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MDR1 Shift Assay
Description:
MDR1 Shift Assay
Trade Name:
Chemicon (Millipore)
Qty/Pk:
50 assays
Product Overview:
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 cell into the extracellular space. The molecular structure of MDR1 consists of 12 transmembrane domains that form a drug-binding pore, and two ATP-binding sites. Subsequently, a family of at least nine proteins related to MDR1 have been characterized and shown to mediate efflux of small molecules from cells (Gottesman et al., 2002). 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. The role of MDR1 in solid tumors has been more difficult to discern, due to variations in methods of detection of MDR1 in tissues by immunohistochemical methods, and efforts have been made to standardize methods for MDR1 detection (Beck et al., 1996). In addition, in vivo imaging of MDR1-mediated efflux with the radiological MDR1 substrate, 99mTc (technetium)-sestamibi, indicates that MDR1 is active in solid tumors of different origin (Gottesman et al., 2002).
MDR1 is also present in several 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).
Efforts to characterize the mechanism of action and to detect active MDR1 have been aided by the production of the UIC2 monoclonal antibody against MDR1. UIC2 inhibits the activity of MDR1 and preferentially recognizes MDR1 that is in the process of transporting substrates, as detected by the flow cytometry method employed in the MDR1 Shift Assay (Mechetner et al., 1997; Figure 1). UIC2 appears to preferentially bind to MDR1 that has completed its catalytic cycle and hydrolyzed ATP, as increased immunoreactivity is also observed in cells that have been depleted of ATP by treatment with sodium azide (Mechetner et al., 1997). Some UIC2 paratopes appear to reside in the extracellular loop between transmembrane segments 5 and 6 (Zhou et al, 1999). As high resolution structural information about MDR1 transport mechanisms is not available, the UIC2 antibody provides a useful tool for probing the mechanism of MDR1-mediated efflux. In addition, the UIC2 antibody has been used to characterize the mechanisms of action of MDR1 inhibitors such as cis-(Z)-flupentixol (Maki et al., 2003), which will aid in the development of drugs for circumventing multidrug resistance in cancer.
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Applications:
This MDR1 Shift Assay provides a convenient method to probe conformational changes in MDR1 that occur upon transport of MDR1 substrates.
Application Notes:
The Millipore MDR1 Shift |