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)

Mechanisms contributing to reduced cytotoxic drug accumulation were studied in two multidrug-resistant (MDR) human lung cancer cell lines without P-glycoprotein expression. In these (non-small cell) SW-1573/2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accumulation of [14C]daunorubicin was 30 and 12%, respectively, of that in the parent cells. When cells, at steady state, were permeabilized with digitonin, the amount of daunorubicin binding increased only in the resistant cells. The reduced accumulation of daunorubicin in the SW-1573/2R120 and GLC4/ADR cells was accompanied by a lower initial (2 min) uptake rate of this drug. No difference in initial efflux rate of daunorubicin from preloaded cells could be detected between sensitive and resistant SW-1573 cells. However, daunorubicin was extruded 5-fold faster from GLC4/ADR cells than from the parental cells. In the presence of the energy metabolism inhibitors sodium azide and deoxyglucose, the reduced daunorubicin accumulations in the SW-1573/2R120 and GLC4/ADR MDR cells were (almost) completely reversed. The effects of these inhibitors on drug uptake were already apparent during the earliest measured time points (less than 15 s). Also, the enhanced efflux of daunorubicin from GLC4/ADR cells was inhibited. In ATP-depleted cells, the intracellular pH was lowered by approximately 0.3 units in resistant as well as in sensitive cells. The lower intracellular pH, however, could not account for the increase in daunorubicin accumulation in the resistant cells. Also, for vincristine and etoposide, the increases in drug accumulation under energy-deprived conditions were more pronounced in the resistant SW-1573/2R120 cells than in the parent SW-1573 cells. These results suggest that accumulation of drugs in the non-P-glycoprotein MDR human lung carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by an energy-dependent drug export mechanism which prevents efficient transport of drug to the target. Since P-glycoprotein expression in lung tumors is generally low, these MDR lung cancer cell lines can be used as a model to study alternative mechanisms leading to multidrug resistance in this tumor type.
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PMID:Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression. 130 22

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.
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PMID:Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts. 135 Feb 80

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro, when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.
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PMID:Mouse-human chimeric antibody MH171 against the multidrug transporter P-glycoprotein. 135 81

In the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (MDR) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent A and ASC systems). P-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in MDR cells. It was our hypothesis that its presence might affect other membrane energy requiring systems such as amino acid transport. On establishing the concentrations of P-glycoprotein by western blotting in the two cell lines to be studied, the kinetics of membrane transport of the neutral amino acids alpha-aminoisobutyric acid (AIB) and serine (SER) were investigated using the CHRC5 multidrug resistant and AUX B1 parental Chinese hamster ovary (CHO) cells. In CHRC5 cells, the amount and rate (Vmax) of accumulated amino acids, was significantly depressed when compared to AUX B1 cells, however, there was no difference in the rates of amino acid efflux between these two cell lines. Using 1,6-diphenyl 1,3,5-hexatriene (DPH) polarization to evaluate the state of membrane fluidity in the two cell lines studied, it was seen that CHRC5 cells showed a slightly lower degree of polarization than that observed in AUX B1 cells. These results suggest, that the P-glycoprotein does not alter amino acid transport directly but may modify the activity or numbers of functional transport carriers.
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PMID:Amino acid transport in multidrug-resistant Chinese hamster ovary cells. 135 38

A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.
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PMID:Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. 135 31

The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism. The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16. The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines. Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells. The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines. In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells. VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45%. However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells. Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells. Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil. These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives.
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PMID:Relationship of VP-16 to the classical multidrug resistance phenotype. 158 2

