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MDR1 Efflux Assay
Description:
MDR1 Efflux Assay
Trade Name:
Chemicon (Millipore)
Qty/Pk:
100 assays
Product Overview:
Introduction
The phenomenon of resistance of tumors to chemically unrelated anticancer drugs, termed multidrug resistance, represents the most formidable challenge to the field of oncology. Multidrug resistance can be present at the time of diagnosis, or can be acquired after initial treatment and remission of a cancer. Although multiple mechanisms mediate multidrug resistance, the first mediator of multidrug resistance to be characterized at the molecular level was MDR1, also known as P-glycoprotein (Pgp) and ABCB1 (Gottesman et al., 2002). MDR1 mediates resistance to various classes of chemotherapeutic agents, including vinca alkaloids (vinblastine and vincristine), anthracyclines, paclitaxel and etoposide, by actively pumping the drugs from the cytosol and plasma membrane into the extracellular space. The molecular structure of MDR1 consists of 12 transmembrane domains that form a drug-binding pore, and two cytoplasmic ATP-binding cassettes. At least nine proteins related to MDR1 have been characterized to date and shown to mediate efflux of small molecules from cells (Gottesman et al., 2002). Two of these MDR1 relatives, multidrug-resistance-associated protein 1 (MRP1, or ABCC1) and breast cancer resistance protein (BCRP, or ABCG2), have also been demonstrated to mediate multidrug resistance in tumor cells. These proteins belong to a larger family of ABC (ATP-binding cassette) proteins that function as transporters of ions, nutrients, and peptides.
The clinical importance of MDR1-mediated multidrug resistance has been best characterized in acute myelogenous leukaemia (Gottesman et al., 2002). The role of MDR1 in solid tumors has been more difficult to discern, due to variations in methods of detection of MDR1 in tissues. Multiple efforts have been made to standardize methods for MDR1 detection using flow cytometry, immunohistochemistry and in situ hybridization (Beck et al., 1996). It has been estimated that at least 50% of human cancers express the MDR1 phenotype. In vivo imaging of MDR1-mediated efflux with the radiological MDR1 substrate, 99mTc (technetium)-sestamibi, indicates that MDR1 is active in several cancer types.
MDR1 activity is also observed in various cell types in normal tissues. Brain microvascular endothelial cells express MDR1, which contributes to the blood-brain barrier. It was proposed that expression of MDR1 in hematopoietic stem cells, intestine, and reproductive tissues (testicular endothelium and placental syncytiotrophoblast) protects these cells from the detrimental effects of xenobiotics. MDR1 tissue distribution suggests that it has a role in cholesterol and steroid metabolism. Several subsets of immune cells also express MDR1 (Gottesman et al., 2002). MRP1 is widely expressed, and has physiological significance in transporting anionic xenobiotics and metabolites in lung, placenta, choroid plexus in the brain, and Sertoli cells in testes.
Assessment of activity of MDR1, MRP1 and BCRP in cultured cells has been facilitated by the observation that several fluorescent small molecules, such as DiOC2(3), rhodamine 123, and calcein AM, serve as substrates for MDR1 and its relatives (Figure 1). DiOC2(3) is highly specific for MDR1, and is not transported by the related multidrug resistance protein, MRP1 (Minderman et al., 1996; Table 1). Rhodamine 123 is effluxed by MDR1 and to a lesser extent by MRP1, and thus serves as a more broad indicator of total cellular efflux activity. Another member of the ABC family, breast cancer resistance protein (BCRP), weakly transports DiOC2(3), but does not transport Rhodamine 123 (Minderman et al., 2002; Table 1). Efflux of dyes can be inhibited by nonfluorescent transport substrates such as vinblastine (Figure 1). Dye efflux assays have proven to be instrume |