Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bunting, K. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bunting, K. D.
Stem Cells 2002;20:11-20 www.StemCells.com
© 2002 AlphaMed Press

CONCISE REVIEW

ABC Transporters as Phenotypic Markers and Functional Regulators of Stem Cells

Kevin D. Bunting

Hematopoiesis Department, American Red Cross Holland Laboratory, Rockville, Maryland, USA

Key Words. Stem cell • Bone marrow transplantation • ABC transporter • Retrovirus vector

Kevin D. Bunting, Ph.D., Hematopoiesis Department, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, Maryland 20855, USA. Telephone: 301-738-0449; Fax: 301-738-0444; e-mail: buntingk{at}usa.redcross.org


   ABSTRACT
Top
 Abstract
Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 
Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product, P-glycoprotein, and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis, with the highest levels in primitive bone marrow stem cell populations that are CD34low and CD34–, respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However, recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia, but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally, the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors, makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings, it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers.


   INTRODUCTION
Top
 Abstract
 Introduction
ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 
The developmental programs that regulate gene expression profiles are tightly controlled and can lead to cancer if perturbed. An increasing number of genes are being characterized that can function as lineage-restricted transcription factors, directing hematopoietic cell differentiation. For example, GATA-1 is required for erythropoiesis, PU.1 for myelopoiesis, and Ikaros for lymphopoiesis, with cross-talk possible between factors modulating differentiation. The differential gene expression patterns that direct hematopoietic stem cell (HSC) self-renewal versus differentiation are less well characterized, but are of great interest. A genetic blueprint for genes that are expressed in highly enriched AA4.1+ fetal liver HSCs has provided a source of starting material upon which gene discovery of interesting candidate regulators of HSC development can be initiated and upon which chips can be designed to apply gene array technology [1]. HSCs are widely studied because of their self-renewal capacity leading to potential utility in therapeutic applications such as bone marrow transplantation and gene therapy. Manipulation of pathways regulating stem cell self-renewal decisions through biochemical methods might provide a means for inducible expansion of HSCs. Gene therapy would benefit greatly from advancements in the methods of culture and maintenance of HSCs. Human gene therapy applications are currently limited by low levels of transduction into HSCs, which is due to a variety of factors that include low receptor expression levels and reduced cell cycle activity required for retroviral vector integration. Specific expansion of transduced HSCs could overcome these limitations and perhaps increase the levels of gene transfer in patients to clinically significant levels.

Stem cell populations can be highly enriched by a variety of methods that typically involve cell surface marker expression or functional characteristics, allowing for better characterization of the biology of these cells. In mice, the HSC can be assayed by reconstitution of lymphoid and myeloid lineages following injection into lethally irradiated recipients. The cells enriched for long-term repopulating activity have been shown to express the Sca-1 antigen, the c-kit receptor tyrosine kinase, but none of a cocktail of differentiation lineage markers [2–5]. Isolation of stem cells based on the efflux of fluorescent dyes has also been an efficient method to further purify stem cells, and it has been demonstrated that Rhodamine123 (Rho123) retention is low in the most primitive hematopoietic cells. While the Rholow fraction provides long-term reconstitution following injection into lethally irradiated mice, the Rhohigh fraction provides only short-term repopulation [6–8]. Hoechst 33342 is another fluorescent dye used for isolation of stem cell fractions. In combination with Rho123 staining, HoechstlowRholow cells are highly enriched for stem cell activity [9,10]. The biological basis for the differential efflux of these dyes in the stem cell compartment has previously been unclear. Decreased retention could be due to decreased mitochondrial activation [8,11] in quiescent cells or the presence of transmembrane proteins capable of pumping these dyes out of the cell. This review will focus on the latter hypothesis, which is attractive considering recent demonstrations of function for ATP-binding cassette (ABC) transporters in hematopoiesis.


   ABC TRANSPORTERS AND HEMATOPOIETIC EXPRESSION
Top
 Abstract
 Introduction
 ABC Transporters and...
Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 
Membrane-associated ABC transporters have been found in two forms, the full-length transporters that are characterized by two identical halves each containing the nucleotide binding domain and the half-transporters that function as homo- or heterodimers (Fig. 1Go). The first identified candidate transporter for the efflux of fluorescent dyes in HSCs was the 1,280 amino acid multidrug resistance (MDR1) P-glycoprotein (P-gp), also known as ABCB1, a glycosylated membrane-associated enzyme that exports a wide range of diverse substrates. Structurally, MDR1 is a duplicated molecule with two identical halves that are separated by a flexible linker region that is critical for the efflux pump function [12]. Each half of the molecule comprises six hydrophobic integral membrane domains, which together form the substrate-binding site that mediates the efflux activity. Various mechanisms for the efflux function of the P-gp have been proposed that include aqueous pore [13] and lipid "flippase" activities [14]. There is still uncertainty about the mechanism of efflux function. The broad substrate specificity is difficult to reconcile with the idea of an aqueous pore of limited size. More likely is that lipophilic molecules already within the membrane are exported. Each half of the molecule also contains an internal nucleotide-binding domain that is required for the hydrolysis of ATP to generate the energy required for export. Blocking the ATPase function through site-directed mutagenesis of the recombinant protein or through treatment with biochemical inhibitors of the enzyme, such as calcium channel blockers or cyclosporin analogs, has been shown to increase the drug sensitivity of expressing cells.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Structural features of the two primary types of membrane-associated ABC transporters.The family of ABC transporters is defined by the presence of the ATP-binding cassette region (ATP), which hydrolyzes ATP to drive the energy-dependent export of substrates from the intracellular cytoplasmic space to the extracellular space. These transporters have classically been shown to contain two mirror image halves separated by a flexible linker region that is essential for the catalytic activity of the transporter (Panel A). Within each half of the molecule is a 6-transmembrane spanning domain (6TM) that docks the transporter protein to the plasma membrane. Transporters that consist of only one of the halves and require membrane association with other half-transporters to form the pore complex have also been described. The predicted membrane topology of the amino acid chain for two half-transporters in close proximity is shown in Panel B. Both the full- and half-transporters have the ability to efflux a wide range of substrates, many of which are chemotherapeutic agents.

 
It was first demonstrated 10 years ago by Chaudhary and Roninson, that the P-gp is highly expressed on CD34+ hematopoietic cells [15], suggesting that efflux pump activity could be responsible for the low retention of Rho123 in primitive subsets of cells. The CD34 gene codes for a transmembrane cell surface phosphoprotein that has been generally accepted as being a stem cell marker based on engraftment following bone marrow transplantation into baboons [16], humans [17], and mice [18]. However, in recent years, this has become a point of controversy in the field. It was first shown by Osawa et al. in 1996 that murine HSCs could be CD34 negative [19]. Soon after, Goodell et al. demonstrated that a highly enriched stem cell fraction termed side-population (SP) cells could be isolated following Hoechst 33342 staining [20]. Human SP cells expressed low to undetectable levels of CD34, and the CD34–38– cells could acquire CD34 during culture [21]. ABC transporter activity is believed to be involved in the differential Hoechst staining, since the SP tail could be eliminated by treatment with verapamil, a known inhibitor of P-gp function. Interestingly, other studies have also demonstrated that both mouse and human CD34+ cells can be derived from CD34– cells [22,23]. Transplantation studies in fetal sheep [24] also showed repopulating activity in the CD34– population. The concept evolved that CD34 expression may be up- or downmodulated in association with "activation" status [23] and that HSCs may therefore be CD34+ or CD34–. Isolation of murine bone marrow with the cell surface marker phenotype of Sca-1+c-kit+lin– (KLS) can be used to enrich for SP cells, thus showing significant overlap between the two populations (Fig. 2Go). A common factor in all of these studies with Hoechst SP/CD34low/+ cells or KLS cells is that primitive hematopoietic cell populations capable of repopulating hosts express ABC transporter activity. It has now become clear that two specific transporters with unique substrate specificity are differentially expressed in this SP fraction during early hematopoietic differentiation.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Hoechst 33342 side-population (SP) analysis identifies primitive hematopoietic stem cells.Functional isolation of hematopoietic stem cell populations based on the efflux of fluorescent dyes such as Rho123 and Hoechst 33342 has been widely used. Cells that exclude these dyes are highly enriched for long-term repopulating activity in transplanted mice and express well-characterized phenotypic markers for hematopoietic stem cells. Phenotypic isolation of stem cells based on the expression of c-kit and Sca-1 and the lack of expression of a panel of lineage markers (Gr-1, Mac-1, B220, CD4, and Ter119) has been characterized and shown to also be highly enriched for hematopoietic stem cell activity. These cells are referred to here as Sca-1+c-kit+lin–or KLS. There is significant overlap between the KLS and SP fractions as shown here. In this typical analysis about 73% of the cells gated for KLS were located within the SP gate.

