MDR1 Efflux Assay; 100 assays

MDR1 Efflux Assay

Chemicon (Millipore)

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 instrumental in screening for compounds that inhibit activity of MDR1 and are thus likely to overcome MDR1-mediated multidrug resistance. In addition, in trials of MDR1 inhibitors in vivo, dye efflux analysis of CD56+ cells isolated from patients after treatment with MDR1 inhibitors permits assessment of intracellular levels of the inhibitor (Robey et al., 1999).

The Multidrug Resistance Direct Dye Efflux Assay Kit includes two of the best characterized and most commonly used multidrug resistance ABC transporter substrates, DiOC2(3) and rhodamine 123. The kit enables researchers to directly assess the functional activity of the MDR1, MRP1 and BCRP membrane pumps in living cells under physiologic conditions by directly measuring the relative fluorescence of cell populations that actively extrude fluorescent multidrug resistance transport substrates.

The Multidrug Resistance Direct Dye Efflux Activity Kit is designed to directly measure the functional activity of MDR1, MRP1 and BCRP by assaying for the ability of the cell to extrude fluorescent transport substrates of these proteins. The kit can be used to measure relative amounts of MDR1, MRP and BCRP activity between different cell populations. In addition, dye efflux assays can be used to assess the ability of unlabeled small molecules to serve as transport substrates for MDR1, MRP1 and BCRP and the ability of candidate inhibitors to block the function of these proteins.

Two conveniently formulated solutions of MDR1, MRP1 and BCRP substrate dyes, DiOC2(3) Solution and Rhodamine 123 Solution, are included in the kit to give the investigator flexibility in the substrate specificity of the efflux probe. In addition, a solution of a nonfluorescent substrate of MDR1 and MRP1, Vinblastine Solution, is provided to serve as a competitive inhibitor of dye efflux. DMSO is included as a diluent control for the Vinblastine Solution. Sterile, concentrated RPMI-1640 and BSA are provided as components for buffer for the loading, efflux and washing steps of the procedure. A sterile, concentrated solution of gentamicin, which has been shown to not be a substrate for MDR1 (Mechetner and Roninson, 1992), is supplied as an optional antibiotic for the buffer. Propidium iodide solution is included to identify dead cells and exclude them from the analysis.

The extent of efflux of fluorescent dyes is best analyzed by flow cytometry. The assay can also be incorporated with immunostaining experiments for multicolor analysis of cell surface marker expression and MDR1, MRP1 and BCRP function. Efflux mediated by MDR1, MRP1 and BCRP can also be quantified by analysis under a fluorescence microscope or on a fluorometric plate reader.

The efflux activity of MDR1 and its relatives is highly temperature sensitive. MDR1 functions optimally near 37ºC, but is effectively inactive at 4ºC. MDR1-expressing cells preloaded with MDR1 fluorescent substrates retain the dye and consequently have high fluorescence when incubated at 4ºC (Figure 1, 4ºC, top panels). Conversely, cells incubated at 37ºC more readily efflux the dye and show reduced fluorescence (Figure 1, 37ºC + DMSO, middle panels). The kit also includes a specific inhibitor vinblastine, which is also a substrate for MDR1 and competitively blocks efflux of DiOC2(3) and Rhodamine 123. Inclusion of excess vinblastine in the efflux reaction at 37ºC therefore results in high fluorescence (Figure 1, 37ºC + vinblastine, bottom panels).

For Research Use Only; Not for use in diagnostic or in vivo procedures

Mammals

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Cancer Kits

• Sterile 5x RPMI-1640 (Part No. 90302) - One bottle containing 200 ml of sterile 5x concentrated RPMI-1640.
• Sterile 30% BSA (Part No. 90303) - One bottle containing 35 ml sterile-filtered 30% bovine serum albumin (BSA) in PBS.
• Gentamicin Solution, 1000x (Part No. 90353) - One vial containing 1 ml sterile-filtered 50 mg/ml gentamicin.
• DiOC2(3) Solution (Part No. 90299) - One vial containing 50 μl of DiOC2(3) (3,3'-diethyloxacarbocyanine iodide) at 1 mg/ml in DMSO.
• Rhodamine 123 Solution (Part No. 90300) - One vial containing 0.5 ml of rhodamine 123 at 1 mg/ml in DMSO.
• Vinblastine Solution (Part No. 90301) - One vial containing 150 μl of 22 mM vinblastine in DMSO.
• DMSO (Part No. 90294) - One vial containing 150 μl DMSO.
• Propidium Iodide Stock Solution (Part No. 90296) - One vial containing 1 ml of 50 μg/ml propidium iodide in PBS.

Kit components should be stored at 2-8°C upon arrival. Product is stable for 1 year after receipt. DiOC2(3) Solution, Rhodamine 123 Solution, Vinblastine Solution, and DMSO will freeze at 2-8°C, and can be thawed and refrozen at least 5 times. Protect DiOC2(3), Rhodamine 123 and Propidium Iodide Solutions from light.

Precautions
Propidium Iodide, Gentamicin, Vinblastine, Rhodamine 123 and DiOC2(3) may have toxic effects if handled improperly. DMSO is absorbed through the skin and can facilitate internalization of toxic agents. Use caution when handling each of these components. Standard precautions include wearing gloves and a lab coat.

Fluorescent

• ABCB1
• PGY1
• MGC163296
• CD243
• GP170
• P-gp
• MDR1
• CLCS
• ABC20
• EC 3.6.3.44

Cell line for testing

Hemocytometer

Sterile water, tissue culture grade

Sterile 1 liter bottle

37°C water bath with a rack to hold test tubes of choice

Ice and ice bucket

Centrifuge capable of 200 x g. A refrigerated centrifuge is recommended but not required

Extra wash buffer (RPMI-1640 or other medium, containing 1% BSA or 2% fetal calf serum), if further staining with an antibody is incorporated into the assay

Flow cytometer or fluorescence plate reader

(Optional) Black-walled 96-well plates, for analysis of samples using fluorescence plate reader.