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Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes
Stephanie E Schleis1, Amy K LeBlanc, Nancy R Neilsen
1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
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.