 
Mice containing homozygous deletions of mdr gene expression were generated to further characterize the physiological roles of this family of endogenous transporters. The phenotypes of knockout mice lacking expression of the two murine mdr1a and mdr1b homologs to the human MDR1 have been relatively mild, with each strain of mice showing normal health and viability [25,26]. mdr1a and mdr1b knockout mice did show altered pharmacokinetics for a number of anticancer agents and hypersensitivity to the pesticide ivermectin. No significant hematologic defects were observed. However, numerous other ABC transporters have been identified in recent years that could serve compensatory endogenous functions. A growing family of MDR-associated proteins, as well as the half-transporters have also been described. A normal percentage of SP cells was recently described in the bone marrow from mdr1a1b–/– mice, suggesting the presence of another transporter that has been previously uncharacterized regarding expression in primitive hematopoietic cells [27]. Further, inhibition of SP cell staining with either 2-deoxyglucose, a general inhibitor of ATP synthesis, or with verapamil in the mdr1a1b–/– background indicated that this activity was due to expression of an ABC transporter. This was not an entirely new concept, since P-gp expression studies in multidrug resistant myeloid leukemia cells from patients showed dissociation of P-gp expression and dye efflux [28,29]. These initial studies strongly suggested that alternative transporters may be active in hematopoietic cells.

A half-transporter known as the breast cancer resistance protein (BCRP), also called ABCG2 [30], was subsequently identified and characterized as a novel stem cell transporter [27]. Like MDR1, enforced overexpression of BCRP in human MCF-7 breast cancer cells conferred a broad spectrum of drug resistance [30], and elevated levels of expression of BCRP have been reported to be associated with acute myeloid leukemia (AML) [31]. Unlike MDR1, wild-type BCRP does not efflux Rho123 [27]. However, it is important to note that a mutant BCRP containing an Arg to Thr change at codon 482 has been described that has the capacity to efflux Rho123 [30]. Codon 482, which is located within the transmembrane 3-region, appears to be critical for the Rho123 substrate specificity of BCRP, as well as other anticancer drug substrates [32]. BCRP is a membrane-associated 663 amino acid transporter protein [33] that is highly expressed in placenta [34] and Hoechst SP/CD34– hematopoietic stem cells [27]. Therefore, one interpretation is that BCRP expression may define primitive quiescent HSCs whereas MDR1 may be expressed in more "activated" repopulating HSCs. However, some overlap in these expression patterns of mdr1a and bcrp has been reported in mouse CD34– stem cells [27]. In human cells, BCRP also appears to be highly expressed in phenotypically defined populations of primitive hematopoietic stem cells [35].


