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)

We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 microM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 microM). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.
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PMID:Polarized efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers. 151 Jun 94

Cellular drug resistance to natural products is often due to the presence of an efflux pump which reduces intracellular drug content and chemosensitivity. A 170 kD cell surface resident P-glycoprotein is believed to act as the efflux pump. In the present report, we have compared three commercially available antibodies C-219, JSB-1, and mdr(Ab-1) for use in flow cytometric detection of P-glycoprotein positive cells. Our data show that C219 gives uniformly good results in a variety of murine and human tumor cell lines for detection of P-glycoprotein positive cells. We have also compared data of C219 stained cells analyzed in parallel on a flow cytometer equipped with a small laser (15 mW) and a large laser (5 watt) cell sorter. Data obtained on these two instruments are comparable. A staining protocol and data on dual staining of cells for DNA content by propidium iodide and P-glycoprotein expression after FITC labeling are also presented.
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PMID:Comparison of three commercially available antibodies for flow cytometric monitoring of P-glycoprotein expression in tumor cells. 168 82

The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins.
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PMID:Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells. 171 35

Acquired multidrug resistance in cultured cells is often due to amplification of pgp genes, which gives rise to overproduction of P-glycoproteins that confer resistance by reducing the intracellular drug accumulation. The size of these amplicons varies between multidrug resistant cell lines and is often much larger than the gene selected for. Amplicons of the multidrug resistant Chinese hamster ovary cell line CHRC5 and its progenitor CHRB3, for example, span at least five different genes besides the pgp genes. Linkage of these gene classes with pgp had been shown by in situ hybridization and by long distance mapping using pulsed field gradient gel electrophoresis. Because the boundaries of the larger amplicons could not be determined, the size of such amplicons is not yet known, even though the six genes span at least 1500 kilobases. In the present study we have determined the amplicon size in B3+, a subclone of CHRB3 with a homogeneously staining region on chromosome 7q+ that harbors the amplified genes. We estimated the amplicon size in revertant clones by correlating the decreased DNA content of the 7q+ homogeneously staining region with the number of lost amplicons. The reduction of the homogeneously staining region DNA that accompanied reversion was determined by flow cytometry of propidium iodide stained chromosome suspensions of the various cell lines. We found that about 107 megabase pairs were lost together with 11-24 P-glycoprotein gene copies, suggesting that the mean amplicon size is in the range of 4.5-10.1 megabase pairs.
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PMID:Determination of the DNA content of a six-gene amplicon in the multidrug-resistant Chinese hamster cell line CHRB3 by flow karyotyping. 197 Feb 76

Expression of the drug efflux pump P-glycoprotein (PGP) was determined by flow cytometry in human lung cancer cell lines and in leukaemic blasts derived from 60 patients with acute myeloid leukaemia (AML). Cells from the PGP-negative parent cell line H69/P and the multidrug resistant (MDR)-variant H69/LX4 could be clearly distinguished by immunostaining with the anti-PGP monoclonal antibody MRK16. In leukaemic blasts, the differences in fluorescence intensities between samples incubated with the idiotypic nonspecific (control sample) and specific antibody (test sample) were small, resulting in nondisjunct distributions. Only in a few leukaemia specimens were PGP-expressing cells detectable by simple subtraction of histograms using a threshold. Therefore, an improved histogram subtraction analysis, based on curve fitting and a statistical test, was applied to distinguish antigen-positive from antigen-negative cells. Moreover, a multiparametric staining procedure employing propidium iodide (PI) and Hoechst 33342 was used to reduce staining artefacts. By this approach, leukaemic cells with low expression of PGP were detected in 39 out of 60 cases. Subpopulations with strong PGP expression, resulting in disjunct fluorescence distributions, were not observed. Only in 5 out of 60 specimens were PGP expressing cells detected by a conventional subtraction of histograms using a threshold. Comparison of data obtained with or without the multiparametric gating procedure indicated that the increase in sensitivity was mainly due to the application of the data analysis. However, exclusion of cell debris using PI and Hoechst staining properties reduced the deviation of data from mean values. No relation between PGP expression and cell cycle position was observed in either cell lines or in leukaemic blasts.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Improved flow-cytometric detection of low P-glycoprotein expression in leukaemic blasts by histogram subtraction analysis. 751 93

