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

  • Vera S. Donnenberg
  • E. Michael Meyer
  • Albert D. Donnenberg
Part of the Methods in Molecular Biology book series (MIMB, volume 568)


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.

Key words

Pre-existing multiple drug resistance ABCB1 activity ABCG2 activity Hoechst 33342 Rhodamine 123 Epithelial tumors Cancer stem cells Flow cytometry 



The authors would like to thank Melanie Pfeifer and Amber McCauslin for technical assistance. This work was supported by grants BC032981 and BC044784 from the Department of Defense, the Hillman Foundation, and the Glimmer of Hope Foundation. Vera Donnenberg is a CDMRP Era of Hope Scholar.


  1. 1.
    Schinkel AH, Mol CA, Wagenaar E, van Deemter L, Smit JJ, Borst P. Multidrug resistance and the role of P-glycoprotein knockout mice. Eur J Cancer 1995;31A:1295–1298.PubMedCrossRefGoogle Scholar
  2. 2.
    Benet LZ, Izumi T, Zhang Y, Silverman JA, Wacher VJ. Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery. J Control Release 1999;62:25–31.PubMedCrossRefGoogle Scholar
  3. 3.
    Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.PubMedCrossRefGoogle Scholar
  4. 4.
    Donnenberg VS, Burckart GJ, Zeevi A, et al. P-glycoprotein activity is decreased in CD4+ but not CD8+ lung allograft-infiltrating T cells during acute cellular rejection. Transplantation 2004;77:1699–1706.PubMedCrossRefGoogle Scholar
  5. 5.
    Udomsakdi C, Eaves CJ, Sutherland HJ, Lansdorp PM. Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamine-123. Exp Hematol 1991;19:338–342.PubMedGoogle Scholar
  6. 6.
    Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:85–94.PubMedCrossRefGoogle Scholar
  7. 7.
    Giangreco A, Shen H, Reynolds SD, Stripp BR. Molecular phenotype of airway side population cells. Am J Physiol Lung Cell Mol Physiol 2004;286:L624–L630.PubMedCrossRefGoogle Scholar
  8. 8.
    Chen J, Hersmus N, Van Duppen V, Caesens P, Denef C, Vankelecom H. The adult pituitary contains a cell population displaying stem/progenitor cell and early embryonic characteristics. Endocrinology 2005;146:3985–3998.PubMedCrossRefGoogle Scholar
  9. 9.
    He DN, Qin H, Liao L, et al. Small intestinal organoid-derived SP cells contribute to repair of irradiation-induced skin injury. Stem Cells Dev 2005;14:285–291.PubMedCrossRefGoogle Scholar
  10. 10.
    Riou L, Bastos H, Lassalle B, et al. The telomerase activity of adult mouse testis resides in the spermatogonial alpha6-integrin-positive side population enriched in germinal stem cells. Endocrinology 2005;146:3926–3932.PubMedCrossRefGoogle Scholar
  11. 11.
    Ling V, Thompson LH. Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J Cell Physiol 1974;83:103–116.PubMedCrossRefGoogle Scholar
  12. 12.
    Wang YC, Juric D, Francisco B, et al. Regional activation of chromosomal arm 7q with and without gene amplification in taxane-selected human ovarian cancer cell lines. Genes Chromosomes Cancer 2006;45:365–374.PubMedCrossRefGoogle Scholar
  13. 13.
    Chen GK, Lacayo NJ, Duran GE, et al. Preferential expression of a mutant allele of the amplified MDR1 (ABCB1) gene in drug-resistant variants of a human sarcoma. Genes Chromosomes Cancer 2002;34:372–383.PubMedCrossRefGoogle Scholar
  14. 14.
    Calcagno AM, Fostel JM, To KK, et al. Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes. Br J Cancer 2008;98:1515–1524.PubMedCrossRefGoogle Scholar
  15. 15.
    Sims-Mourtada J, Izzo JG, Ajani J, Chao KS. Sonic Hedgehog promotes multiple drug resistance by regulation of drug transport. Oncogene 2007;26:5674–5679.PubMedCrossRefGoogle Scholar
  16. 16.
    Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci U S A 2007;104:4048–4053.PubMedCrossRefGoogle Scholar
  17. 17.
    Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science 2004;304:1755–1759.PubMedCrossRefGoogle Scholar
  18. 18.
    Johnstone RW, Ruefli AA, Tainton KM, Smyth MJ. A role for P-glycoprotein in regulating cell death. Leuk Lymphoma 2000;38:1–11.PubMedGoogle Scholar
  19. 19.
    Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A 2006;103:11154–11159.PubMedCrossRefGoogle Scholar
  20. 20.
    Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 2005;65:6207–6219.PubMedCrossRefGoogle Scholar
  21. 21.
    Harris MA, Yang H, Low BE, et al. Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res 2008;68:10051–10059.PubMedCrossRefGoogle Scholar
  22. 22.
    Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+ alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer Res 2007;67:6796–6805.PubMedCrossRefGoogle Scholar
  23. 23.
    Mitsutake N, Iwao A, Nagai K, et al. Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology 2007;148:1797–1803.PubMedCrossRefGoogle Scholar
  24. 24.
    Lichtenauer UD, Shapiro I, Geiger K, et al. Side population does not define stem cell-like cancer cells in the adrenocortical carcinoma cell line NCI h295R. Endocrinology 2008;149:1314–1322.PubMedCrossRefGoogle Scholar
  25. 25.
    Donnenberg VS, Luketich JD, Landreneau RJ, DeLoia JA, Basse P, Donnenberg AD. Tumorigenic epithelial stem cells and their normal counterparts. Ernst Schering Found Symp Proc 2006;5:245–263.PubMedCrossRefGoogle Scholar
  26. 26.
    Donnenberg VS, Landreneau RJ, Donnenberg AD. Tumorigenic stem and progenitor cells: implications for the therapeutic index of anti-cancer agents. J Control Release 2007;122:385–391.PubMedCrossRefGoogle Scholar
  27. 27.
    Lou H, Dean M. Targeted therapy for cancer stem cells: the patched pathway and ABC transporters. Oncogene 2007;26:1357–1360.PubMedCrossRefGoogle Scholar
  28. 28.
    Donnenberg VS, Donnenberg AD. Therapeutic index and the cancer stem cell paradigm. In: Bagley R, Teicher B, eds. Stem Cells and Cancer Series: Cancer Drug Discovery and Development. New York: Springer, Humana Press; 2009.Google Scholar
  29. 29.
    Bertoncello I, Williams B. Hematopoietic stem cell characterization by Hoechst 33342 and rhodamine 123 staining. Methods Mol Biol 2004;263:181–200.PubMedGoogle Scholar
  30. 30.
    Turgeon ML. Clinical hematology: theory and procedures (4th ed). Philadelphia, PA: Lippincott Williams & Wilkins; 2005.Google Scholar
  31. 31.
    Ibrahim SF, Diercks AH, Petersen TW, van den Engh G. Kinetic analyses as a critical parameter in defining the side population (SP) phenotype. Exp Cell Res 2007;313:1921–1926.Google Scholar
  32. 32.
    Donnenberg AD, Donnenberg VS. Understanding clinical flow cytometry. In: O’Gorman MR, Donnenberg AD, eds. Handbook of Human Immunology (2nd ed). Boca Raton: CRC Press Taylor and Francis; 2008:181–220.CrossRefGoogle Scholar
  33. 33.
    Donnenberg AD, Donnenberg VS. Rare-event analysis in flow cytometry. Clin Lab Med 2007;27:627–652.Google Scholar
  34. 34.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983–3988.Google Scholar
  35. 35.
    Kane S, Reinhard D, Fordis M, Pastan I, Gottesman M. A new vector using the multiple drug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells. Gene 1989;84:439–446.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Vera S. Donnenberg
    • 1
  • E. Michael Meyer
    • 1
  • Albert D. Donnenberg
    • 1
  1. 1.Division of Hematology and Oncology, Department of Medicine, University of Pittsburgh School of MedicineHillman Cancer CenterPittsburghUSA

Personalised recommendations