Analysis of P-glycoprotein-mediated membrane transport in human peripheral blood lymphocytes using the UIC2 shift assay.
BACKGROUND
During transport-associated adenosine triphosphate hydrolysis, P-glycoprotein (Pgp) undergoes conformation transitions detected by UIC2, a functional anti-Pgp monoclonal antibody. A newly developed UIC2 shift assay is based on increased UIC2 reactivity in the presence of Pgp substrates. All peripheral blood leukocytes express low Pgp levels. The existing antibody-based detection methods are limited in their sensitivity and require additional techniques to simultaneously analyze Pgp expression and efflux, making it difficult to ascertain the physiologic role of Pgp-mediated transport.


METHODS
We validated the UIC2 shift assay against UIC2 immunostaining and DiOC(2) efflux. The UIC2 shift assay was then used to characterize Pgp functional expression and its physiologic substrates in peripheral blood leukocytes.


RESULTS
A strong correlation was observed between the UIC2 shift assay versus immunostaining and dye efflux tests. The UIC2 shift assay showed improved sensitivity (compared with conventional UIC2 staining) and allowed for simultaneous detection of Pgp expression and function. Using this assay, we identified several new Pgp substrates, including monensin and retinol, and confirmed that interleukin-2 and interferon-gamma can be transported by Pgp.


CONCLUSIONS
Our findings validate the use of the UIC2 shift assay in MDR1 detection and support the idea that Pgp plays a physiologic role in immunoregulation.
Park
Suk W
SW
Ingenex, Inc., Menlo Park, California, USA.
Lomri
Noureddine
N
Simeoni
Luiz A
LA
Fruehauf
John P
JP
Mechetner
Eugene
E
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology; D002232:Carbocyanines; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D008985:Monensin / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology; D014801:Vitamin A / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D002232:Carbocyanines; D007376:Interleukin-2; D014801:Vitamin A; C081554:3,3'-diethyloxacarbocyanine; D007371:Interferon-gamma; D008985:Monensin