   BCRP AS A NOVEL STEM CELL MARKER
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 
The expression pattern of bcrp is not limited to primitive mouse and human hematopoietic stem cell populations. Bcrp expression has been shown to be a determinant of the Hoechst 33342 SP phenotype in cells from diverse sources [27] (Fig. 3Go). Bcrp expression was exclusive to SP cells identified from murine skeletal muscle and rhesus bone marrow as compared with non-SP cells. Interestingly, SP cells were also found within embryonic stem (ES) cells, indicating that ES cells are not homogenous. Sorted SP+ ES cells showed greater ability to contribute to chimeric offspring mice than non-SP ES cells. However, the lack of a correlation between bcrp expression and the SP phenotype in murine ES cells indicated that, in a cell line, other characteristics, such as cell cycle status or the presence of other transporters, could also be involved in the SP phenotype. It will be important to isolate ES cells based strictly on bcrp expression to determine whether the blastocyst contribution is more highly associated with bcrp+ SP cells or bcrp– SP cells. The potential plasticity of SP cells in development makes this an attractive population to study. SP cells isolated from satellite skeletal muscle cells were reported to reconstitute hematopoiesis following intravenous transplantation [36], however, this result has recently been challenged [37]. The ability of transplanted bone marrow (BM)-derived SP cells to regenerate muscle in the mdx mouse, a model for Duchenne muscular dystrophy [38], and to regenerate cardiomyocytes, in a myocardial infarct model [39], has been demonstrated. Therefore, bcrp expression may prove to be a marker for positive selection of pluripotent stem cells from adults. Recently, two studies isolated bcrp in enriched neural stem/progenitor populations using subtractive hybridization and array analysis technologies [40,41]. Studies showing contribution of BM cells to neuronal tissue and vice versa have been reported. The demonstration of bcrp as a common gene expressed in hematopoietic stem cells and neural stem cells, suggests that bcrp might have a functional role in stem cells that could contribute to plasticity between brain and blood or between muscle and blood.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 3. Bcrp is expressed on sorted SP cells from various tissue sources.RNA was extracted from cells sorted by a fluorescence-activated cell sorter (FACS) for the SP phenotype and from a non-SP gate as a control. Reverse transcriptase-polymerase chain reaction (PCR) analyses were performed on SP cells that were sorted from a diverse range of sources, which included rhesus monkey BM (panel A), mouse skeletal muscle (panel B), and murine ES cells (panel C). Determination of the relative transcript levels for both bcrp1 and ß -actin was performed by PCR [27]. Since the total number of starting SP cells varied from each source, the total number of cycles shown for each bcrp1 sample was chosen based on the equivalent signal intensity for amplification of the ß-actin control. In the primary monkey BM and mouse skeletal muscle, bcrp1 expression was highly associated with the SP phenotype. A close association between bcrp1 and the SP phenotype was not observed in murine ES cells, possibly due to additional cellular determinants. Reproduced by permission from Nature America Inc. Zhou et al. [27].

 

   FUNCTIONAL CHARACTERIZATION OF ABC TRANSPORTERS BY OVEREXPRESSION IN MICE
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
Possible Physiological Functions...
 Concluding Remarks
 References
 
Gene therapy strategies for protection of BM from the dose-limiting toxicity of chemotherapeutic agents have been investigated for many years. As a consequence of these studies, expression of ABC transporters to protect against multiple drugs has been actively pursued. Methods for optimizing gene transfer and expression of a splice-corrected MDR1 vector [42] led to high-level expression of the human P-gp in murine stem cells. It was first reported by Bunting et al. that Harvey murine sarcoma virus (Ha)MDR1-transduced BM cells showed a significant engraftment advantage relative to those BM cells transduced with a dihydrofolate reductase (DHFR) mutant containing a leucine to tyrosine mutation at codon 22 (HaDHFRL22Y) [43]. The MDR1-transduced BM graft persisted long term in primary recipients of expanded cells in nonmyeloablated hosts and in lethally irradiated secondary recipients. This observation was then followed up by competitive repopulation studies that confirmed that the MDR1 graft did contain a greatly enhanced repopulating ability, to the degree that it could outcompete freshly harvested BM cells [43,44]. The expansion occurred serially over time in the 12-day cultures. Further experiments tested the possibility that expansion of MDR1-transduced HSCs could occur in vivo as well. For these experiments BM cells were transduced with either HaMDR1 or HaDHFRL22Y vectors and combined 50:50 in competitive repopulation assays. With time, the MDR1 graft overtook the competitor graft indicating that expansion could also occur in vivo [45]. Therefore, the effects of MDR1 were generalizable, beyond the in vitro liquid suspension culture conditions. Further experiments demonstrated that a deletion mutant MDR1 (MDR1{Delta}34) vector lacking the efflux pump activity was not sufficient to provide the expansion phenotype. While these studies were performed with whole BM cells, the results were consistent with effects on early cells with repopulating activity. However, to conclusively demonstrate that these effects can occur at the HSC level, it will be necessary to perform similar studies using enriched HSC populations.

