Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Dubin-Johnson syndrome is characterized by an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into the bile. We have recently identified the membrane protein mediating the adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronate conjugates as a multidrug-resistance protein (MRP) and localized it to the canalicular as well as to the lateral hepatocyte plasma membrane. In the present study we show the selective absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syndrome by double-label immunofluorescence and confocal laser scanning microscopy using antibodies directed against MRP and dipeptidyl-peptidase IV (DPPIV). Another isoform of MRP was detected, however, in the lateral hepatocyte membrane of the patient. Moreover, MRP was present on immunoblots of erythrocyte membranes from Dubin-Johnson syndrome and normal humans. These findings are analogous to our recent observations on the localization of the rat homolog of MRP and its canalicular isoform, cMrp, in normal and transport-deficient GY/TR- Wistar rat liver. The elucidation of the selective absence of an isoform of MRP and from the canalicular membrane domain in conjunction with the defined substrate specificity of the MRP and cMRP gene-encoded conjugate export pumps contributes to the molecular definition of the transport defect in Dubin-Johnson syndrome.
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PMID:Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. 862 Nov 34

The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.
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PMID:Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity. 862 16

Multidrug resistance (MDR) is frequently associated with decreased cellular drug accumulation resulting from enhanced drug efflux. This is correlated with the presence of a membrane protein, the P-glycoprotein, which pumps a wide variety of drugs out of cells, reducing their intracellular concentration and thus their toxicity. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the P-glycoprotein-medicated efflux. Four anthracycline derivatives, doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin, which have the same amino sugar, were used to analyze the respective contribution of the kinetics of uptake and the P-glycoprotein-mediated efflux in their impaired accumulation in MDR cells. The kinetics of uptake of the four drugs vary over a very large range: the kinetics of uptake of daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin are 16, 200, and 400 times higher than that of doxorubicin, respectively. However, the four drugs are extruded by P-glycoprotein at comparable rates. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic drug concentrations at half-maximal velocity for the cell lines used, were approximately 2.2 microM for daunorubicin and and approximately 1 microM for idarubicin and 8-(S)-fluoroidarubicin.
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PMID:Relation among the resistance factor, kinetics of uptake, and kinetics of the P-glycoprotein-mediated efflux of doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin in multidrug-resistant K562 cells. 864 93

The mammalian P-glycoprotein (Pgp) is a approximately 170-kDa membrane protein that mediates multidrug resistance in many chemotherapy-resistant tumors by effluxing toxic compounds from the cell. Pgp homologs are expressed in many organisms, from bacteria to yeast and mammals. Previous studies established a model system to analyze the function of murine, human, and Plasmodium falciparum Pgp by heterologous expression in the yeast Saccharomyces cerevisiae. However, such studies have been hampered by the inherent resistance of yeast cells to chemotherapeutic agents. We find that an erg6 mutation, which blocks the final synthetic step of the membrane sterol ergosterol, renders yeast sensitive to anthracyclines and dactinomycin, clinically relevant Pgp substrates. We demonstrate that expression of the murine mdr3 gene confers dactinomycin resistance in both the erg6 mutant yeast strain and in an erg6 rad52 DNA repair mutant yeast strain. Similarly, murine mdr3 expression confers resistance to the immunosuppressants cyclosporin A (CsA) and FK506 in a CsA-FK506-sensitive vph6 mutant yeast strain. CsA and FK506 are known to partially overcome Pgp-mediated drug resistance, suggesting the targets of these drugs might regulate Pgp function. We find that both murine mdr3 and the yeast Pgp homolog STE6 function in yeast mutants lacking the CsA target proteins cyclophilin A and calcineurin. In contrast, murine mdr3 function was severely compromised in yeast mutants lacking the FK506/rapamycin target protein FKBP12. Both wild-type FKBP12 and an F43Y FKBP12 mutant with reduced prolyl isomerase activity supported mdr3 function. Our results support the model that immunosuppressants reverse multidrug resistance by competing with other Pgp substrates but reveal that inhibition of FKBP12-dependent Pgp function may also contribute to reversal of multidrug resistance by FK506 and rapamycin.
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PMID:Immunosuppressant target protein FKBP12 is required for P-glycoprotein function in yeast. 870

