Ranking of P-glycoprotein substrates and inhibitors by a calcein-AM fluorometry screening assay

Tiberghien, Françoise; Loor, Francis

doi:10.1097/00001813-199607000-00012

Cited by 176 publications

(159 citation statements)

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“…Our findings, however, are consistent with one report that used another P-gp-specific mAb (MRK16) (31). Moreover, our finding of functional inhibition of calcein-AM dye efflux from purified CD4 ϩ T cells and monocytes by specific (Hyb-241 mAb) and pharmacologic P-gp inhibitors, a well-described function of P-gp in other cell types (30,32), confirmed that P-gp is functional in CD4 ϩ T cells and monocytes.…”

Section: Discussionsupporting

confidence: 93%

“…1B). We next examined whether the specific anti-P-gp mAb Hyb-241 and the pharmacologic P-gp inhibitor tamoxifen could inhibit the P-gp-mediated efflux of calcein-AM dye in purified CD4 ϩ T cells and CD14 ϩ APCs, a characteristic function of P-gp in other cell types (30). Serial fluorometry analyses of calcein-AM-loaded purified CD4 ϩ T cells over 90 min following calcein-AM loading revealed a mean cellular fluorescence loss of 47% in untreated or isotype mAbtreated controls vs 15% in tamoxifen (5 M)-and Hyb-241 mAb (20 g/ml)-treated cultures (Fig.…”

Section: Human T Cells and Apcs Express Functional Surface P-gpmentioning

confidence: 99%

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Specific MDR1 P-Glycoprotein Blockade Inhibits Human Alloimmune T Cell Activation In Vitro

Frank

Denton

Alexander

et al. 2001

The Journal of Immunology

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MDR1 P-glycoprotein (P-gp), the multidrug resistance-associated transmembrane transporter, is physiologically expressed by human peripheral immune cells, but its role in cell-mediated immunity remains poorly understood. Here, we demonstrate a novel role for P-gp in alloantigen-dependent human T cell activation. The pharmacologic P-gp inhibitor tamoxifen (1–10 μM) and the MDR1 P-gp-specific mAb Hyb-241 (1–20 μg/ml), which detected surface P-gp on 21% of human CD3+ T cells and 84% of CD14+ APCs in our studies, inhibited alloantigen-dependent, but not mitogen-dependent, T cell proliferation in a dose-dependent manner from 40–90% (p < 0.01). The specific inhibitory effect on alloimmune T cell activation was associated with >85% inhibition (p < 0.01) of IL-2, IFN-γ, and TNF-α production in 48-h MLR coculture supernatants. Addition of recombinant human IL-2 (0.1–10 ng/ml) restored proliferation in tamoxifen-treated cocultures. Pretreatment of purified CD4+ T cells with Hyb-241 mAb before coculture resulted in inhibition of CD4+ T cellular IFN-γ secretion. Also, blockade of P-gp on allogeneic APCs inhibited IL-12 secretion. Taken together these results demonstrate that P-gp is functional on both CD4+ T cells and CD14+ APCs, and that P-gp blockade may attenuate both IFN-γ and IL-12 through a positive feedback loop. Our results define a novel role for P-gp in alloimmunity and thus raise the intriguing possibility that P-gp may represent a novel therapeutic target in allograft rejection.

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Section: Discussionsupporting

confidence: 93%

Section: Human T Cells and Apcs Express Functional Surface P-gpmentioning

confidence: 99%

Specific MDR1 P-Glycoprotein Blockade Inhibits Human Alloimmune T Cell Activation In Vitro

Frank

Denton

Alexander

et al. 2001

The Journal of Immunology

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“…5 Because P-gp functions as a pump, its expression can be evaluated quickly and easily using a fluorescent substrate such as calcein-AM, which has been used to evaluate P-gp activity. 33,53 In a study comparing several different assays of P-gp, calcein-AM extrusion was found to be as reliable as immunological assays or drug sensitivity assessments in evaluating the drug resistance in several tumor cell lines. Our results indicate that liposomal MDR1 asODN formulated in CCL and stabilized by PEG-DSPE do not demonstrate significant activity against P-gp expression.…”

Section: Discussionmentioning

confidence: 99%

“…Calcein-acetoxymethylester (calcein-AM) (Molecular Probes, Eugene, Ore) is a lipid-soluble fluorogenic dye that has been shown to be a substrate for P-gp. 33 In non-P-gpexpressing cells, calcein-AM easily penetrates the cell membrane; once inside the cell, esterases cleave the ester bond, releasing calcein, which is hydrophilic and highly fluorescent. In P-gp expressing cells, calcein-AM is rapidly extruded from the plasma membrane, resulting in a decrease in the accumulation of fluorescent calcein.…”

Section: Down-regulation Of P-gp Activitymentioning

confidence: 99%

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1

2000

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The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo. Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung. We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle. The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN. The biodistribution and pharmacokinetics of an 18-mer 125 I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice. The majority of the asODN was cleared from blood, with a half-life of Ͼ10 hours compared with Ͻ1 hour for free asODN. When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein. No inhibition was found for nontargeted formulations or for free asODN. A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN. Cancer Gene Therapy (2000) 7, 466 -475

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“…[15][16][17] SDZ PSC 833 which is the most extensively studied of these, has proven to be well tolerated in phase I trials, [18][19][20] and is currently being investigated as an adjunct to cancer chemotherapy in phase II and III trials.…”

Section: Introductionmentioning

confidence: 99%

Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents

et al. 1999

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The multidrug transporter P-glycoprotein (Pgp), which is frequently overexpressed in multidrug resistant leukemia, has many proposed physiological functions including involvement in transmembraneous transport of certain growth-regulating cytokines. Therefore, we studied cell growth of three pairs of drug resistant and sensitive leukemia cell lines (KG1a, K562 and HL60) exposed to three different inhibitors of Pgp. The resistant KG1a and K562 sublines, which expressed high levels of Pgp, responded to low doses of the cyclosporin SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhibition. In the resistant variants of KG1a and K562 cells the mean half-maximal growth inhibitory doses (GI 50 ) of SDZ PSC 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280-446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 M SDZ PSC 833 resulted in tetraploidization, cytokinesis failure and apoptosis of the KG1a and K562 MDR variants. Conversely, parental cells with no or low levels of Pgp and the non-Pgp resistant variant of HL60 cells were not receptive to these cytotoxic effects. We conclude that inhibition of Pgp may exercise selective cytotoxicity in Pgp-rich leukemia cells indicating a possible therapeutic target in multiresistant leukemia.

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Ranking of P-glycoprotein substrates and inhibitors by a calcein-AM fluorometry screening assay

Cited by 176 publications

References 0 publications

Specific MDR1 P-Glycoprotein Blockade Inhibits Human Alloimmune T Cell Activation In Vitro

Specific MDR1 P-Glycoprotein Blockade Inhibits Human Alloimmune T Cell Activation In Vitro

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1

Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents

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