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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp). Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp. Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil. Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively. Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone. Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil. These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.
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PMID:Antiestrogens and steroid hormones: substrates of the human P-glycoprotein. 791 5

The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.
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PMID:Modulation of P-glycoprotein by protein kinase C alpha in a baculovirus expression system. 791 39

In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.
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PMID:Fluorescent cellular indicators are extruded by the multidrug resistance protein. 810 40

The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
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PMID:Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein. 856 33

Multidrug resistance (MDR) to chemically unrelated therapeutic anticancer agents in mammalian cells is mediated by the overexpression of an ATP-dependent 150- to 180-kD membrane glycoprotein P-glycoprotein (P-gp). Although the complete physiological role of P-gp is unknown, it is proposed to function in cellular detoxification of xenobiotics. In this study, we investigated whether the organophosphorus insecticide chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate) or its metabolites interact with P-gp. Immunohistochemical analysis of tissues from male Fischer 344 rats administered chlorpyrifos (7.6 mg/kg gavage) showed increased P-gp expression in the kidney, adrenal, liver, jejunum, and stomach (tissues associated with elimination of xenobiotics), compared to control tissues. The most prominent increase was detected in the large bile ducts of the liver and the proximal tubule region of the kidney. P-gp expression was increased throughout the adrenal medulla and cortex, while a moderate increase was detected in the epithelial layers of the stomach and jejunum. To examine further the interaction between chlorpyrifos and P-gp, we evaluated whether chlorpyrifos or its active metabolite, chlorpyrifos oxon, could inhibit [3H]azidopine labeling of P-gp in MDR1 baculovirus-infected insect Sf9 cells. A concentration-dependent inhibition of [3H]azidopine labeling of P-gp was detected with chlorpyrifos oxon, while significant inhibition was not detected with chlorpyrifos. To correlate the binding of chlorpyrifos oxon to P-gp with a biochemical effect, we examined its ability to stimulate P-gp-mediated ATPase activity in these Sf9 cells. Chlorpyrifos oxon stimulated P-gp ATPase activity 1.75 times that of the positive control (10 microM verapamil). Taken together, these results suggest that chlorpyrifos oxon interacts with P-gp, and support the hypothesis that P-gp may play a role in the cellular detoxification of insecticides in mammalian tissues. To our knowledge this is the first report of an organophosphorus insecticide interacting with and increasing the expression of P-gp.
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PMID:Chlorpyrifos oxon interacts with the mammalian multidrug resistance protein, P-glycoprotein. 860 Feb 91

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

The MDR1 protein (P-glycoprotein) is a membrane ATPase whose expression results in resistance to several anti-tumor drugs. It has been proposed that the MDR1 protein, in addition to its pumplike properties, can function as (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54:618-622, 1994) or mediate the activity of (Hardy et al. EMBO J. 14: 68-75, 1995) a hypotonic stress-induced Cl- current. In addition, one study found that drug transport and Cl- channel-associated functions of MRD1 were separable and mutually exclusive and that, when cells were swelled, the MDR1 protein could not transport substrate. This hypothesis was tested in four pairs of isogenic cell lines with MDR1 transfectants expression 8,000-55,000 MDR1 antibody binding sites per cell. Cytoplasmic exclusion of rhodamine 123 was used as an indicator of MDR1 function to measure the effect of hypotonic stress, MDR1 inhibitors, and Cl- channel blockers on MRD1 transport function. It was found that MDR1 activity and its inhibition by cyclosporine A or flufenamic acid were unaffected by hypotonicity alone or in combination with Cl- channel blockers.
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PMID:MDR1/P-glycoprotein function. I. Effect of hypotonicity and inhibitors on rhodamine 123 exclusion. 896 46

VX-710 or (S)-N[2-Oxo-2-(3,4,5-trimethoxyphenyl)acetyl]-piperidine-2-carboxylic acid 1,7-bis(3-pyridyl)-4-heptyl ester, a novel non-macrocyclic ligand of the FK506-binding protein FKBP12, was evaluated for its ability to reverse P-glycoprotein-mediated multidrug resistance in vitro. VX-710 at 0.5-5 microM restored sensitivity of a variety of multidrug resistant cells to the cytotoxic action of doxorubicin, vincristine, etoposide or paclitaxel, including drug-selected human myeloma and epithelial carcinoma cells, and human MDR1 cDNA-transfected mouse leukemia and fibroblast cells. Uptake experiments showed that VX-710 at 0.5-2.5 microM fully restored intracellular accumulation of [14C]doxorubicin in multidrug resistant cells, suggesting that VX-710 inhibits the drug efflux activity of P-glycoprotein. VX-710 effectively inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine or [125I]iodoaryl azidoprazosin with EC50 values of 0.75 and 0.55 microM. Moreover, P-glycoprotein was specifically labeled by a tritiated photoaffinity analog of VX-710 and unlabeled VX-710 inhibited analog binding with an EC50 of 0.75 microM. VX-710 also stimulated the vanadate-inhibitable P-glycoprotein ATPase activity 2- to 3-fold in a concentration-dependent manner with an apparent k(a) of 0.1 microM. These data indicate that a direct, high-affinity interaction of VX-710 with P-glycoprotein prevents efflux of cytotoxic drugs by the MDR1 gene product in multidrug resistant tumor cells.
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PMID:Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro. 907 9

The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl- channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl- currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced activation of the Cl- currents in both cells. The relation between whole-cell Cl- currents and cell size measured simultaneously showed that volume sensitivity of the Cl- channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase in volume sensitivity of the Cl- channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein does not represent the volume-sensitive Cl- channel but that its expression modulates volume sensitivity of the Cl- channel in a manner independent of its ATPase activity or of the protein kinase C activity.
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PMID:Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl- channel. 914 59

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.
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PMID:Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region. 916 12


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