A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate Efflux.
Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).
University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA.
Dominique
D
Perez
University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA.
Peter C
PC
Simons
University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA.
Yelena
Y
Smagley
University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA. LSklar@salud.unm.edu.
Larry A
LA
Sklar
University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA.
Alexandre
A
Chigaev
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007938:Leukemia / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D000242:Cyclic AMP
ABC transporters; Cyclic AMP; Efflux inhibitors; Fluorescent substrates; High-throughput flow cytometry