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Detection of the M_r 190,000 Multidrug Resistance Protein, MRP, with Monoclonal Antibodies¹

Cancer Research Laboratories [D. R. H., S. D. G., R. G. D., S. P. C. C.] and Department of Pathology [D. R. H., R. G. D., S. P. C. C.], Queen's University, Kingston, Ontario, Canada K7L 3N6

MRP is a M_r 190,000 integral membrane phosphoglycoprotein that is overexpressed in some drug-selected resistant cell lines and has been shown to cause multidrug resistance in transfected cells. Five murine hybridoma cell lines (QCRL-1, QCRL-2, QCRL-3, QCRL-4, and QCRL-6) have been generated which secrete monoclonal antibodies (MAbs) that react specifically with membrane proteins of MRP-overexpressing, multidrug-resistant, drug-selected H69AR cells and MRP-transfected HeLa cells (T5) but not the respective parental (H69) and vector-transfected (C1) cells. The ability of three of these MAbs (QCRL-1, QCRL-2, and QCRL-3) to selectively immunoprecipitate a M_r 190,000 protein from ³⁵S-labeled H69AR and T5 membranes indicates that these MAbs are specific for MRP. MAb QCRL-1 is also capable of detecting the low levels of MRP present in revertant H69PR cells by immunoblot analysis. Indirect immunofluorescence analyses show that MAbs QCRL-1, QCRL-2, and QCRL-3 strongly and differentially react with fixed T5 and H69AR cells but not with unfixed cells, suggesting that these MAbs recognize intracellular MRP epitopes. The availability of reagents for the specific and sensitive immunodetection of MRP should greatly facilitate biological and clinical studies of this novel drug resistance protein.

¹ Supported by grants from the Medical Research Council of Canada (to S. P. C. C. and R. G. D.) and the Cancer Research Society (to S. P. C. C. and R. G. D.). D. R. H. is the recipient of a studentship from the Medical Research Council of Canada and was supported in part by a Queen's University Graduate Award. R. G. D. is the Stauffer Research Professor of Queen's University, and S. P. C. C. is a Career Scientist of the Ontario Cancer Foundation.

² To whom requests for reprints should be addressed, at Cancer Research Laboratories, Queen's University, 3rd Floor, Botterell Hall, Kingston, Ontario, Canada K7L 3N6.

Received 9/ 8/94. Accepted 10/ 6/94.

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