D. 750 93

The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution. The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.
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PMID:Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein. 751 Feb 82

Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.
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PMID:MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase. 751 Oct 43

Drug resistance, be it intrinsic or acquired, is a major problem in cancer chemotherapy. In vitro, one well characterised form of resistance against many different cytotoxic drugs is caused by the MDR1 P-glycoprotein, a large plasma membrane protein that protects the cell by actively pumping substrate drugs out. Available evidence suggests that this protein may cause drug resistance in at least some clinical tumours. Drugs inhibiting the MDR1 P-glycoprotein activity are, therefore, co-administered during chemotherapy of these tumours. To predict the biological and pharmacological effects of the blocking of this protein, we have generated mice with a genetic disruption of the drug-transporting mdr1a P-glycoprotein. These mice are overall healthy, but they accumulate much higher levels of substrate drugs in the brain, and have markedly slower elimination of these drugs from the circulation. For some drugs, this leads to dramatically increased toxicity, indicating that P-glycoprotein inhibitors should be used with caution in patients.
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PMID:Multidrug resistance and the role of P-glycoprotein knockout mice. 757 39

P-glycoprotein (P-gp), a membrane protein that was originally found to be involved in the efflux of cytotoxic drugs out of the tumor cells, is also present in a variety of normal human and animal tissues, such as the adrenal cortex. The function of P-gp in the adrenal cortex has not been defined yet. The aim of our study was to determine whether the blockade of P-gp by cyclosporine A (CsA) dissolved in Cremophor EL (Crem) inhibits cortisol secretion in rabbits. In 14 rabbits, the baseline and ACTH stimulated serum cortisol levels were measured before and after CsA treatment. Seven rabbits were treated with 2 x 30 mg/kg CsA and seven with 2 x 90 mg/kg CsA injected s.c. Serum cortisol levels were determined by radioimmunoassay adjusted for expected values. The whole blood CsA levels were determined by a commercially available fluorescence polarization immunoassay. Serum cortisol levels, both baseline and ACTH stimulated, significantly increased after both low and high dose CsA treatment. The increase was dose dependent. The mean baseline cortisol levels increased from 5.7 (SD = 6.3) to 15.0 nmol/l (SD = 7.2) in the low dose group and from 7.7 (SD = 4.9) to 44.9 nmol/l (SD = 13.8) in the high dose group. The mean cortisol levels 8 h after ACTH stimulation increased from 53.3 (SD = 34.5) to 106.0 nmol/l (SD = 33.0) in the low dose group and from 47.7 (SD = 12.2) to 153.0 nmol/l (SD = 55.1) in the high dose group.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cyclosporine A increases serum cortisol levels in rabbits. 757 69

Chinese hamster pgpl 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 and as a chloride channel. Previously, we have shown that hamster pgpl 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. Different topological structures of Pgp may be involved in different functions. In this study, we examined the role of cytoplasmic components in cell-free translation systems in modulating the topologies of Pgp. By using rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE) expression systems, we showed that WGE contains a soluble, heat-labile, high molecular weight fraction that regulates the membrane topology of truncated Pgp molecules. These results and our previous findings indicate that the membrane topology of a mammalian polytopic membrane protein may be regulated both by the amino acid sequence of the protein and by soluble cytoplasmic component(s). We speculate that Pgp expressed in various cell types may have different topological structures modulated by specific cytoplasmic factors.
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PMID:Involvement of cytoplasmic factors regulating the membrane orientation of P-glycoprotein sequences. 761 15

P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein. 762 71


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