Flow cytometric analysis of breast cancer resistance protein expression and function.
BACKGROUND
The breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) half-transporter that mediates energy-dependent drug efflux. Assessing the clinical relevance of the BCRP will require sensitive and specific methods for detecting its expression and function that allow high-volume specimen throughput and employ widely available instrumentation.


METHODS
The BXP-34 and BXP-21 monoclonal antibodies were evaluated for flow cytometric detection of BCRP expression. The modulation of efflux of rhodamine-123, 3,3'-diethyloxacarbocyanine iodide, doxorubicin, and mitoxantrone by fumitremorgin C was studied as an assay for BCRP function in BCRP-overexpressing cell lines and controls.


RESULTS
BXP-34 and BXP-21 allowed detection of BCRP expression by flow cytometry in all BCRP-expressing cell lines. Mitoxantrone was the only substrate transported by BCRP in all lines, and with mitoxantrone at a 3-microM concentration, light emission (>670 nm) caused by excitation at 488 nm was sufficiently intense to allow detection of differences in retention associated with low levels of BCRP expression.


CONCLUSIONS
Immunophenotyping with BXP-21 or BXP-34 and fumitremorgin C modulation of mitoxantrone retention allow detection of BCRP expression and function by flow cytometry with standard instrumentation. These assays will facilitate determination of the role of BCRP in clinical drug resistance.
Minderman
Hans
H
Leukemia Section, Department of Medicine, Roswell Park Cancer Institute, Science Building 616, Elm and Carlton Streets, Buffalo, NY 14263, USA. hans.minderman@roswellpark.org
Suvannasankha
Attaya
A
O'Loughlin
Kieran L
KL
Scheffer
George L
GL
Scheper
Rik J
RJ
Robey
Robert W
RW
Baer
Maria R
MR
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D000970:Antineoplastic Agents / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D008942:Mitoxantrone / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000276:immunology; D014407:Tumor Cells, Cultured
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D008942:Mitoxantrone