Using the same model system, it was later shown that the SP cell phenotype could be modulated by MDR1 expression resulting in an increased percentage of SP cells. It is important to note that while under physiological conditions, many stem cells fall within the SP fraction, not all SP cells are stem cells. To address this issue, an absolute increase in hematopoietic repopulating activity following limiting dilution analysis of sorted MDR1-overexpressing SP cells was confirmed. When doing experiments with isolated SP cells, it is critical to remember that the SP fraction can also be contaminated with lineage positive cells such as natural killer lymphocytes. Therefore, the lineage negative fraction must be stringently isolated, and the SP cell phenotype per se cannot be used as proof of stem cell identity without additional supporting functional analysis.

In addition to the profound expansion of repopulating activity that was observed with wild-type MDR1, mice receiving MDR1-transduced cells also developed a myeloproliferative disease characterized by high peripheral white blood cell counts and splenomegaly due to myeloid progenitor accumulation at both sites. The disease phenotype was also reported from a second retroviral vector [44] that encoded a bicistronic HaMDR1-IRES-DHFRL22Y, indicating that it was not specific to a particular ecotropic producer cell clone. Elevated numbers of peripheral blood CD11b (Mac-1)+GR-1+ and Mac-1+GR-1– cells were found in transplanted mice (Fig. 4Go). The disease did not appear to be caused by transformation of the MDR1-transduced cells because no tumors were formed following transplant of these abnormal cells into CB.17 SCID mice and no gross chromosomal translocations could be identified. Also, the abnormal peripheral blood cells remained dependent on growth factors for survival following culture ex vivo. Morphological analysis of Wright/Giemsa-stained peripheral blood smears showed an accumulation of early myeloid cells, similar to chronic myelomonocytic leukemia.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Myeloid-specific cell surface marker expression on peripheral blood leukocytes following HaMDR1 gene transfer. C57Bl/6 BM cells were transduced with the HaMDR1 vector and transplanted into irradiated mice (HaMDR1 BMT). Two mice showing evidence of myeloproliferative disease were analyzed by FACS for Gr-1 and CD11b (Mac-1) myeloid differentiation marker expression. Two nontransplanted wild-type mice are shown as a control (Wild-type). Mice overexpressing MDR1 in the myeloid lineage showed an increased percentage of myeloid progenitors in the peripheral blood and spleen. Associated with the disease progression was an increase in the percentage of Mac-1+Gr-1–peripheral blood cells, which are normally a low percentage of the blood composition. This suggests that ectopic MDR1 expression in the myeloid lineage might also confer specific effects on myeloid differentiation in addition to the described effects on BM repopulating activity.

 
It should be noted that when trying to extrapolate these findings to the nonhuman primate model, HaMDR1 gene transfer did not result in detectable adverse consequences even with ex vivo expansion prior to infusion of marked cells [46]. Differences in gene transfer efficiency into mouse versus rhesus HSCs might explain this result, since the oligoclonal expanded stem cell population found in mice showed high proviral copy number. Furthermore, no abnormal hematopoiesis has been observed in MDR1 clinical trials [47,48], although the marking levels have been much lower than could be achieved in a mouse model. Another difference is that for the murine and rhesus studies a wild-type MDR1 vector was expressed whereas, in the human studies, a codon 185 variant vector was used. Mutation of codon 185 from glycine to valine alters substrate specificity relative to wild-type MDR1. At least in terms of known P-gp substrates, this mutation increases the capacity to efflux colchicine but decreases the ability to efflux taxol and vinblastine. The unknown substrates being modulated in stem cells could be affected by this specific mutation, and currently it is not possible to eliminate this possibility. Furthermore, the VL-30 containing Harvey murine sarcoma virus backbone might play a role in the effects of MDR1 on bone marrow cells, although direct comparison with a control vector lacking MDR1 has demonstrated specificity for the MDR1 transgene. Expression of MDR1 from other vector designs and in mouse strains other than C57Bl/6 will be required to fully demonstrate the generalizability of these findings.

