• 1.

    MacEwen EG. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer Metastasis Rev 1990;9:125136.

  • 2.

    Moore A, Leveille C, Reimann K, et al. The expression of P-glycoprotein in canine lymphoma and its association with multidrug resistance. Cancer Invest 1995;13:475479.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Page RL, Hughes C, Huyan S, et al. Modulation of P-glycoprotein-mediated doxorubicin resistance in canine cell lines. Anticancer Res 2000;20:35333538.

    • Search Google Scholar
    • Export Citation
  • 4.

    Vail DM. Hematopoietic tumors. In: Ettinger SJ, Feldman EC, eds. Textbook of veterinary internal medicine. 5th ed. Philadelphia: WB Saunders Co, 2000;507523.

    • Search Google Scholar
    • Export Citation
  • 5.

    Thomas H, Coley H. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting P-glycoprotein. Cancer Causes Control 2003;10:159165.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Conrad S, Vietelhaus A, Orzechowski A, et al. Sequencing and tissue distribution of the canine MRP2 gene compared with MRP1 and MDR1. Toxicology 2000;156:8191.

    • Search Google Scholar
    • Export Citation
  • 7.

    Cui Y, Konig J, Buchholz J, et al. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 1999;55:929937.

    • Search Google Scholar
    • Export Citation
  • 8.

    Culmsee K, Gruber A, Von Samson-Himmerlstjerna G, et al. Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcriptase quantitative polymerase chain reaction. Res Vet Sci 2004;77:223229.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Ginn PE. Immunohistochemical detection of P-glycoprotein in formalin-fixed and paraffin-embedded normal and neoplastic canine tissues. Vet Pathol 1996;33:533541.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Mealey KL, Bentjen S, Gay J, et al. Dexamethasone treatment of a canine, but not human, tumor cell line increases chemoresistance independent of P-glycoprotein and multidrug resistance-related protein expression. Vet Comp Oncol 2003;1:6775.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    van der Kolk D, de Vries E, van Putten W, et al. P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia. Clin Cancer Res 2000;6:32053214.

    • Search Google Scholar
    • Export Citation
  • 12.

    Ma L, Pratt S, Cao J, et al. Identification and characterization of the canine multidrug resistance-associated protein. Mol Cancer Ther 2002;1:13351342.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lee J, Hughes C, Fine R, et al. P-glycoprotein expression in canine lymphoma: a relevant, intermediate model of multidrug resistance. Cancer 1996;77:18921898.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Bergman P, Ogilvie G, Powers B. Monoclonal antibody C219. Immunohistochemistry against P-glycoprotein: sequential analysis and predictive ability in dogs with lymphoma. J Vet Intern Med 1996;10:354359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Steingold SF, Sharp N, McGahan M, et al. Characterization of canine MDR1 mRNA: its abundance in drug resistance cell lines and in vivo. Anticancer Res 1998;18:393400.

    • Search Google Scholar
    • Export Citation
  • 16.

    Brugger D, Herbart H, Gekeler V, et al. Functional analysis of P-glycoprotein and multidrug resistance associated protein related multidrug resistance in AML-blasts. Leuk Res 1999;23:467475.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Dogan A, Legrand O, Faussat A-M, et al. Evaluation and comparison of MRP1 activity with three fluorescent dyes and three modulators in leukemia cell lines. Leuk Res 2004;28:619622.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Egashira M, Kawamata N, Sugimoto K, et al. P-glycoprotein expression on normal and abnormal expanded natural killer cells and inhibition of p-glycoprotein function with cyclosporine A and its analogue, PSC833. Blood 1999;93:559606.

    • Search Google Scholar
    • Export Citation
  • 19.

    Laupeze B, Amiot L, Courtois A, et al. Use of the anionic dye carboxy-2', 7'-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity. Int J Oncol 1999;15:571576.

    • Search Google Scholar
    • Export Citation
  • 20.

    Meaden E, Hoggard P, Khoo S, et al. Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo. J Immunol Methods 2002;262:159165.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Rhaaijimakers H, van den Bosch G, Boezeman J, et al. Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors. Cytometry 2002;49:135142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Barrand MA, Bagrij T, Neo SY. Multidrug resistance-associated protein: a protein distinct from P-glycoprotein involved in cytotoxic drug expulsion. Gen Pharmacol 1997;28:639645.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Williamson G, Aeberli I, Miguet L, et al. Interaction of positional isomers of quercetin glucuronides with the transporter ABCC2 (cMOAT, MRP2). Drug Metab Dispos 2007;35:12621268.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Gekeler V, Ise W, Sanders KH, et al. The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Biophys Res Commun 1995;208:345352.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Fan TM. Lymphoma updates. Vet Clin North Am Small Anim Pract 2003;33:455471.

  • 26.

    Ruslander D, Gebhard D, Tompkins M, et al. Immunophenotypic characterization of canine lymphoproliferative disorders. In Vivo 1997;11:169172.

    • Search Google Scholar
    • Export Citation

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Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes

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  • 1 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.
  • | 2 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.
  • | 3 Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.
  • | 4 Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

Abstract

Objective—To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance–associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes.

Sample Population—Nodal lymphocytes from 21 clinically normal dogs.

Procedures—Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors.

Results—Expression of P-gp was (mean ± SEM) 50.62 ± 13.39 (n = 21) and that of MRP was 2.16 ± 0.25 (13). Functional activity was 1.27 ± 0.06 (n = 21) for P-gp and both inhibitors and 21.85 ± 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated.

Conclusions and Clinical Relevance—Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.

Abstract

Objective—To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance–associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes.

Sample Population—Nodal lymphocytes from 21 clinically normal dogs.

Procedures—Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors.

Results—Expression of P-gp was (mean ± SEM) 50.62 ± 13.39 (n = 21) and that of MRP was 2.16 ± 0.25 (13). Functional activity was 1.27 ± 0.06 (n = 21) for P-gp and both inhibitors and 21.85 ± 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated.

Conclusions and Clinical Relevance—Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.

Contributor Notes

Supported by Novartis/Pharma Incorporated by the generous gift of PSC833.

Address correspondence to Dr. Casey LeBlanc.