P-glycoprotein (Pgp)1 is a polytopic membrane protein and functions as an energy-dependent drug efflux pump. It is responsible for multidrug resistance (MDR) in cancer cell lines. Recently, the topological structure of Pgp has been investigated. However, the results are in dispute. A major question concerning the Pgp topology is the membrane orientation of the loop linking TM4 and TM5 (loop 4) and the loop linking TM8 and TM9 (loop 8). In this study, we generated polyclonal antibodies specific to these two loops. In combination with a panel of other well-characterized site-specific polyclonal- and monoclonal antibodies of Pgp, we tested the membrane orientation of these two loops of Pgp in multidrug-resistant cells using immunocytochemistry and proteolysis/membrane protection assay. Our results showed that (1) both loops 4 and 8 are located extracellularly whereas other domains, such as the ATP-binding sites, are in the cytoplasm and (2) proteolysis of Pgp is not a random event and the trypsin-sensitive sites are cleaved in orders. Since the Pgp was not genetically manipulated in this study, in contrast to previous studies, we believe that naturally expressed Pgp molecules have an unconventional topology. We speculate that this alternate topology of Pgp may represent a different functional state of the protein. Further detailed analysis of Pgp topology will help to understand the fundamental mechanism of drug transport by Pgp.
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PMID:Topological folding and proteolysis profile of P-glycoprotein in membranes of multidrug-resistant cells: implications for the drug-transport mechanism. 870 44

Multidrug resistance (MDR) is a phenomenon by which tumor cells exposed to a single anti-proliferative agent acquire resistance to other structurally and functionally unrelated drugs. The classical form of MDR is caused by a plasma-membrane protein currently named P-glycoprotein or P-170 encoded by the human mdr-1 gene in its functional isoform. In vitro cell lines expressing P-170 usually also present phenotypic and functional alterations. In the present study we report that the cytotoxicity mediated by tumor necrosis factor alpha (TNF alpha) in MDR variants of the human T-lymphoblastoid CEM cell line is associated with apoptosis (programmed cell death). Susceptibility of MDR cells to apoptosis was increased upon cycloheximide + TNF alpha sequential treatment, whereby the impairment of protein synthesis due to the former agent was followed by the effect of cytokine exposure. Massive apoptosis of P-170-positive cells, but not of controls, was also obtained by depletion of nutrients (i.e., serum starvation). In contrast, TNF-alpha exerted a similar apoptotic effect in epithelial (MCF-7) or myeloma (S8226) drug-sensitive/ -resistant cell pairs. However, the MDR variant of myeloma S8226 was more sensitive to the cytostatic effect of TNF alpha than the parental drug-sensitive cell line. These results suggest that the presence of the MDR phenotype may be associated with increased histotype-dependent cell susceptibility to specific, protein-synthesis-independent, apoptotic pathways.
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PMID:Tumor necrosis factor alpha is a powerful apoptotic inducer in lymphoid leukemic cells expressing the P-170 glycoprotein. 876 May 94

Multidrug resistance in cancer cells, in cell culture and in the clinic, is often associated with a membrane protein (the multidrug resistance pump or P-glycoprotein) that pumps out anti-cancer drugs as fast as they enter the cell. This pump is blocked by a range of well-known pharmaceuticals that reverse drug resistance. We have investigated whether effective reversal of drug resistance could be achieved by using many reversers together, each at a low dose relative to its maximal tolerated plasma level. We measured in cell culture, using resistant P388 cells in suspension, the extent of reversal of the accumulation of two labeled cytotoxins (vinblastine and daunomycin). We fitted the data to a modified Michaelis-Menten equation and extracted the half-inhibition constants for 18 reversers acting on the pump. We measured also the reversal of resistance in a cell growth assay using incorporation of labeled thymidine. We showed that these drugs in groups of up to 18 together, each drug being at a low dose, in many cases well-tolerated in humans, had additive effects so that the combination was as effective as any of the drugs present singly. This was the case both for reversal of cell accumulation and for the effects of cytotoxins on cell growth. Our data show that a low-dose multidrug approach to saturation reversal of the multidrug pump is feasible in cell culture and provide the initial experimental basis for the development of an effective regime of such combination reversal therapy.
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PMID:Saturation reversal of the multidrug pump using many reversers in low-dose combinations. 884 84