The mdr1a1b–/– mouse model has proven useful in studies by Zhou et al. to identify the endogenous transporter activity present in SP cells lacking expression of known mdr1 homologs. Molecular studies revealed that the bcrp transporter was expressed in a highly regulated manner with the most expression in primitive cells and sharp downregulation following commitment to differentiation. In contrast to Harvey/MDR1 overexpression studies, Harvey/BCRP overexpression significantly blocked hematopoietic development and resulted in less progeny in the bone marrow and peripheral blood [27]. The intriguing possibility is that BCRP expression may play a role in early stem cell self-renewal by partially blocking differentiation. MDR1 may alternatively promote differentiation and engraftment. Further genetic studies characterizing mice deficient and mice overexpressing both bcrp and mdr1ab will be required to fully characterize the complex and possibly overlapping roles of these transporters in stem cells. Additionally, studies using purified HSC populations from these mice will be needed to determine whether HSC commitment is modulated or whether the effects are at the level of differentiated progeny cells.


   POSSIBLE PHYSIOLOGICAL FUNCTIONS OF ABC TRANSPORTER ACTIVITY
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
Concluding Remarks
 References
 
The normal physiological functions of ABC transporters are quite diverse, and drug transport appears not to be the important evolutionarily conserved function of the transporter activity [49]. Perhaps MDR1 could keep cells from undergoing apoptosis long enough for subsequent cytokine stimulation to promote differentiation. MDR1 has been implicated in the protection of cells against apoptotic cell death induced by a variety of methods including growth factor deprivation, UV irradiation, ionizing radiation, or tumor necrosis factor-{alpha} treatment. In addition, sensitivity to apoptosis-inducing treatment was significantly increased following treatment with PSC-833, a highly potent inhibitor of P-gp [50]. A potential mechanism whereby P-gp could protect cells against apoptosis would be at the early stages of phosphatidylserine (PS) externalization. A lipid "flippase" activity for P-gp has been described [14], although it is unclear whether this activity is dependent on the well-characterized ATPase-dependent drug efflux activity of the P-gp. A link between a rare disease of PS externalization and lack of MDR gene expression also suggests that PS could be a substrate for MDR1 [51]. A recent report showed a correlation between P-gp function in primary AML samples and reduced levels of apoptosis following suspension culture [52]. Further, study of these P-gp+ AML cells revealed that the effects of P-gp expression were due to reduced levels of sphingomyelin on the cell surface, implicating ceramide as a potential downstream intermediate modulated by P-gp activity [53]. Ceramide is a well-characterized intracellular mediator of apoptosis signals, and it has recently been demonstrated to be an important signaling intermediate in regulating cell fate decisions by primitive hematopoietic cells [54]. In addition to ceramide, which is produced intracellularly, retinoids are exogenous fat-soluble vitamins and potential P-gp substrates that are important for terminal differentiation of myeloid cells. Vitamin A-starved mice develop an expansion of myeloid cells and lower levels of apoptosis [55]. Reversal of retinoid acid-resistant acute promyelocytic leukemia differentiation in the presence of MDR1 inhibitors [56] supports a role for alteration of retinoid metabolism by MDR1. Whether MDR1 expression directly modulates myeloid differentiation and whether any of these mechanisms are relevant remain to be demonstrated.

Data supporting a role for MDR1 in modulating signal transduction pathways should also be considered as another mechanism for providing a survival function. MDR1 can be phosphorylated by protein kinase C, but the significance of this phosphorylation is unclear due to conflicting reports that the phosphorylation is important for the efflux function of the protein. For instance, phosphorylation defective mutants of P-gp do not appear to have reduced enzyme activity [57], yet human breast cancer cells show induction of drug efflux following phorbol ester induction [58]. Secreted factors are classically described as having roles in directing early embryonic development, and picomolar changes in concentration that change receptor binding can have dramatic consequences. A well-studied example of this type of signaling is activin, a member of the transforming growth factor-ß family. The mechanism whereby P-gp expression could have effects on signal transduction pathways could involve removal from the cell of some key substrate that is itself a regulator of differentiation and in which subtle changes in concentration can have large effects. Differences in the substrate specificity between BCRP and MDR1 may also be responsible for the differences in function observed in overexpression studies.