The characteristics of volume-activated chloride currents, drug transport function and levels of P-glycoprotein (PgP) expression were compared between two human chronic erythroleukemia cell lines: a parental (K562) cell line and a derivative obtained by vinblastine selection (K562 VBL400). Parental K562 cells showed no detectable P-glycoprotein expression, measured at the protein level (immunofluorescence labeling with monoclonal antibodies), and had very low levels of MDR-1 mRNA expression (RT-PCR analysis), when compared with levels measured in K562 VBL400. Differences in Pgp-mediated transport were estimated by comparing the rates of Fluo3 accumulation. The higher drug-transport function of K562 VBL400 cells (e.g., lower Fluo3 accumulation) correlated with their elevated levels of MDR-1. The rate of dye transport was sensitive to verapamil but was not affected by the tonicity of the extracellular medium. In contrast to the clear differences in transport function, the characteristics of chloride currents induced by cell swelling were indistinguishable between the two cell lines. Currents measured in the whole-cell configuration were outwardly rectifying, had a higher permeability to iodide than to chloride (SCN- > I- > Cl- > gluconate), were potently blocked by NPPB and were unresponsive to verapamil. The percentage of responding cells and the mean current density were nearly identical in both cell lines. In addition, activation of the volume-sensitive current was not prevented during whole-cell recordings obtained with pipettes containing high concentration of cytotoxic drugs (vincristine or vinblastine). These results do not lend support to the previously reported association between Pgp expression and volume-sensitive chloride channels, and suggest that a different protein is responsible for this type of chloride channel in K562 cells.
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PMID:Drug-transport and volume-activated chloride channel functions in human erythroleukemia cells: relation to expression level of P-glycoprotein. 763 88

To date, all reported measurements of multidrug resistance have been semiquantitative. The purpose of the present study is to establish and validate the self-competitive binding assay technique utilizing monoclonal antibody for quantitative estimation of multidrug resistance in tumor cells. This technique is used for P-glycoprotein concentration measurement in BE(2)-C human neuroblastoma cell line and its sublines with primary resistance to colchicine and actinomycin D. Monoclonal antibody MRK-16 was used in this study. It was labeled with iodine-125 (125I) to trace the concentration of antibody-antigen complexes. The binding data were obtained by varying the concentration of the unlabeled antibody. The results were fitted to a model equation to estimate the number of binding sites and antibody-antigen dissociation constant. The P-glycoprotein concentration was significantly higher in the resistant sublines than in the sensitive line. The highest levels were achieved in actinomycin D-resistant cells: 2.1 x 10(6) binding sites/cell versus 5.4 x 10(4) binding sites/cell in the sensitive cells. The consistency of the results was verified by repeating the study three times for each cell line. The binding assay results were confirmed by Western blot experiments performed on the same cell lines.
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PMID:Measurement of P-glycoprotein expression in multidrug-resistant human neuroblastoma cell lines using self-competitive binding assay. 866 May 14

Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine-123 (Rh-123), and 3,3'-diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP-expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant lines became apparent and even more pronounced after 24h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC2(3) can not.
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PMID:DiOC2(3) is not a substrate for multidrug resistance protein (MRP)-mediated drug efflux. 887 50

Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). The MDR phenotype can be characterized either by use of monoclonal antibodies raised against P-gp or with functional tests based on the intracellular accumulation of fluorescent molecules. The aim of the present study was to compare the effectiveness of functional tests performed by flow cytometry including uptake of daunorubicin (DNR) (2 micrograms/ml), Hoechst 33342 (5 micrograms/ml), or rhodamine 123 (RH 123) (0.1 microgram/ml); and to evaluate the effect of cell death induced by heating at 60 degrees C for 2 h on incorporation of DNR and RH 123. Sensitive and resistant human hematopoietic K 562 cells expressing P-gp were identified by monoclonal antibodies C 219 and MRK-16. Fluorescence of the dyes was always higher in sensitive than in resistant cells. However, DNR and Hoechst 33342 produced a slight incorporation in resistant cells, while RH 123 showed lack of incorporation in resistant cells. Thus, RH 123 allows sensitive and resistant cells to be clearly distinguished. In case of cell death, accumulation of RH 123 and DNR were different. With RH 123, fluorescence intensity strongly decreased in sensitive cells. With DNR, fluorescence intensity was enhanced in resistant cells. Thus, when the MDR phenotype is defined by uptake of DNR or RH 123, artifactual results due to cell death may be avoided by using a dye such as propidium iodide to eliminate dead cells.
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PMID:Flow cytometry evaluation of the multidrug-resistant phenotype with functional tests involving uptake of daunorubicin, Hoechst 33342, or rhodamine 123: a comparative study. 892 22

The ATP binding cassette transporter ABC1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death. Since members of this family of proteins such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator share the ability to transport anions, we have investigated the transport capability of ABC1 expressed in Xenopus oocytes using iodide efflux and voltage-clamp techniques. We report here that ABC1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABC1 is up-regulated by orthovanadate, cAMP, protein kinase A, and okadaic acid. In other ABC transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABC1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, we conclude that ABC1 is a cAMP-dependent and sulfonylurea-sensitive anion transporter.
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PMID:ABC1, an ATP binding cassette transporter required for phagocytosis of apoptotic cells, generates a regulated anion flux after expression in Xenopus laevis oocytes. 900 6


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