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)

Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established to detect low expression level of P-glycoprotein (P-gp) using a monoclonal antibody MRK16, which recognizes a cell surface epitope of P-gp. With K-562 and U-937 cell lines, which are known to express low levels of P-gp and hence routinely used as negative control cell lines in conventional flow cytometry, both assays gave significantly positive reactivities indicating improved specificity and sensitivity of these assays. The findings in the dilution test, where P-gp-positive cells were added to P-gp-negative cells at various ratios, demonstrated that the MRK16-MACS assay is quantitative and capable of detecting small numbers of P-gp-positive cells as few as 2.5% of the total cells tested. Furthermore, specific enrichment of P-gp-expressing cells in magnetic cell sorting assays was verified by reverse transcription-polymerase chain reaction (RT-PCR) analysis and functional assay for P-gp with Rhodamine 123. The availability of such magnetic cell sorting assays may offer an approach to quantitate low level of P-gp expression.
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PMID:Establishment and evaluation of MRK16-magnetic cell sorting assays for detecting low expression of multidrug resistance P-glycoprotein using human leukemia cell lines and peripheral blood cells from healthy donors. 749 Apr 49

Previous data showing the correlation of multidrug resistance (MDR) and differentiation in tumor cell populations (Melloni et al. 1988; Stavrovskaya et al. 1990) suggest that: 1) isolation of MDR cells by cytostatic drugs leads to the selection of more differentiated cell variants and 2) in more differentiated cell variants the activity of MDR-related P-glycoprotein (Pgp) is more prominent than in less differentiated cells. Here we used human melanoma cell line mS and two variants selected from mS population: a) MDR variant of mS selected by colchicine (mS-0.5) and b) mS-trRAR/2--variant obtained by introduction of expressing retinoic acid receptor RAR-alpha cDNA into mS cell. The differentiation status, expression of MDR1 gene and Pgp functioning were compared in wild-type cells and mS variants. Electron microscopic examination of melanosomes showed that the mS-0.5 subline comprised more differentiating cells in the population than parental mS cultures and that these cells were at later stages of melanogenesis. The increase in the degree of differentiation in mS-0.5 population coincided with MDR1 gene overexpression, occurrence of Pgp molecules on the cell membrane and acceleration of Pgp-mediated Rhodamine 123 (Rh123) efflux. mS-trRAR/2, proved to be more differentiated than mS cells. The MDR1 mRNA level and Rh123 efflux were not elevated in mS-trRAR/2 cells, however, retinoic acid (RA) treatment increased both the degree of differentiation and Rh123 efflux in mS-trRAR/2 to a greater extent than in mS cultures. Thus, the data obtained in this study are in favor of the suppositions mentioned above. The mechanisms of coordinated alterations of differentiation and Pgp activity in MDR cells are discussed.
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PMID:Alterations of melanin synthesis in human melanoma cells selected in vitro for multidrug resistance. 758 Jan 2

We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.
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PMID:Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function. 762 41

Multidrug resistant (MDR) phenotype is characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). MDR phenotype can be characterized either with monoclonal antibodies raised against P-gp or with functional tests, most often based on the incorporation of fluorescent compounds. In the present study, data obtained with the monoclonal antibodies C219, JSB1 and MRK16 are compared to those of functional tests performed by flow cytometry including uptake of daunorubicin (DNR), Rhodamine 123 (Rh 123) or Hoechst 33342. Sensitive and resistant cell lines K562S, K562R, KBA1 and KB31, derived either from a human chronic myeloid leukemia or from a human epithelial carcinoma, were used. In resistant cells, P-gp expression was revealed with either the monoclonal antibodies C219, JSB1 or MRK-16. The most specific results were obtained with MRK-16. With functional tests, no matter which dyes were used, the fluorescence was always stronger in sensitive than in resistant cells. However, with DNR and Hoechst 33342, an incorporation of these dyes was exhibited in resistant cells. This phenomenon was not observed with Rh 123, which makes it possible to distinguish clearly between sensitive and resistant cells and to detect as few as 1% of resistant cells. Because of its high sensitivity, the functional test involving incorporation of Rh 123 was successfully used in acute myeloid leukemia to detect multichemoresistant cells.
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PMID:Evaluation of multidrug resistant phenotype by flow cytometry with monoclonal antibodies and functional tests. 765 50

The neoplasias resistance to chemotherapy is mainly due to multidrug resistance phenomenon (MDR) mediated by an ATP-dependent efflux pump called P-glycoprotein. This function can be reversed by many multidrug reversing agents so numerous chemotherapy regimens have been initiated in malignancies. To make sure the success of these protocols it is necessary to detect as soon and as certain as possible the presence of resistant cells among malignant population. We have chosen to evaluate functional test using rhodamine 123 in this aim in view. We have made various mixtures of resistant ans sensitive cells of three cell lines. Rhodamine 123 allows to detect 1% of resistant cells among sensitive cells. Influence of dead cells on the interpretation is discussed.
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PMID:[Value of rhodamine 123 in the detection of minor amounts of multidrug resistant cells]. 774 18

In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.
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PMID:Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines. 776 42

