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 calcium channel blocker verapamil has been shown to reverse multidrug resistance (T. Tsuruo et al., Cancer Res. 41: 1967-1972, 1981), but the mechanism of action of this agent has not been fully elucidated. A radioactive photoactive analogue of verapamil, N-[benzoyl-3,5-3H-(+/-)-5-[(3,4-dimethoxyphenetyl)methylamino]-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylpentylamine, was used to label the plasma membranes of a human myelogenous leukemia cell line (K562), a multidrug-resistant subline selected for resistance to Adriamycin (K562/ADM) and its revertant cell (R1-3). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled Mr 170,000-180,000 protein in the membranes from K562/ADM but not from the drug-sensitive parental K562 and revertant R1-3 cells. The Mr 170,000-180,000 verapamil acceptor was immunoprecipitated by monoclonal antibody MRK16 specific for P-glycoprotein associated with multidrug resistance, indicating that P-glycoprotein in the plasma membrane is a major target of verapamil in K562/ADM cells. The photolabeling of P-glycoprotein with N-[benzoyl-3,5-3H]-(+/-)-5-[(3,4-dimethoxyphenetyl)methylamino]-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylphentylamine was significantly blocked by other calcium channel blockers, nicardipine and diltiazem, that have been shown to overcome multidrug resistance. In addition, the photolabeling was partially blocked by Adriamycin, vincristine, and colchicine, suggesting that the specific binding sites for verapamil on P-glycoprotein are closely related to the binding sites for these calcium channel blockers and antitumor agents. To determine whether verapamil could be a substrate for P-glycoprotein, the cellular accumulation of [3H]verapamil into K562 and K562/ADM was evaluated. The accumulation of [3H]verapamil in the multidrug-resistant cells was 30% of K562 cells and increased when K562/ADM cells were treated with vincristine and nicardipine at 5 microM, indicating that the P-glycoprotein transports verapamil as well as other antitumor agents in the multidrug-resistant cells. These results suggest that verapamil enhances antitumor agent retention through competition for closely related binding sites on P-glycoprotein.
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PMID:Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ADM cells. 256 30

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.
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PMID:Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells. 288 31

A radioactive photoactive dihydropyridine calcium channel blocker, [3H]azidopine, was used to photoaffinity label plasma membranes of multidrug-resistant Chinese hamster lung cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled 150-180-kDa doublet in the membranes from drug-resistant but not from the drug-sensitive parental (DC-3F) cells. A similar radiolabeled doublet was barely detected in a drug-sensitive partial revertant (DC-3F/ADX-U) cell line. The 150-180-kDa doublet exhibited a specific half-maximal saturable photolabeling at 1.07 X 10(-7) M [3H]azidopine. The dihydropyridine binding specificity was established by competitive blocking of specific photolabeling with nonradioactive azidopine as well as with nonphotoactive calcium channel blockers nimodipine, nitrendipine, and nifedipine. In addition, [3H]azidopine photolabeling was blocked by verapamil and diltiazem but was stimulated by excess prenylamine and bepridil suggesting a cross-specificity for up to four different classes of calcium channel blockers. The 150-180-kDa calcium channel blocker acceptor co-electrophoresed exactly with the 150-180-kDa surface membrane glycoprotein (gp150-180 or P-glycoprotein) Vinca alkaloid acceptor from multidrug-resistant cells and was immunoprecipitated by polyclonal antibody recognizing gp150-180. [3H]Azidopine photolabeling of the 150-180-kDa component in the presence of excess vinblastine was reduced over 90%, confirming the identity or close relationship of the calcium channel blocker acceptor and the gp150-180 Vinca alkaloid acceptor. The [3H]azidopine photolabeling of gp150-180 also was reduced by excess actinomycin D, adriamycin, or colchicine, demonstrating a broad gp150-180 drug recognition capacity. The ability of gp150-180 to recognize multiple natural product cytotoxic drugs as well as calcium channel blockers suggests a direct function for gp150-180 in the multidrug resistance phenomenon and a role in the circumvention of that resistance by calcium channel blockers.
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PMID:Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. 303 8

Some characteristics of doxorubicin-resistant CHO cell line (RC1) were studied by means of cell biological methods and SDS-PAGE electrophoresis. The resistance factor was 16.5-fold, and RC1 revealed cross-resistances to colchicine, actinomycin and harringtonine. By indirect immunofluorescence assay, P-glycoprotein was not detected. Compared with CHO, the doxorubicin (Dox) uptake and accumulation of RC1 decreased, but the membrane fluidity of RC1 increased. The reduction in drug accumulation was correlated with increase in membrane fluidity. Dox was mainly distributed in the cell nucleus of CHO, but in both cytoplasm and nucleus of RC1. This suggested that Dox was transported more slowly in RC1 cytoplasm than in CHO cytoplasm, resulting in less Dox entrance into the cell nucleus of RC1 than into that of CHO. We also found that a 30-40 kDa nuclear protein which was expressed normally in CHO disappeared in RC1.
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PMID:[Drug resistance of doxorubicin-resistant CHO cell line]. 751 12

