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)

In previous work [Luz et al. (1994) Biochemistry 33, 7239-7249; Roepe et al. (1994) Biochemistry 33, 11008-11015] we measured changes in Cl- and HCO3--dependent pHi regulation for LR73 Chinese hamster ovary fibroblasts overexpressing mu MDR 1 protein. However, only one clonal cell line overexpressing the protein but not previously exposed to chemotherapeutic drug (i.e., a "true" transfectant) was examined, since very few MDR cell lines of this nature have been constructed. Recently [Hoffman et al. (1996) J. Gen. Physiol. 108, 295-313] we derived a series of true LR73/hu MDR 1 transfectants that are valuable for defining the MDR phenotype mediated by MDR protein alone, without the additional complexities introduced by exposing cells to chemotherapeutic drugs. Several independently derived clones from these and additional transfection experiments exhibit expression of MDR protein that is higher than that found in other true transfectants, and that is similar to the highest level of overexpression yet recorded for drug selected MDR cells. We examined altered Cl--dependent pHi regulation for these clones using improved single-cell photometry (SCP) techniques. Short-term isotonic Cl- substitution experiments performed in the presence of CO2/HCO3- reveal that mild overexpression of hu MDR 1 protein alters anion exchange (Cl-/HCO3- exchange or AE) for LR73 cells, as expected on the basis of previous work [Luz et al. (1994) Biochemistry 33, 7239-7249]. Interestingly, we now find that several independently selected high-level MDR 1 overexpressing clones acidify quite extensively upon isotonic exchange of Cl- and then rapidly alkalinize upon restoring normal [Cl-]. These data suggest that MDR protein may effectively compete against AE. The MDR protein effect is not dependent on HCO3-/CO2 or K+, is partially inhibited by verapamil, is completely inhibited by substituting K+ or N-methylglucamine (NMG+) for Na+ in the SCP perfusate but is not affected by 100 microM levels of amiloride, bumetanide, chlorothiazide, or stilbene. ATP depletion inhibits the MDR 1 effect. We are unable to restore normal AE activity for the MDR clones via manipulation of Cl- or HCO3- gradients. We thus suggest that MDR protein overexpression provides a novel Na+- and Cl--dependent pathway for transmembrane H+ transport. From analysis of ion dependency and inhibitor sensitivities, we conclude the transport is not via altered regulation of any known K+/H+, Na+/H+, or Cl-/HCO3- antiporters, Na+:K+:2Cl-, Na+:K+:2HCO3-, K+:HCO3-, or Na+:HCO3- co-transporters, or any combination of these. Thus, it appears to represent a novel ATP and Na+-dependent Cl-/H+ antiport process that (1) may be directly mediated by the MDR protein, (2) may represent the modulation of one or more currently unidentified ion transport proteins by MDR protein, (3) may be due to some combination of direct ion transport and regulation of ion transport, or (4) may represent unusual passive H+ movement in response to a novel Cl--dependent electrical perturbation that occurs during our Cl- substitution protocol. The results have important implications for understanding drug resistance mediated by MDR 1 overexpression, as well as the physiologic function of endogenously expressed MDR protein.
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PMID:Analysis of ion transport perturbations caused by hu MDR 1 protein overexpression. 928 58

A population of NG108-15 neuroblastoma cells resistant to doxorubicin (NG/DOXR) was established. The cells exhibited a multidrug resistance phenotype with cross-resistance to vinblastin and colchicine, overexpression of a 170 kDa membrane protein identified as P-glycoprotein and reversal of resistance by verapamil and quinine. Compared with NG108-15 cells, NG/DOXR cells showed an increase in Na+ current density and a decrease in cyclic-AMP-activated Cl- current density with no change in K+- and volume-sensitive Cl- current densities. As previously observed in NG108-15 cells, the vacuolar-type H+-ATPase inhibitors bafilomycin A1 and nitrate induced membrane depolarizations in NG/DOXR cells. The resting potentials of sensitive and resistant cells were not significantly different, but the depolarizations evoked by these agents were significantly larger in NG/DOXR than in NG108-15 cells. The resting membrane potential of NG/DOXR cells, but not that of NG108-15 cells, was depolarized by verapamil, and this effect was abolished by bafilomycin. The volume-sensitive Cl- currents of drug-sensitive and drug-resistant cells were inhibited by a decrease in intracellular pH from 7.3 to 6.8. Whereas bafilomycin prevents activation of Cl- currents in both drug-sensitive and drug-resistant cells, verapamil inhibited the Cl- current only in NG/DOXR cells. The results are discussed in terms of the roles of cytoplasmic pH and membrane potential in multidrug resistance.
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PMID:Alterations of ionic membrane permeabilities in multidrug-resistant neuroblastoma x glioma hybrid cells. 939 Sep 33

Multiple drug resistance (MDR) mechanisms are known to limit the effectiveness of some cancer chemotherapies, probably through enhancing P-glycoprotein-mediated drug efflux from mammalian cells. Similar mechanisms appear to act in other organisms, including bacteria, and may affect not only the toxicity but also the mutagenicity of certain chemicals. At least in some experimental situations, MDR can be overcome through concomitant treatment of the cells with various types of inhibitors. Two MDR inhibitors, verapamil, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, were assayed for their ability to modulate the potency of nine mutagens with varying mechanisms of action in various Salmonella typhimurium his- strains. Neither verapamil nor trifluoperazine affected the direct mutagenicity of sodium dichromate and 2-methoxy-6-chloro-9[3-(2-chloroethyl)amino-propyl-amino] dihydrochloride (ICR 191) or the S9-mediated mutagenicity of benzo[a]pyrene and 2-amino-3,4-dimethyl-amidazo[4,5-f]quinoline (MeIQ). Both modulators enhanced the direct mutagenicity of doxorubicin. Moreover, trifluoperazine sharply increased the S9-mediated mutagenicity of cyclophosphamide and 2-aminofluorene, while it consistently decreased the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The contrasting effect towards the aromatic amine 2-aminofluorene and the heterocyclic amine Trp-P-2, representative of important chemical families responsible for the bacterial mutagenicity of cigarette smoke, may explain the observed lack of influence of trifluoperazine on the mutagenicity of a cigarette smoke condensate. These observations extend the known range of chemical types whose mutagenicity can be modulated by inhibitors of MDR and suggest that there may be value in adding MDR inhibitors, especially trifluoperazine, to optimize the detection of mutagenicity by certain types of chemicals in the Salmonella/mammalian microsome mutagenicity test.
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PMID:Modulation of the potency of promutagens and direct acting mutagens in bacteria by inhibitors of the multidrug resistance mechanism. 941 96

Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400+/-70 microm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100+/-50 microm), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 microm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 microm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids.
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PMID:Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids. 950 30

P-glycoprotein (Pgp) is an inducible transmembrane protein that functions as an ATP-dependent efflux pump. Pgp is normally expressed in two types of cells: specialized epithelial cells with secretory/excretory functions (e.g., proximal renal tubules) and specialized endothelial cells (e.g., the capillary endothelial cells of the blood-brain barrier). In normal tissues, Pgp could exert a cytoprotective effect by facilitating excretion of drugs. It follows that inhibition of Pgp would alter the pharmacokinetics of drugs, like doxorubicin, in cells that express Pgp. The purpose of this study was to determine whether or not inhibition of Pgp by cyclosporin A (CsA) facilitated the transport of certain drugs across the blood tissue barriers of the brain and testes (barriers tissues expressing Pgp). 120 retired male breeder CD Fisher rats were randomly assigned to groups of 4 rats each. They were given either CsA, CsA vehicle, or saline followed by doxorubicin (Dox), cisplatin (CDDP), Evan's blue (EB), sodium fluorescein (NaF), or horseradish peroxidase (HRP). There was a CsA dose dependent increase in the tissue concentration of doxorubicin in brain and testes, but platinum (Pt) concentrations, derived from CDDP, were unaffected. Unlike CDDP, Dox, can be effluxed by Pgp. These increases in Dox concentrations were not due to altered vascular permeability as a result of CsA treatment as determined by lack of EB. NaF, or HRP in brain parenchyma. Modulation of Pgp function may prove to be useful for improving chemotherapy efficacy for patients with malignancies affecting tissues with blood-tissue barriers.
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PMID:Modulation of doxorubicin concentration by cyclosporin A in brain and testicular barrier tissues expressing P-glycoprotein in rats. 952 37

Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose. In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells. Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators. However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp.
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PMID:pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells. 961 76

P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.
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PMID:Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis. 963 Jul 1

The characteristics of doxorubicin handling have been studied in the cultured kidney epithelial cell line LLC-PK1, which has structure and function similar to those of renal tubular cells and expresses P-glycoprotein. The uptake of doxorubicin by LLC-PK1 cells was time dependent, reaching a steady state at about 4 hr, and reduced at low temperature; the initial uptake was saturable. The efflux of doxorubicin from LLC-PK1 cells was also temperature dependent but, even at 37 degrees C, a significant percentage of the drug remained associated with the cells after 180 min, which suggests a strong cellular binding, and the fluorescence microscopy revealed that the drug was concentrated in intracellular organelles. Substances that are substrates for P-glycoprotein, such as verapamil, vinblastine, vincristine and quinidine, significantly increased doxorubicin concentrations in LLC-PK1 cells. Similar results were obtained with the metabolic inhibitors sodium metavanadate and 2,4-dinitrophenol. On the other hand, the uptake was not affected by the classic organic cation transport drugs cimetidine, decynium 22 or decynium 24, nor by the organic anion drug probenecid. These results indicate that, in LLC-PK1 cells, doxorubicin enters by passive diffusion, is trapped in intracellular organelles and then is extruded from cells by a mechanism that probably involves P-glycoprotein. On the contrary, substances that interfere with the renal organic cation or anion secretory system have no effect on doxorubicin net accumulation in these cells.
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PMID:Handling of doxorubicin by the LLC-PK1 kidney epithelial cell line. 965 98

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.
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PMID:Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica. 980 32

Phenylbutyrate (PB), a novel lead compound for prostate cancer therapy, has molecular activities distinct from its metabolite, phenylacetate (PA). Both PB and PA promote differentiation in human prostate cancer cell lines, yet little data exist comparing the cytotoxic effects of each drug. We found that PB is more potent than PA in vitro; PB is 1.5-2.5 times more active at inhibiting growth and inducing programmed cell death than PA at clinically achievable doses against each human prostate cancer line studied. PB is equipotent to sodium butyrate, which induces apoptosis and differentiation through multiple mechanisms. Exposure of prostate cancer cell lines to PB reduces their DNA synthesis, leads to fragmentation of genomic DNA, and causes 50-60% of cells to undergo apoptosis. These PB-induced effects are 2-10 times greater than those of the control or PA. The stereotypical changes of apoptosis can be seen with sodium butyrate at similar concentrations, but not with PA. Prostate cancer cell lines overexpressing P-glycoprotein or possessing heterogeneous molecular alterations, including p53 mutations, are also sensitive to the effects of PB. In vivo, Copenhagen rats treated with oral PB had delayed growth of the androgen refractory Dunning R-3327 MAT-LyLu prostate cancer subline by 30-45% in a dose-dependent manner. These results demonstrate that PB induces cytotoxicity via apoptosis in human prostate cancer, in addition to its differentiating properties.
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PMID:Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate. 981 81


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