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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    ABSTRACT: Currently we are more and more improving our knowledge about the characteristics and the role of cancer stem cells in human cancer. Particularly we have realized that self-renewing ovarian cancer stem cells (CSCs) or ovarian cancer-initiating cells, and mesenchymal stem cells (SCs) too, are probably implicated in the etiopathogenesis of epithelial ovarian cancer (EOC). There is clear evidence that these cells are also involved in its intra- and extra-peritoneal diffusion and in the occurrence of chemo-resistance. In assessing the molecular characteristics of ovarian CSCs, we have to take note that these cellular populations are rare and the absence of specific cell surface markers represents a challenge to isolate and identify pure SC populations. In our review, we focused our attention on the molecular characteristics of epithelial ovarian CSCs and on the methods to detect them starting from their biological features. The study of ovarian CSCs is taking on an increasingly important strategic role, mostly for the potential therapeutic application in the next future.
    Journal of Cancer 03/2014; 5(5):301-310. DOI:10.7150/jca.8610 · 3.27 Impact Factor
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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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    ABSTRACT: Tumor cells can persist undetectably for an extended period of time in primary tumors and in disseminated cancer cells. Very little is known about why and how these tumors persist for extended periods of time and then evolve to malignancy. The discovery of cancer stem cells (CSCs) in human tumors challenges our current understanding of tumor recurrence, drug resistance, and metastasis, and opens up new research directions on how cancer cells are capable of switching from dormancy to malignancy. Although overlapping molecules and pathways have been reported to regulate the stem-like phenotype of CSCs and metastasis, accumulated evidence has suggested additional clonal diversity within the stem-like cancer cell subpopulation. This review will describe the current hypothesis linking CSCs and metastasis and summarize mechanisms important for metastatic CSCs to re-initiate tumors in the secondary sites. A better understanding of CSCs' contribution to clinical tumor dormancy and metastasis will provide new therapeutic revenues to eradicate metastatic tumors and significantly reduce the mortality of cancer patients.
    Frontiers in Endocrinology 10/2012; 3:125. DOI:10.3389/fendo.2012.00125
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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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    ABSTRACT: Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.
    PLoS ONE 03/2012; 7(3):e33253. DOI:10.1371/journal.pone.0033253 · 3.23 Impact Factor
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