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.
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PMID:Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells. 764 Feb 9

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723-20729, 1989; Mol Cell Biol 10:3020-3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr(R) and murine melanoma B16/Adr(R) cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.
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PMID:Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells. 769 26

Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM). These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of [3H]vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles. However, profound inhibition of ATP-dependent [3H]VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0.02 nM). The addition of 1 microM Ca2+ reduced the inhibition of [3H]VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+. Binding of CLM, itself, to P-gp was demonstrated in two ways. The P-gp content of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment. Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of [3H]VBL, down-modulates the translocation of this agent in the presence of ATP.
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PMID:Functional modulation of multidrug resistance-related P-glycoprotein by Ca(2+)-calmodulin. 774 32

Multidrug-resistant tumor cells can be resensitized by combined application of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker. Since clinical trials on the circumvention of multidrug resistance (MDR) with chemosensitizers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination. Thus, the Adriamycin(ADR)-selected, P-glycoprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.05 microgram/ml ADR. The initial CsA concentration (plus 0.05 microgram/ml ADR) to inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 microgram/ml. By continuous incubation for more than 6 months, the IC50 for CsA (at constant ADR) was elevated to 3.6 micrograms/ml (90-fold), thus generating the variant F4-6RADR-CsA. The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA. As shown by immunocytochemistry as well as by the polymerase chain reaction and by Western blotting including densitometry, P-glycoprotein was preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis could detect no new proteins in F4-6RADR-CsA as compared to F4-6RADR. Interestingly, resistance of F4-6RADR-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by different pharmacodynamic mechanisms. Transport studies with [14C]ADR, performed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resistance found in proliferation assays. Cellular accumulation of [3H]cyclosporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-CsA cells, but remained at the level of controls in F4-6RADR cells. Results indicate that increased amounts of the P-glycoprotein--besides other, perhaps more important mechanisms that are as yet unknown--partially mediate CsA resistance in F4-6RADR-CsA cells. We have designated this new form of resistance "secondary combined resistance" (SCR). The results suggest that at least some clinical cases of insensitivity to chemosensitizers or of relapse after reversing therapy could be explained by SCR, and that resensitizing treatment of tumor patients should be based on the consideration of several chemosensitizers of different pharmacodynamics.
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PMID:Secondary combined resistance to the multidrug-resistance-reversing activity of cyclosporin A in the cell line F4-6RADR-CsA. 790 33

P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells. The functional form of P-gp in its native state is not known. Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes [Boscoboinik et al. (1990) Biochim. Biophys. Acta 1027, 225-228]. In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines. The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells. When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers. In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization. The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido[alpha-32P]ATP) or the drug [3H]azidopine in membrane preparations. P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function. When MDR cells were labeled with [32P]orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct. The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation. Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.
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PMID:Detection of oligomeric and monomeric forms of P-glycoprotein in multidrug resistant cells. 790 29

We previously isolated and characterized a partially purified preparation of ATPase-active P-glycoprotein, the multidrug transporter (Doige, C.A., Yu, X. and Sharom, F.J. (1992) Biochim. Biophys. Acta 1109, 149-160). The effect of various detergents and membrane phospholipids on the ATPase activity of P-glycoprotein has now been investigated. P-Glycoprotein ATPase activity was most stable in CHAPS, with over 50% of the activity retained at a concentration of 8 mM. Octyl glucoside in the low mM range also supported the ATPase, while deoxycholate destroyed all activity at 1 mM. Digitonin and SDS inhibited ATPase activity at very low concentrations. Triton X-100 at 2-10 microM stimulated the ATPase almost 2-fold, while higher levels inhibited activity. Although P-glycoprotein ATPase was sensitive to thermal inactivation, full activity was preserved in the presence of asolectin, but not phosphatidylcholine species. Further studies revealed that asolectin, both saturated and unsaturated phosphatidylethanolamines, and phosphatidylserine, were best able to maintain ATPase activity at 23 degrees C. Saturated phosphatidylethanolamine species activated P-glycoprotein ATPase up to 40% at 23 degrees C, and 80% at 4 degrees C. Following detergent delipidation, various lipids were able to restore P-glycoprotein ATPase activity. Unsaturated phosphatidylcholine and phosphatidylserine were most effective, while saturated species were not able to restore catalytic activity. These results indicate that membrane lipids are necessary for catalytic activity of the ATPase domains of P-glycoprotein. P-Glycoprotein has well-defined lipid preferences, with saturated phosphatidylethanolamines both activating the ATPase and providing protection from thermal inactivation, while fluid lipid mixtures are able to restore activity following delipidation.
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PMID:The effects of lipids and detergents on ATPase-active P-glycoprotein. 809 61


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