Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies.
We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
A H
AH
Schinkel
E M
EM
Roelofs
P
P
Borst
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002999:Clone Cells; D003429:Cross Reactions; D015342:DNA Probes; D003630:Daunorubicin / Q000378:metabolism / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics*; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D008545:Melanoma; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D015342:DNA Probes; D008562:Membrane Glycoproteins; D003630:Daunorubicin