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)

Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo-1AM was hindered by the low level of accumulation of indo-1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P-glycoprotein (P-gp). Rhodamine-123, which is a known substrate of P-gp, was used to confirm the presence of P-gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulation of indo-1 in these cells and therefore allowed for improved intracellular calcium measurements. P-gp overexpressing colchicine-resistant CHO cells (CHRC5) also displayed enhanced indo-1AM loading with P-gp inhibitors. Nondetectable levels of P-gp activity were found in wild-type CEM-CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo-1AM loading in the presence or absence of P-gp inhibitors. Loading of a second calcium fluorescent probe fluo-3AM was improved in CHO cells by P-gp inhibition, whereas the structurally related pH probe BCECF-AM was minimally affected. Because low levels of P-gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes.
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PMID:Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes. 787 42

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.
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PMID:Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A. 790 14

We have established a model of human renal cell carcinoma, Kgg2, transplanted into athymic nude mice which expressed P-glycoprotein (P-gp) (detected by flow cytometry) and a high level of mRNA transcript of mdr1 gene (Northern blot analysis). We have evaluated the antitumor activity of a new highly potent vinca-alkaloid derivative, S 12363, in comparison with the activity of the reference compound vinblastine (VLB), when used alone or in combination with verapamil (VRP). The influence of the calcium influx blocker verapamil on the activity of the combination of S 12363 with adriamycin (ADR) was also determined. The results showed that S 12363 at a dose of 0.05 mg/kg/day, administered alone by intraperitoneal route daily on days 1 to 5, induced a tumoral regression of 50% during the first days after treatment. This effect was potentialized by simultaneous treatment with verapamil at 20 mg/kg/day for 5 days, leading to a long-term reduction of 70% of tumor growth. Vinblastine at a dose of 0.4 mg/kg/day administered alone or in combination with verapamil, using the same protocol, was less efficient. The association of S 12363 at 0.075 mg/kg/day (on days: 1-5, 11, 21 and 31), adriamycin at 2 mg/kg/day (on days: 11, 21 and 31) and verapamil at 20 mg/kg/day (on days: 0-5, 11, 21 and 31) induced an important reduction of tumor growth of 80% at the end of the experiment. In conclusion, the new vinca-alkaloid derivative S 12363 could present a therapeutic advantage over the reference compound vinblastine in the treatment of renal cell carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antitumor activity of the new vinca-alkaloid S 12363 alone or in combination with verapamil on a human multidrug resistant renal carcinoma xenograft. 790 53

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

Resistance to the intracellular Ca2+ pump inhibitor thapsigargin (TG) is associated with overexpression of both Ca2+ transport ATPase and the multidrug resistance (mdr) transporter P-glycoprotein (pgp). This is supported by increased resistance to TG following transfection of a functional pgp1 cDNA, and reversal of TG resistance with known inhibitors of pgp function. However, pgp is unlikely to represent the only mechanism of resistance to TG. Cell lines selected for high levels of resistance to TG (250-fold) show only a 3.7-fold increase in pgp expression and a 2-fold increase in cross-resistance to other drugs of the mdr class. Overexpression of endogenous Ca2+ transport ATPase may represent a second mechanism of resistance to TG. Increased Ca2+ ATPase expression (3-fold) is seen in cells made resistant to TG, and TG resistance increases with the transfection of a specific Ca2+ ATPase cDNA into DC-3F cells. If these transfectants are then made resistant to TG, both the endogenous Ca2+ ATPase and the exogenously transfected Ca2+ ATPase become overexpressed. These studies suggest that while TG may be a substrate for pgp, acquired resistance to TG can involve alterations in both pgp and Ca2+ ATPase expression. Additional, as yet unidentified, mechanisms of resistance may be involved in resistance to TG.
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PMID:Alterations in Ca2+ transport ATPase and P-glycoprotein expression can mediate resistance to thapsigargin. 790 87

Multidrug-resistance (MDR) is a cellular mechanism, which in certain tumors reduces the chemosensitivity of cells to a characteristic group of structural different cytostatic drugs, as anthracyclines, Vinca alkaloids and others, and correlates with a unfavourable clinical prognosis. In MDR-cells the intracellular concentration of cytostatic drugs is reduced due to the action of the mdr-1-gene-encoded P-glycoprotein (P-gp/gp 170), which functions as drug efflux pump with broad substrate specificity. Many calcium channel blockers of all subclasses (phenylalkylamine, dihydropyridine and benzothiazepine type) and other calcium antagonists inhibit the P-gp-mediated drug efflux and represent modulators of MDR (resistance modifiers, chemosensitizers). Since the sensitized tumor cells express no voltage-gated calcium channels, the induction of changes in intracellular free calcium showed no effect on MDR and the MDR-activity of antagonists is not correlated with their cardiovascular effects, the chemosensitization by these drugs is independent from their action on calcium channels and Ca(++)-regulation. Photoaffinity labelling with reactive derivatives proved the direct competitive interaction of calcium antagonists with the binding site of P-gp for cytostatic drugs as mechanism of action. The MDR-modulation of the doxorubicin or Vinca alkaloid resistance of tumor cells in vitro by verapamil and other calcium channel blockers was confirmed in vivo as increased survival length in mice with tumor transplants in combination with cytostatic therapy. The clinical application of calcium antagonists is limited by their severe cardiovascular side effects associated with the high concentrations required for successful reversal of MDR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Calcium antagonists as modulators of multi-drug resistant tumor cells]. 790 28

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.
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PMID:Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. 790 31

A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.
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PMID:Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. 791 80

We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.
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PMID:GTP-stimulated phosphorylation of P-glycoprotein in transporting vesicles from KB-V1 multidrug resistant cells. 791 30

A human lung-cancer PC-9 subline with acquired resistance to vincristine (VCR), a chemotherapeutic agent, was established with incremental increases of the drug. The resistant PC-9 subline (PC-9/VCR) shows a 12-fold increase in resistance to VCR and a unique cross-resistance pattern: high cross-resistance to the potent VCR analogue colchicine (6.9-fold) and vinblastine (2.5-fold); lower cross-resistance to actinomycin D (1.8-fold), cisplatin (1.2-fold) and adriamycin (1.3-fold) and a sensitivity to melphalan and VP-16 which is similar to that of the parental cell line. A reduced accumulation of VCR in the resistant cells was demonstrated. Interestingly, the VCR resistance of the PC-9/VCR cell line was partially reversed by ascorbic acid, and the drug uptake was enhanced. In contrast, ascorbic acid had no effect on drug tolerance and drug accumulation was not observed in either PC-9 parental cells or known multidrug-resistant (MDR) cells, suggesting that VCR resistance in PC-9/VCR cells results essentially from reduced drug accumulation. It is worth noting that, whereas reduced drug accumulation in the PC-9/VCR cells was susceptible to modulation by ascorbic acid, the increased efflux rate characteristic of the resistant cells was not. Further, there was a higher efflux rate in resistant cells than in parental cells. DNA Southern- and RNA Northern-blot hybridization analyses indicate that PC-9/VCR cells do not contain amplified mdr genes or overexpress P-glycoprotein. In addition, the calcium-channel blocker verapamil, which acts as a competitive inhibitor of drug binding and efflux, did not affect the resistant phenotype of PC-9/VCR cells. These findings suggest an ascorbic acid-sensitive drug uptake mechanism which is important in mediating VCR resistance per se in human lung-cancer cells; this differs from the P-glycoprotein-mediated MDR mechanism.
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PMID:Ascorbic acid increases drug accumulation and reverses vincristine resistance of human non-small-cell lung-cancer cells. 791 1


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