Soft tissue sarcomas comprise a heterogeneous group of mesenchymal tumors accounting for less than one-percent of adult neoplasms. In the last few years, the use of adjuvant chemotherapy has been proposed for the treatment of these lesions in order to obtain a better systemic control, but its usefulness is still controversial. In this study, we evaluated whether P-glycoprotein, a membrane protein strictly associated with multidrug resistance, is overexpressed in soft tissue sarcomas. By using human multidrug resistant sarcoma cell lines as controls, we analyzed P-glycoprotein expression in 34 primary and in 23 relapsed soft tissue sarcomas of the extremities. Overexpression of P-glycoprotein was found in 6 out of 34 primaries (18%) and in 8 out of 23 relapses (35%). In particular, in malignant fibrous histiocytoma, the most frequent soft tissue sarcoma of adults, P-glycoprotein overexpression was found in 23% of primary untreated cases, in agreement with the reported relapse rate of this tumor after surgery and chemotherapy. These data suggest that, in soft tissue sarcomas, overexpression of P-glycoprotein may be of prognostic value and that the assessment of P-glycoprotein expression may be useful for the design of chemotherapy protocols.
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PMID:Evaluation of P-glycoprotein expression in soft tissue sarcomas of the extremities. 886 15

Expression of P-glycoprotein, a phylogenetically conserved integral plasma membrane protein, is implicated as one of the most important factors contributing to tumor cell multidrug resistance. Formalin-fixed, paraffin-embedded normal and neoplastic canine tissues were studied using an avidin-biotin complex technique employing three murine monoclonal antibodies (C494, C219, JSB-1) to different epitopes of the P-glycoprotein molecule. Evaluation of immunostaining of normal canine tissues revealed positive labeling detected by each antibody in the liver, proximal renal tubular epithelium, adrenal cortex, colonic epithelium, and capillary endothelial cells of the brain. A total of 166 tumors of epithelial or mesenchymal origin were evaluated for P-glycoprotein immunoreactivity. Hepatomas (4/4), colorectal adenomas (7/7), colorectal carcinomas (4/4), adrenal cortical adenomas (3/3), hemangiopericytomas (15/15), apocrine gland adenocarcinomas (4/5, 80%), and transitional cell carcinomas (2/2) consistently labeled with at least one of the antibodies. Histiocytomas (0/10), cutaneous plasma cell tumors (0/10), fibromas (0/3), fibrosarcomas (0/4), and leiomyomas (0/4) were uniformly negative with all antibodies. Malignant lymphomas (6/22, 27.3%), malignant melanomas (4/13, 30.8%), leiomyosarcomas (3/6, 50%), mammary gland carcinomas (12/19, 63.2%), mammary gland adenomas (3/9, 33.3%), squamous cell carcinomas (8/10, 80%), basal cell tumors (5/7, 71.4%), apocrine gland adenomas (1/2, 50%), cholangiocarcinomas (2/3, 66.7%), and thyroid gland carcinomas (2/4, 50%) gave variable results. The antibodies C494, JSB-1, and C219 labeled 66/166 (39.8%), 53/166 (31.9%), and 38/166 (22.9%) of all tumors studied, respectively. A total of 26/166 (15.7%), 22/166 (13.3%), and 37/166 (22.6%) of tumors were labeled by all three, just two, or one antibody alone, respectively. The antibody C494 was the only antibody labeling 28/166 (16.9%) of the cases. JSB-1 alone labeled 9/166 (5.4%) of the tumors. C219 failed to label any tumors not also labeled by either C494 or JSB-1. Labeling by C494 was more intense and specific than labeling by the other two antibodies. Results indicate that P-glycoprotein can be detected in routinely processed canine tissues. The detection of P-glycoprotein within canine liver, kidney, adrenal gland, and colon and within tumors arising from these tissues is consistent with that reported in the literature for human tissues. Variable labeling results of other tumors such as malignant lymphoma and mammary gland carcinomas also is consistent with reports of human studies. Detection of multidrug resistance markers such as P-glycoprotein in canine tissues may provide additional information upon which to base a prognosis or to design treatment regimens for canine tumors.
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PMID:Immunohistochemical detection of P-glycoprotein in formalin-fixed and paraffin-embedded normal and neoplastic canine tissues. 888 80

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.
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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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