Multidrug Resistance

The phenomenon of resistance of tumors to chemically unrelated anticancer drugs, termed multidrug resistance, represents the most formidable challenge in the field of oncology. Multidrug resistance can be present at the time of diagnosis, or can be acquired after initial treatment and during remission of a cancer. Although multiple mechanisms mediate multidrug resistance, 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). MDR1 mediates resistance to various classes of chemotherapeutic agents, including vinca alkaloids, anthracyclines, paclitaxel and etoposide, by actively pumping drugs from the cytosol through the plasma membrane into the extracellular space. 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.

MultiDrugQuant™ Assay

Discriminate between MDR1 and MRP1 positive and negative populations
Examine drug effects on MDR mechanisms
Flow cytometry platform provides quantitative results

The MultiDrugQuant Assay* is designed for the functional determination of multidrug resistance in living tumor cells. The kit provides a fast, sensitive and quantitative method for measuring the drug transport activity of the clinically most important multidrug resistance proteins: MDR1 (P-glycoprotein) and MRP1. The assay is based on determining fluorescence intensities using a flow cytometer, after a short in vitro incubation of the cell suspension with the dye calcein-acetoxymethyl ester (calcein AM), in the presence or absence of selective inhibitors of MDR1 and MRP1. This testing approach provides a separate measure of multidrug resistance for both MDR1 and MRP1.

Kit Components

Calcein AM
Inhibitor 1 (an inhibitor for both
MDR1 and MRP1-mediated
dye extrusion)
Inhibitor 2 (a specific inhibitor
of MRP1-mediated
dye extrusion)
Propidium Iodide
DMSO • Reaction Buffer
NaHC03 powder

	Due to the transport activity of MDR proteins, low intracellular dye accumulation is observed in MDR expressing cells.
	Inihibition of the Calcein AM extrusion by MDR inhibitor or substrate in excess results in higher intracellular dye accumulation.

MultiDrug Resistance Direct Dye Efflux Assay

Measure end-point dye efflux activity of MDR1, MRP, and BCRP transporters
Flow cytometry platform provides quantitative results

The Multidrug Resistance Direct Dye Efflux Assay is designed to assess the functional activity of the three major MDR mechanisms using fluorescent dyes transported by these pumps as differential flow cytometry probes. This assay directly measures the relative levels of MDR1, MRP1, and BCRP efflux activity in living cells, as well as the ability of potential MDR modulators to interfere with the function of these ABC transporters. The major advantage of this assay is that it provides a good approximation of in vivo conditions in normal tissues and tumors where multiple MDR membrane pumps are simultaneously expressed. The kit includes two of the best characterized multidrug resistance ABC transporter substrates, DiOC₂(3) and Rhodamine 123, enabling researchers to measure relative levels of MDR1, MRP, and BCRP activity in different cell populations. The kit also includes a specific inhibitor vinblastine, which is a substrate for MDR1, that competitively blocks efflux of DiOC₂(3) and Rhodamine 123.

Kit Components

Sterile RPMI-1640
Sterile 30% BSA
Gentamicin
DiOC2(3)
Rhodamine 123
Vinblastine
DMSO
Propidium Iodide

MDR1 Shift Assay

Selective quantitation of MDR1 among other MDR phenotypes
Screening and characterization of MDR1 substrates and modulators

To address the problem of discriminatory MDR1 detection on the background of the other MDR phenotypes, Millipore offers the MDR1 Shift Assay. Based on the increased reactivity of a functional monoclonal antibody, UIC2, in the presence of MDR1 transport substrates, this assay allows for simultaneous quantification of P-glycoprotein (Pgp) expression and functional activity without detecting nonspecific signals from other members of the ABC superfamily. UIC2 is a conformation-specific antibody that inhibits the activity of Pgp and preferentially recognizes Pgp that is in the process of transporting substrates. The UIC2 mouse monoclonal antibody (Millipore cat. no. MAB4334) against human Pgp is also available separately and is suitable for immunohistochemistry, flow cytometry, and functional studies.

Kit Components

Anti-MDR1 Monoclonal UIC2
Mouse Isotype Control
Anti-Mouse IgG-PE Secondary Antibody
Vinblastine • DMSO Sterile PBS
Sterile 30% BSA Propidium Iodide


MDR1-expressing cells in the absence of an MDR1 transport substrate have Pgp in a “closed” conformation that binds but does not hydrolyze ATP. The UIC2 antibody binds to this Pgp conformation with relatively low affinity, resulting in lower fluorescent staining.	MDR1-expressing cells in the presence of the nonfluorescent MDR1 transport substrate vinblastine (triangles) have Pgp in an “open” conformation that hydrolyzes ATP. The UIC2 antibody binds to Pgp in this conformation with higher affinity. As a result, the cells demonstrate higher fluorescence.