IntroductionThe ability of early hematopoietic progenitors to efflux certain fluorescent dyes such as rhodamine 123 and Hoechst 33342 has long been appreciated and exploited for the isolation of these cells by fluorescence-activated cell sorting (FACS). [1][2][3] More recently, one method has proven to be especially useful for the isolation of the most primitive bone marrow cells from a number of different species. 4 This method relies on incubating the target cell population with the fluorescent dye Hoechst 33342 and subsequent FACS analysis of dual-wavelength Hoechst fluorescence with gating on a specific side population displaying low red and low blue fluorescence. Hence, the isolated cells are termed side-population (SP) cells. This low-staining SP is lost after treatment with verapamil, which has led to the assumption that the MDR1-encoded adenosine triphosphate-binding cassette (ABC) transporter, P-glycoprotein (P-gp), is responsible for Hoechst dye efflux in these cells. 4 Recently, excitement has been generated by the finding that putative stem cells from solid tissues may also share this SP phenotype. [5][6][7] Moreover, the degree of efflux activity seems to correlate with the maturation state, such that cells exhibiting the highest efflux activity are the most primitive. 8 Aside from its value in stem cell purification, the dye efflux phenomenon raises 2 important questions: (1) what are the biochemical mechanisms by which it is produced in SP cells and (2) what physiologic roles do such export activities play in stem cells?The multidrug-resistance gene, MDR1, is the best-studied member of the ABC transporter super-family of genes. The products of these genes are transmembrane proteins involved in energy-dependent transport of a wide spectrum of substrates across membranes. 9,10 Currently, more than 30 members have been identified in humans. 11 To date, most drug efflux phenomena have been ascribed to expression of MDR1. 12 Due to known Hoechst transport activity of MDR1, 13 and to the expression of MDR1 in early hematopoietic progenitor cells, 14,15 the export activity in stem cells has largely been attributed to MDR1. 4,14,16,17 MDR1 is only one of many candidate transporters for Hoechst. Members of the B, C, and G families of the ABC transport super-family are all associated with multidrug resistance and exhibit overlapping substrate specificities. Of particular importance here, they possess the ability to efflux broad and overlapping ranges of lipophilic molecules, a category that includes Hoechst. 18 A careful review of the literature revealed that the efflux activity of rhodamine in murine hematopoietic stem cells is only partially blocked by the MDR1 inhibitor, verapamil. In fact, the murine long-term repopulating cells, considered to be the most primitive stem cells, are isolated on the basis of rhodamine efflux (Rho Ϫ ) in the presence of verapamil. 16 Thus, it seems probable that another transporter may contribute to the SP phenotype associated with stem cells. Materials and methods Cell cu...

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.

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The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dosedependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [ 125 I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.