A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate Efflux
| Autor: | Alexandre Chigaev, Peter C. Simons, Yelena Smagley, Dominique Perez, Larry A. Sklar |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cell Drug Evaluation Preclinical ATP-binding cassette transporter Article Flow cytometry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cell Line Tumor medicine Cyclic AMP Humans Cyclic adenosine monophosphate Fluorescent Dyes Leukemia medicine.diagnostic_test Chemistry Biological Transport Flow Cytometry High-Throughput Screening Assays Cytolysis 030104 developmental biology medicine.anatomical_structure Biochemistry Cytoplasm 030220 oncology & carcinogenesis Cancer cell ATP-Binding Cassette Transporters Efflux |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493936717 |
| ISSN: | 1940-6029 |
| Popis: | 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 pump 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 (1). 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). |
| Databáze: | OpenAIRE |
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