Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.
BACKGROUND
To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively.


METHODS
ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter.


RESULTS
ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays.


CONCLUSIONS
Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters.
García-Escarp
Marta
M
Unitat de Diagnòstic i Teràpia Molecular, Centre de Transfusió i Banc de Teixits, Barcelona, Spain.
Martínez-Muñoz
Vanessa
V
Sales-Pardo
Irene
I
Barquinero
Jordi
J
Domingo
Joan Carles
JC
Marin
Pedro
P
Petriz
Jordi
J
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007624:KB Cells; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins