By Lucas Armstrong, Richard Sullivan and Eugene Mechetner
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4°C
• MDR1 inactive
• Dye (circles) retained
• High fluorescence |
DiOC2(3)
Rhodamine 123
 |
|
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37°C
• MDR1 active
• Dye efflux
• Low fluorescence |
DiOC2(3)
Rhodamine 123
 |
|
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37°C + vinblastine
• MDR1 active
• Excess vinblastine (triangles) competitively blocks dye efflux
• High fluorescence |
DiOC2(3)
Rhodamine 123
 |
|
| Figure 1. Direct Dye Efflux Assay. MDR1-overexpressing K562/I-S9 cells were loaded with DiOC2(3) or rhodamine 123 and incubated at 4 C, or at 37 C in the presence or absence of vinblastine. After washing, cells were analyzed by flow cytometry. Cells incubated at 4 C exhibit high fluorescence, as MDR1 is inactive at low temperatures and the cells retain the dye. Cells incubated at 37 C with DMSO (diluent control) have low fluorescence, as MDR1 is active and cells efflux the dye. Cells incubated at 37 C in the presence of vinblastine, a specific MDR1 inhibitor, have high fluorescence, as vinblastine competes with the dye for efflux by MDR1. |
Intrinsic and acquired drug resistance to various classes of chemically diverse chemotherapy agents (referred to as "MultiDrug Resistance", MDR) represents the major obstacle in modern cancer chemotherapy. The effects of the majority of the most commonly used chemotherapy agents are hindered by MDR mechanisms expressed in cancer cells. Although multiple mechanisms mediate MDR, the first mediator of clinical multidrug resistance to be characterized at the molecular level was the MDR1 gene product, also known as P-glycoprotein (Pgp). Pgp consists of 12 transmembrane domains that form a drug-binding pore, and two cytoplasmic ATP-binding domains. This molecular structure is typical for the members of the ATP-binding cassette (ABC) protein family. Over fifty ABC proteins have been characterized to date. Two of these proteins, MRP1 and BCRP, are also involved in clinical resistance to chemotherapy drugs. With over 1.3 million new cancer cases expected in the US in 2004, over 530,000 patients will succumb to the disease, primarily due to the failure of chemotherapy protocols that involve one or, more often, several anti-tumor agents. Overall, the MDR1 phenotype is found in approximately 50% of cancer cases across the board. That is why MDR research focused on the development of new anti-tumor pharmaceuticals and treatment protocols is currently one of the focal points of attention of the international cancer research and biotechnology communities.
To further facilitate studies in this important area, Chemicon International (Temecula, Calif.) has recently launched a series of three flow cytometry assays for the detection and quantitative characterization of the functional activity exhibited by different MDR membrane pumps.
The main features of these functional MDR assays are user friendliness and reproducibility. To attain low inter- and intra-test variability and eliminate discrepant results from different research laboratories, all the necessary reagents and buffers are included in the kits, as well as detailed protocols and troubleshooting instructions. Comprehensive descriptions of the expected patterns of substrate specificity for the known members of the ABC superfamily of membrane transporters are also included. Another important characteristic of these kits is their compatibility with other methods of MDR detection, e.g. multicolor flow cytometric detection of the expression of various MDR proteins using surface immunostaining techniques. Finally, the assays are time- and cost efficient and formatted for immediate use in any research facility equipped with a flow cytometer.
Direct Dye Efflux Assay The Multidrug Resistance Direct Dye Efflux Assay was designed to assess the functional activity of the three major clinical MDR mechanisms (MDR1, MRP, BCRP) using fluorescent dyes transported by these pumps as differential flow cytometry probes. Chemicon's Direct Dye Efflux Assay can be used to determine relative levels of MDR mediated functional activities in living cells, as well as the ability of potential MDR modulators to interfere with the efflux function of these ABC transporters (see Figure 1).
The major advantage of this assay is that its end-point, direct drug efflux from living cells, is the best approximation of the in vivo situation in normal tissues and tumors that may simultaneously express multiple MDR mechanisms in one cell.
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Figure 2. Principle of MultiDrugQuant™ Assay. Due to the transport activity of MDR proteins, low intracellular dye accumulation is observed in MDR expressing cells (A). Inhibition of the Calcein AM extrusion by MDR inhibitor or MDR substrate in excess results in higher intracellular accumulating of the dye (B).
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Figure 3. Analysis of transport substrate-induced conformational change of MDR1 with the MDR1 Shift Assay. MDR1-overexpressing K562/I-S9 cells were incubated at 37 C in the presence of vinblastine or vehicle control DMSO. Control IgG2a or conformation selective anti-MDR1 monoclonal UIC2 were added and further incubated at 37 C. Cells were then incubated with phycoerythrin labeled anti-mouse IgG and washed. Binding of UIC2 was evaluated by flow cytometry. Cells incubated with control IgG2a (thin solid line) serve as a background control. Cells incubated with DMSO (diluent) and UIC2 (dotted line) show specific staining with the UIC2 antibody, while cells incubated in vinblastine and UIC2 (thick solid line) display higher fluorescence due to increased binding of this antibody to MDR1 in its active conformation. |
The MultiDrugQuant™ Assay The MultiDrugQuant™ dye accumulation assay was developed for the quantitative flow cytometric analysis of MDR1 (ABCB1) and MRP1 (ABCC1) resistance phenotypes. In this test, living target cells are exposed to a non-fluorescent compound, calcein-acetoxymethylester (calcein AM), which crosses the cell membrane by passive diffusion and is converted into a fluorescent dye, calcein, by cytoplasmic and mitochondrial esterases. In the absence of efflux activity, free calcein accumulates within the cell resulting in a 100- to 500-fold increase in the intracellular concentration of the dye and bright fluorescence exhibited by living target cells. If calcein AM is extruded from the cell by a membrane transporter, the intracellular concentration of the dye is decreased, depending on the activity of the efflux pump (see Figure 2). This phenomenon enables discrimination between MDR-positive and MDR-negative cell populations. Furthermore, if a drug present in the tested medium inhibits the efflux function, the trapped calcein is accumulated in the cytoplasm resulting in enhanced cellular fluorescence, thereby making possible the examination of drug effects on MDR mechanisms.
Conformational MDR Shift Assay for Simultaneous Quantification of MDR1 Expression and Function To address the problem of discriminatory MDR1 detection on the background of the other MDR phenotypes, Chemicon has introduced a new test, the MDR1 Shift Assay. This flow cytometry assay allows for highly selective quantitative detection of Pgp and function in human MDR1 cells. The test is based on the increased reactivity of a functional monoclonal antibody, UIC2, in the presence of MDR1 transport substrates at physiologic conditions (see Figure 3).
Based on a conformation-specific UIC2 antibody, Chemicon's MDR1 Shift Assay is unique in that it can be used for simultaneous determination of MDR1 expression and functional activity without detecting any nonspecific signals from other members of the ABC superfamily. It is ideal for screening and characterizing MDR1 substrates and modulators. Another advantage of this test is its enhanced sensitivity of P-glycoprotein expression, as compared with the conventional immunostaining.
About the authors The authors are with Chemicon International. Lucas Armstrong is a Senior Scientist, Richard Sullivan is a Research Assistant, and Eugene Mechetner is Senior Director of Corporate Development and Scientific Affairs. He also serves as an Adjunct Associate Professor in the Department of Radiological Sciences of the University of California, Irvine.
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