To optimize the immunohistochemical detection of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp) in chronic lymphoid disorders, the authors compared the sensitivity of three different monoclonal antibodies (MoAb) directed against P-gp (C219, JSB-1, and MRK 16) by using the APAAP technique on four tissue preparations obtained from lymphoid tumors: Cryostat sections, ModAMEX processed sections, frozen cytospin preparations, and fresh cytospin preparations. Tumor samples were obtained from patients with previously treated chronic lymphocytic leukemia (6 cases) or non-Hodgkin's malignant lymphoma (4 cases). Lymph nodes (n = 9), spleen (n = 3), and blood (n = 5) were analyzed. JSB-1 MoAb detected P-gp in 4 of 12 cases (33.3%) on either frozen sections or ModAMEX processed sections, and in 6 of 17 cases (35.3%) on frozen cytospin preparations. The sensitivity of JSB-1 was significantly improved when fresh cytospin preparations were used with an incidence of P-gp positive samples as high as 70.6% (P < .05). C219 MoAb was unreactive with lymphoid cells whatever the technique used, whereas this antibody stained stromal cells. MRK 16 MoAb was equally reactive to JSB-1 on fresh cytospin preparations, but unreactive when the other preparations were used. The specificity of JSB1 MoAb was confirmed by both Western blot analysis and Rhodamine 123 efflux assay. The authors used JSB-1 MoAb on fresh cytospin smears prepared from 28 CLL patients. Overall incidence of P-gp positive cases was 39.2%. Univariate analysis showed that P-gp expression was correlated with prior therapy, refractoriness to treatment, Rai stratification, and time of tissue storage after diagnosis. The authors recommend the use of JSB-1 on fresh cytospin preparations for the immunocytochemical detection of P-gp in chronic lymphoid disorders.
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PMID:Optimization of immunohistochemical detection of P-glycoprotein in chronic lymphoid disorders. 780 2

Rhodamine 123 (Rh123) is a fluorescent dye which locates in the mitochondria of cells. It is a substrate for P-glycoprotein (Pgp) and can, therefore, be used as a molecular probe in studies of the multidrug resistance (MDR) phenotype. However, not all MDR cells overexpress Pgp. In some, the MDR phenotype is associated with expression of an alternative transporter molecule, the multidrug resistance-associated protein (MRP). We have studied the accumulation and efflux of Rh123 in MDR cells having both Pgp-mediated and MRP-associated phenotypes. In the mouse tumour parental cell line, EMT6/P, Rh123 accumulates rapidly to reach plateau levels by 90 min. Confocal microscopy confirms a localisation to the mitochondria. In the MDR subline, EMT6/AR1.0, which overexpresses Pgp and which is 10-fold resistant to Rh123 cytotoxicity, accumulation is dramatically reduced. Efflux of Rh123 from both resistant and parental lines is rapid but can be inhibited by reduced temperature or by the presence of cyclosporin A (5 micrograms/ml). Efflux from the parental line is probably due to the presence of very low, but detectable, levels of Pgp but the existence of other mechanisms cannot be ruled out. In contrast, the human lung cancer parental cell line COR-L23/P, and its MRP-associated (but Pgp-negative) MDR subline, COR-L23/R (which is 23-fold resistant to Rh123 cytotoxicity), accumulate Rh123 at similar rates for the first 30 min. The curves then diverge so that, at 180 min, the resistant cells contain only 70% of the Rh123 of parental cells. Confocal microscopy demonstrates a similar distribution of fluorescence in resistant and parental cells. Essentially no efflux of Rh123 occurs from parental cells, whereas 70% of the content is lost from resistant cells over a period of 150 min. Such efflux may again be inhibited by reduced temperature but cyclosporin A (5 micrograms/ml) has little effect. These observations should be borne in mind when interpreting Rh123 efflux data in terms of MDR mechanisms.
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PMID:A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes. 799 26

Rhodamine 123 is a fluorescent dye that localizes in mitochondria, is a substrate for the multidrug resistance pump, and is retained for long periods of time by carcinoma cells. 17 beta-Estradiol causes GH4C1 cells (rat pituitary tumor cells) to lose rhodamine 123 fluorescence faster than untreated cells. We found that estradiol induces accumulation of the mRNA for the multidrug resistance pump 3-5-fold, with maximum induction occurring within 1 day at 10(-9) M estradiol. Immunoblot analysis demonstrated that estradiol induces a protein of 150 kDa that reacts with an antibody to P-glycoprotein, the multidrug resistance pump. The reduced retention of rhodamine 123 caused by estradiol is prevented by verapamil and cyclosporin, inhibitors of the pump. A clone resistant to the effects of estradiol on rhodamine 123 has greatly reduced levels of mRNA for the pump. The effect of estradiol is more marked on rhodamine 123 retention than it is on that of rhodamine 110 or tetramethylrhodamine methyl ester. We conclude that estradiol enhances rhodamine 123 efflux by inducing the multidrug resistance gene. The specificity for rhodamine 123, compared with other analogs, may be caused by differences in accessibility to the pump.
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PMID:Estradiol induction of rhodamine 123 efflux and the multidrug resistance pump in rat pituitary tumor cells. 842 69

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts. 852 64


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