   CONCLUDING REMARKS
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
References
 
HSCs have great potential for a wide range of clinical applications that include: A) umbilical cord blood transplants to adults where the stem cell numbers are limiting; B) ex vivo gene modification for gene therapy, and C) in vitro generation of mature blood cell products. Defining the key regulatory genes or biochemical pathways that are active at the stem cell level remains an important goal for research toward the treatment of blood diseases. Initial functional characterization of ABC transporter family members has provided a model for studying regulation of hematopoietic stem cell self-renewal and differentiation processes. It has also generated many new questions about how these molecules might function and what evolutionarily conserved substrates may be modulated. Since ABC transporter function is associated with both normal and aberrant hematopoiesis, it will be important to fully characterize the function of this class of transporter proteins in hematopoietic cell differentiation and to define the underlying mechanisms. Furthermore, the possible role of ABC transporter function in the SP cell phenotype found in other tissues, such as described in muscle, might indicate a more global function in recently described adult pluripotent stem cells capable of differentiating into various tissues such as liver, muscle, and brain. Additionally, the lines between embryonic and organ-restricted stem cells have become less clear since SP cells are common to both stem cell types. The many potential applications for the treatment of disease using adult stem cells without utilization of fetal tissue warrant continued studies. It is possible that certain ABC transporters may play a unique role in developmental stem cell biology, and that identification of regulatory substrates may reveal novel targets for enhancing future stem-cell-based therapies.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 
The author thanks Brian Sorrentino for helpful comments and suggestions and Heath Bradley and Teresa Hawley for technical assistance with flow cytometry analyses.


   REFERENCES
Top
 Abstract
 Introduction
 ABC Transporters and...
 Bcrp as a Novel...
 Functional Characterization of...
 Possible Physiological Functions...
 Concluding Remarks
 References
 

  1. Phillips RL, Ernst RE, Brunk B et al. The genetic program of hematopoietic stem cells. Science 2000;288:1635–1640.[Abstract/Free Full Text]

  2. van de Rijn M, Heimfeld S, Spangrude GJ et al. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci USA 1989;86:4634–4638.[Abstract/Free Full Text]

  3. Uchida N, Jerabek L, Weissman IL. Searching for hematopoietic stem cells. II. The heterogeneity of Thy-1.1(lo)Lin(-/lo)Sca-1+ mouse hematopoietic stem cells separated by counterflow centrifugal elutriation. Exp Hematol 1996;24:649–659.[Medline]

  4. Orlic D, Fischer R, Nishikawa S et al. Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 1993;82:762–770.[Abstract/Free Full Text]

  5. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol 1995;11:35–71.[CrossRef][Medline]

  6. Zijlmans JM, Visser JW, Kleiverda K et al. Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability. Proc Natl Acad Sci USA 1995;92:8901–8905.[Abstract/Free Full Text]

  7. Spangrude GJ, Brooks DM, Tumas DB. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 1995;85:1006–1016.[Abstract/Free Full Text]

  8. Spangrude GJ, Johnson GR. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci USA 1990;87:7433–7437.[Abstract/Free Full Text]

  9. Wolf NS, Kone A, Priestley GV et al. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol 1993;21:614–622.[Medline]

  10. Leemhuis T, Yoder MC, Grigsby S et al. Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and Rhodamine 123. Exp Hematol 1996;24:1215–1224.[Medline]

  11. Kim M, Cooper DD, Hayes SF et al. Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 1998;91:4106–4117.[Abstract/Free Full Text]

  12. Hrycyna CA, Airan LE, Germann UA et al. Structural flexibility of the linker region of human P-glycoprotein permits ATP hydrolysis and drug transport. Biochemistry 1998;37:13660–13673.[CrossRef][Medline]

