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)

Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.
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PMID:Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123. 748 25

The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.
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PMID:Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis. 764 31

The detection of multidrug resistance (MDR) in clinical samples is still a topic for discussion. One method, proven extremely useful for detection of membrane proteins in patients with hematological malignancies is the flow cytometrical analysis of individual tumor cells. Recently an assay was described based on the labeling of the P-glycoprotein (P-gp) with the monoclonal antibody MRK16, combined with detection of active daunorubicin (DNR) extrusion. In order to improve the specificity of the assay, on line with the results obtained by Wall et al., we exploited staining with Fluo-3. Both assays prove to be able to discriminate between drug-resistant and drug-sensitive cells. A major drawback of labeling with Fluo-3 in combination with the monoclonal antibody MRK16 is the important overlap of emission spectra of both fluorochromes. Moreover, using Fluo-3 for the detection of MDR might be complicated by the fact that differences in fluorescence intensities are not solely dependent on the presence of P-gp, but also on the activity of cytosolic esterases and the intracellular calcium concentration. Combination of the detection of structural and functional aspects of the MDR-associated protein may lead to a more precise detection of the MDR-positive patient.
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PMID:Comparison of daunorubicin and Fluo-3 for detection of multidrug resistance in human tumor cells. 904 67

Human lung adenocarcinoma A549 cells sensitive and A549/DDP cells resistant to Cis-dichlorodiammine platinum[II] (cisplatin) exhibit different intracellular free calcium and calcium fluorescence images labeled with Fura-2/AM and Fluo-3/AM as judged by dual-excitation fluorescence assay, Miracal Imaging and Laser Scanning Confocal Microscopy (LSCM) of single cells. The concentration of intracellular free calcium of the resistant A549/ DDP cells is one third that of the sensitive A549 cells. The efflux of Rhodamine 123 in resistant A549/DDP cells is faster than that in sensitive A549 cells. In addition, A549/DDP cells have an increase of Phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity in the plasma membrane. So it is tentatively suggested that the increase in PtIns 4-kinase activity resulting from lower intracellular Ca2+ concentration leads to an increase of its enzymatic products--PIP and PIP2, which may stimulate the activity of P-glycoprotein.
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PMID:Intracellular free calcium concentration and cisplatin resistance in human lung adenocarcinoma A549 cells. 1109 13

P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) are members of the ATP-binding cassette (ABC) family of transporter proteins. Both molecules are membrane-associated, energy-dependent efflux pumps with different substrate selectivity and they may play a role in the activation, differentiation and function of haematopoietic cells. Mouse haematopoietic cells are characterized by the expression of the cell surface molecules c-kit and Sca-1. Herein, the presence and activities of Pgp and MRP1 in mouse bone marrow mononuclear cells (BMMC) and their relationship with the proteins c-kit and Sca-1 were evaluated. Pgp and MRP activities were measured based on the extrusion of rhodamine 123 (for Pgp) and Fluo-3 (for MRP). Cell populations were assessed by cytometry using anti-c-kit and anti-Sca1 antibodies. Pgp activity was present in 5% of BMMC while 50% of BMMC cells showed MRP activity. These findings agreed with the proportion of cells expressing the MRP1 surface molecule (51.3 +/- 4.17%). About 14% of BMMC were positive for c-kit and/or Sca-1 (9.3% c-kit- Sca-1+, 4.2% c-kit+ Sca-1- and 0.9% c-kit+ Sca-1+). Among these subpopulations only c-kit- Sca-1+ cells presented Pgp activity (21.36%). On the other hand, MRP activity was present in all three subpopulations. Most cells (82.5%) of the c-kit+ Sca-1- subpopulation presented MRP1 activity compared to only 54.1% of c-kit+ Sca-1+ and 38.8% of c-kit- Sca-1+. This study demonstrates the expression and activity of MRP1 in BMMC. While only a small proportion of precursor cells had Pgp activity, MRP1 activity was present among different subpopulations of precursor cells. Further studies are necessary to establish the role of these transporters in haematopoietic cells.
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PMID:Expression of c-kit and Sca-1 and their relationship with multidrug resistance protein 1 in mouse bone marrow mononuclear cells. 1742 3

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P<0.05) and a two-fold increase in Fluo-3 fluorescence (P<0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DeltaPsim, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis.
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PMID:Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential. 1750 81

Human cancers are characterized by a high degree of drug resistance. The multidrug resistance transporters MDR1-P-glycoprotein (ABCB1) and ABCC2 (MRP2) are expressed in a variety of human cancers, including hepatocellular carcinoma (HCC). The ABCC2 gene encodes a membrane protein involved in the ATP-dependent transport of conjugates of lipophilic substances. In this study we analyzed the effect of an ABCC2 antisense construct on the chemosensitization of HepG2 cells. Adenoviral vectors were constructed to allow an efficient expression of anti-ABCC2 antisense constructs. The effective target sequence comprised nucleotides 2543-2942 of the human ABCC2 cDNA. Adenoviral delivery of the ABCC2 antisense construct resulted in a reduced IC(50) for doxorubicin (12-fold), vincristine (50-fold), cisplatin (25-fold) and etoposide (VP-16) (25-fold). The adenoviral delivery of the ABCC2 antisense construct was so efficient that chemosensitization of HepG2 cells could even be demonstrated in mass cell cultures without a selection of transduced cells for single ABCC2 antisense-expressing HCC cell clones. After transfection of the ABCC2 antisense-expressing construct, HepG2 cells had significantly reduced ABCC2 mRNA and ABCC2 protein levels. Transduction of the ABCC2 antisense-expressing construct into HepG2 cells resulted in the accumulation of the high-affinity ABCC2 substrate Fluo-3. HepG2 tumors stably transfected with an anti-ABCC2 antisense construct regressed significantly in nude mice upon vincristine treatment. In addition, significant tumor regression was also observed when adenovirus-expressing anti-ABCC2 antisense construct was directly injected into HepG2 tumors in nude mice. Our study demonstrates the specific reversal of ABCC2-related drug resistance in adenovirus-transduced HepG2 cells and in HepG2 tumors in nude mice expressing this ABCC2 antisense construct.
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PMID:Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-ABCC2 antisense constructs. 1770 53