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)

Regulation of cell volume is essential for every cell and is accomplished by the regulated loss or gain of intracellular ions or other osmolytes. Regulatory volume decrease often involves the parallel activation of potassium and chloride channels. Overexpression of P-glycoprotein leads to volume-activated Cl- currents but its physiological importance for volume regulation is unclear. CIC-2 is a ubiquitously expressed Cl- channel activatable by non-physiologically strong hyperpolarization. We now show that CIC-2 can be activated by extracellular hypotonicity, which suggests that it has a widespread role in volume regulation. Domains necessary for activation by both voltage and volume are localized to the amino terminus. Mutations in an 'essential' region lead to constitutively open channels unresponsive to medium tonicity, whereas deletions in a 'modulating' region produce partially opened channels responsive to both hypo- and hypertonicity. These domains can be transplanted to different regions of the protein without loss of function.
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PMID:Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. 133 33

Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.
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PMID:Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. 197 54

1. P-glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP-dependent transporter activity. It has been suggested that P-glycoprotein may also function as a volume-regulated chloride channel or chloride channel regulator. To assess the chloride channel function of P-glycoprotein, we examined swelling-activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA. 2. Functional expression of P-glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P-glycoprotein substrate, calcein AM. 3. Endogenous, swelling-activated chloride conductances were virtually absent by the time P-glycoprotein expression was confirmed. Thus, this expression system afforded the advantage of assessing putative MDR1-associated chloride currents in the absence of background currents. 4. The currents activated by hypotonic shock (50%) in both MDR1-injected and control (water-injected) oocytes were not significantly different. The swelling response was due in part to the activation of a potassium-selective conductance which could be inhibited by barium. No chloride-selective currents were activated by hypotonic shock in the presence or absence of barium. Therefore, we conclude that P-glycoprotein expression does not produce a swelling-activated chloride conductance in the Xenopus oocyte expression system.
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PMID:Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity. 747 31

Cation-transport properties were compared in a human leukemic cell line (K562) and its vincristine-selected, mdr1-gene-expressing sublines (K562/Vcr30 and K562/Vcr150) by the capacity of the cells to accumulate the potassium analogue thallium (201Tl). Determination of the time course of thallium accumulation in the absence and presence of ouabain, an inhibitor of sodium-potassium adenosine triphosphatase (ATPase), showed that the initial (at 20 min) rate of ouabain-resistant uptake was about 70% higher in the K562/Vcr30 cells than in the parental line. The maximal rate (Vmax) of ouabain-resistant uptake was 78 mmol/h for K562 cells and 115 mmol/h for K562/Vcr30 cells, and the Michaelis constant (Km) was 0.37 and 0.18 mmol, respectively. Bumetanide (50 microM), a specific inhibitor of ouabain-resistant Na-K-Cl cotransport, inhibited the elevated 201Tl uptake in K562/Vcr150 cells but had no effect on cellular vincristine accumulation. Incubation with different multidrug resistance (MDR)-reversing agents (verapamil as well as cyclosporin A and its analogue PSC833) had no significant effect on 201Tl uptake. Membrane depolarization by an elevation of the potassium concentration in the incubation medium did not affect vincristine accumulation in any cell line, which indicated that the changed drug-transport properties in mdr1-gene-expressing cells were not due to membrane hyperpolarization. It was concluded that P-glycoprotein-positive cells have a more efficient ouabain-resistant cation-transport mechanism than to cells without P-glycoprotein. A functional relationship between this phenomenon and MDR was not identified.
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PMID:Increased cation transport in mdr1-gene-expressing K562 cells. 772 Jan 83

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.
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PMID:Microfluorometric evaluation of calcein acetoxymethyl ester as a probe for P-glycoprotein-mediated resistance: effects of cyclosporin A and its nonimmunosuppressive analogue SDZ PSC 833. 791 May 63

Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal ("re-reversal"), exhibits a sharp extracellular potassium concentration ([K+(0)]) dependence. It is observed at [K+(0)] > 2-4 mM and not at [K+(0)] greater than or equal to 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that "re-reversal" is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+(0)]-dependence similar to that of the "re-reversal." These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells.
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PMID:Reversal of multidrug resistance by valinomycin is overcome by CCCP. 860 82

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.
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PMID:The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease. 888 37

Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRP-overexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dose-dependent manner, through inhibition of cellular export of the dye; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 microM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function.
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PMID:Reversal of MRP-mediated multidrug resistance in human lung cancer cells by the antiprogestatin drug RU486. 1032 17

Sestamibi imaging is used to assess the staging of nasopharyngeal carcinoma (NPC), and to monitor response to therapy. Sestamibi was added to single-cell suspensions of the NPC cell lines CNE-1 and CNE-2Z, and aliquots were removed over 60 min and centrifuged to determine cell-associated radioactivity. Sestamibi accumulation reached similar plateau values in both cell lines within 30 min of addition. Saturating concentrations of P-glycoprotein (Pgp) modulators increased accumulation by 1.4-1.7 fold over controls. Hyperpolarization of the mitochondrial membrane with nigericin increased accumulation by 2.6-3.4 fold. In contrast, depolarization of the plasma membrane with isotonic high potassium buffer reduced accumulation to 70-75% of control values, and additional depolarization of the mitochondrial membrane with valinomycin further reduced accumulation to 21-29% of control levels. These results indicate that both cell lines contain a modest level of Pgp activity and that both are capable of further polarization of the mitochondria. This suggests that mitochondrial hyperpolarization is not the complete explanation for high accumulation of sestamibi in NPC.
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PMID:Sestamibi accumulation in human nasopharyngeal carcinoma cell lines in vitro. 1081 Mar 39

Although some authors have suggested that sestamibi imaging is useful in evaluation of patients with lymphoma, others have obtained equivocal results. This discrepancy has been further investigated in vitro using two patient-derived non-Hodgkin's lymphoma cell lines, OCI-Ly3 and OCI-Ly18. Sestamibi (0.2 MBq/ml) was added to a suspension of OCI-Ly3 or OCI-Ly18 cells and aliquots were removed over 1 h and centrifuged to determine cell-associated radioactivity. Further experiments studied the effect of addition of a P-glycoprotein (Pgp) modulator or alteration in plasma and/or mitochondrial membrane potentials. Accumulation of sestamibi reached plateau values within 30 min, but these values were 6-fold higher in OCI-Ly3 than in OCI-Ly18. Inhibition of Pgp function with GG918 or PSC833 did not affect OCI-Ly3 cells but increased accumulation in OCI-Ly18 cells 3-fold, indicating a moderate level of Pgp. However, both cell lines responded similarly to membrane potential alterations: hyperpolarization of the mitochondrial membrane with nigericin had little effect on accumulation: in contrast, depolarization of the plasma membrane with an isotonic high potassium buffer reduced accumulation of sestamibi to 52% of control and additional depolarization of the mitochondrial membrane with valinomycin further reduced accumulation to 12% of control levels. These studies suggest that there can be wide differences in accumulation between cell lines, in part due to Pgp-mediated efflux, but that both of these cell lines have highly polarized mitochondria with little further capacity for hyperpolarization.
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PMID:Accumulation of sestamibi in lymphoma cell lines in vitro. 1095 81


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