Measurement of Multiple Drug Resistance Transporter Activity in Putative Cancer Stem/Progenitor Cells

Hillman Cancer Center, Pittsburgh, PA, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 568:261-79. DOI: 10.1007/978-1-59745-280-9_17
Source: PubMed


Multiple drug resistance, mediated by the expression and activity of ABC-transporters, is a major obstacle to antineoplastic therapy. Normal tissue stem cells and their malignant counterparts share MDR transporter activity as a major mechanism of self-protection. Although MDR activity is upregulated in response to substrate chemotherapeutic agents, it is also constitutively expressed on both normal tissue stem cells and a subset of tumor cells prior to the initiation of therapy, representing a built-in obstacle to therapeutic ratio. Constitutive and induced MDR activity can be detected in cellular subsets of disaggregated tissues, using the fluorescent substrates Rhodamine 123 and Hoechst 33342 for ABCB1 (also known as P-gp and MDR1) and ABCG2 (BCRP1). In this chapter, we will describe the complete procedure for the detection of MDR activity, including: (1) Preparing single-cell suspensions from tumor and normal tissue specimens; (2) An efficient method to perform cell surface marker staining on large numbers of cells; (3) Flow cytometer setup and controls; (4) Simultaneous measurement of Hoechst 33342 and Rhodamine123 transport; and (5) Data acquisition and analysis.

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Available from: Albert Donnenberg, Jul 22, 2014
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    • "Drug effluxion: some of most frequently studied drug transporters, associated with acquisition of resistance in normal SCs as well as in CSCs, are multifunctional efflux transporters from the ABC gene family 95. These contribute to tumor resistance by actively transporting drugs across cell membranes through ATP hydrolysis 96. "
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    • "Evidence supporting this model comes from studies showing CSCs have increased drug resistance capacity. For example, it has been shown that stem-like subpopulation of cancer cells express high levels of ATP-binding cassette (ABC) transporters that can actively efflux drugs and shield them from the adverse effects of chemotherapeutic insult (Pardal et al., 2003; Lou and Dean, 2007; Dean, 2009; Donnenberg et al., 2009; Ding et al., 2010; Moitra et al., 2011). In addition to an increased drug efflux capacity, CSCs also exhibit intrinsic resistance to apoptosis. "
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    • "R123 is not the only chemical that has been proposed for use as a tracer dye for MDR1. The most commonly used alternatives are currently Hoechst 33342 [38], 3,39-diethyloxacarbocyanine iodide and, calcein-AM [39], with commercially systems such as eFluxx-ID also being recently released (Enzo Lifesciences; [40]). However, it should be noted that Prochazkova and colleagues recently demonstrated that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate were all substrates for MDR1, and hence could represent potential tracer dyes [41]. "
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