Luminal epithelium from mouse small intestine was fractionated into four distinct components ranging from the most differentiated (absorptive epithelial cells, fraction I) to the least differentiated (proliferative crypt cells, fraction IV). Immunoblot analysis of these fractions with monoclonal antibodies to P-glycoprotein, C219, and HYB-241, indicated that the amount of P-glycoprotein increased in cells as they progressed in level of differentiation from crypt to the villar surface of the lumen of the small intestine. P-glycoprotein in these normal intestinal cells functioned as a transporter of vincristine, one of a group of cancer chemotherapeutic agents to which cells can develop multidrug resistance. Transport ability was shown by efflux studies with vincristine as substrate. Efflux increased with differentiation and correlated with the increase in the amount of P-glycoprotein. Fraction I cells effluxed vincristine about seven times more rapidly than fraction IV cells. Efflux of vincristine was almost entirely inhibited from fraction I cells after treatment with antibody HYB-241, which recognizes an external epitope of P-glycoprotein. This finding would appear to demonstrate that P-glycoprotein was directly responsible for at least the majority of efflux of vincristine in these cells. The observed Mr of P-glycoprotein in fraction I cells was 140,000 when cells were solubilized with sodium dodecyl sulfate at room temperature before electrophoresis and immunoblot analysis. When solubilized fraction I protein was heated with 2-mercaptoethanol before electrophoresis, P-glycoprotein was seen by immunochemical procedures as an Mr 50,000-55,000 protein. No Mr 170,000 or 180,000 forms of P-glycoprotein (forms that are found in multidrug-resistant cell lines and are stable to heat or sulhydryl agents) were detected in any fraction of these epithelial cells under the conditions used. Functional P-glycoprotein was, therefore, associated with differentiation of luminal epithelium in small intestine, and the P-glycoprotein produced by these cells may be a type not previously described.
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PMID:P-glycoprotein content and mediation of vincristine efflux: correlation with the level of differentiation in luminal epithelium of mouse small intestine. 167 12

In this paper the biochemical properties of the antigens detected by six murine monoclonal antibodies (MAbs) are described. These MAbs react selectively with the multidrug-resistant small cell lung cancer (SCLC) cell line, H69AR, compared to its sensitive parent cell line, H69 (Mirski & Cole, 1989). Because H69AR cells do not overexpress P-glycoprotein, the antigens detected by these MAbs may be markers for non-P-glycoprotein-mediated mechanisms of resistance. We found that the 36 kDa protein precipitated by MAb 3.186 is phosphorylated and has a pI of approximately 6.7. The 55 kDa protein precipitated by MAb 3.50 is also phosphorylated and has a pI of approximately 5.7. Several observations suggest that MAbs 3.80, 3.177 and 3.187 recognise the same 47 kDa molecule and hence only MAb 3.187 was characterised further. This MAb precipitates an acidic protein which runs as a streak on isoelectric focusing gels. The 25 and 22.5 kDa cell surface proteins precipitated by MAb 2.54 both have a pI of approximately 7.6. Treatment of immunoprecipitates with glycosidase F indicated that none of the proteins detected by MAbs 2.54, 3.187, 3.50 and 3.186 have large N-linked carbohydrates. The peptide nature of the epitopes detected by MAbs 2.54 and 3.186 was unequivocally demonstrated by precipitation from in vitro translation products of H69AR RNA. The antigens detected by MAbs 3.50 and 3.187 were not detectable in immunoprecipitates of translation products but the epitopes are probably peptides because they were destroyed by boiling in sodium dodecyl sulphate. When the reaction of the MAbs with a panel of 15 paired drug-sensitive and -resistant cell lines was examined in a cell enzyme-linked immunosorbent assay, only a few resistance associated reactions were observed. Most of the reactions were either negative or not resistance-associated. When tested with three SCLC cell lines, MAb 3.187 reacted in a manner consistent with the relative resistance of the cell lines. Antigens that had similar electrophoretic mobility to those from H69AR cells were precipitated from extracts of five human cell lines of various tumour types. These data indicate that the cross-reactivities of the MAbs are due to antigens shared among the cell lines and not just the expression of common epitopes on different proteins. Resistance-associated proteins with the biochemical properties of the antigens described in this paper have not been reported previously and they remain potential markers for the as yet to be determined mechanisms of drug resistance in SCLC and other human malignancies.
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PMID:Multidrug resistance-associated antigens on drug-sensitive and -resistant human tumour cell lines. 167 58

P-glycoprotein, a hydrophobic 170-kDa integral protein overexpressed in the plasma membrane of multidrug-resistant cells, is proposed to function as an ATP-dependent drug efflux pump. Plasma membrane preparations highly enriched in P-glycoprotein were isolated from multidrug-resistant cells by discontinuous sucrose gradient and Ca2+ precipitation methods. Several strategies were used for P-glycoprotein purification, with the goal being to achieve both good yields and purity, while keeping experimental manipulation to a minimum. P-glycoprotein was solubilized from the plasma membrane using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Immunoaffinity chromatography using C219 monoclonal antibody produced low yields of moderately pure protein. Sequential lectin affinity chromatography on RCA-120 followed by lentil lectin resulted in a P-glycoprotein preparation that showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A fraction of P-glycoprotein did not bind to RCA-120, most likely as a result of heterogeneous glycosylation. A combination of chromatography on RCA-120 followed by immunoaffinity chromatography on C219 resulted in low yields of very pure P-glycoprotein.
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PMID:Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary 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


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