  13. Altenberg GA, Vanoye CG, Horton JK et al. Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane. Proc Natl Acad Sci USA 1994;91:4654–4657.[Abstract/Free Full Text]

  14. van Helvoort A, Smith AJ, Sprong H et al. MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 1996;87:507–517.[CrossRef][Medline]

  15. Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:85–94.[CrossRef][Medline]

  16. Berenson RJ, Andrews RG, Bensinger WI et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 1988;81:951–955.

  17. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991;77:1717–1722.[Abstract/Free Full Text]

  18. Krause DS, Ito T, Fackler MJ et al. Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells. Blood 1994;84:691–701.[Abstract/Free Full Text]

  19. Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34–low/negative hematopoietic stem cell. Science 1996;273:242–245.[Abstract]

  20. Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.[Abstract/Free Full Text]

  21. Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337–1345.[CrossRef][Medline]

  22. Nakamura Y, Ando K, Chargui J et al. Ex vivo generation of CD34(+) cells from CD34(-) hematopoietic cells. Blood 1999;94:4053–4059.[Abstract/Free Full Text]

  23. Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells. Blood 1999;94:2548–2554.[Abstract/Free Full Text]

  24. Zanjani ED, Almeida-Porada G, Livingston AG et al. Human bone marrow CD34– cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells. Exp Hematol 1998;26:353–360.[Medline]

  25. Schinkel AH, Smit JJ, van Tellingen O et al. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 1994;77:491–502.[CrossRef][Medline]

  26. Schinkel AH, Mayer U, Wagenaar E et al. Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci USA 1997;94:4028–4033.[Abstract/Free Full Text]

  27. Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.[CrossRef][Medline]

  28. Xie XY, Robb D, Chow S et al. Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia. Leukemia 1995;9:1882–1887.[Medline]

  29. Leith CP, Kopecky KJ, Chen IM et al. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood 1999;94:1086–1099.[Abstract/Free Full Text]

  30. Doyle LA, Yang W, Abruzzo LV et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA 1998;95:15665–15670.[Abstract/Free Full Text]

  31. Ross DD, Karp JE, Chen TT et al. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood 2000;96:365–368.[Abstract/Free Full Text]

  32. Honjo Y, Hrycyna CA, Yan QW et al. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in mxr/ bcrp/abcp-overexpressing cells. Cancer Res 2001;61:6635–6639.[Abstract/Free Full Text]

  33. Rocchi E, Khodjakov A, Volk EL et al. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem Biophys Res Commun 2000;271:42–46.[CrossRef][Medline]

  34. Allikmets R, Schriml LM, Hutchinson A et al. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res 1998;58:5337–5339.[Abstract/Free Full Text]

  35. Scharenberg CW, Harkey MA, Torok-Storb BJ. The transporter ABCG2 effluxes Hoechst and its expression is restricted to the most primitive human hematopoietic cells. Blood 2000;96:666a.

  36. Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 1999;96:14482–14486.[Abstract/Free Full Text]

  37. Kawada H, Ogawa M. Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle. Blood 2001;98:2008–2013.[Abstract/Free Full Text]

  38. Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401:390–394.[CrossRef][Medline]

  39. Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.[CrossRef][Medline]

  40. Terskikh AV, Easterday MC, Li L et al. From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc Natl Acad Sci USA 2001;98:7934–7939.[Abstract/Free Full Text]

  41. Geschwind DH, Ou J, Easterday MC et al. A genetic analysis of neural progenitor differentiation. Neuron 2001;29:325–339.[CrossRef][Medline]

  42. Sorrentino BP, McDonagh KT, Woods D et al. Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice. Blood 1995;86:491–501.[Abstract/Free Full Text]

  43. Bunting KD, Galipeau J, Topham D et al. Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice. Blood 1998;92:2269–2279.[Abstract/Free Full Text]

  44. Bunting KD, Galipeau J, Topham D et al. Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture. Ann N Y Acad Sci 1999;872:125–40.[Abstract/Free Full Text]

  45. Bunting KD, Zhou S, Lu T et al. Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo. Blood 2000;96:902–909.[Abstract/Free Full Text]

  46. Sellers SE, Tisdale JF, Agricola BA et al. The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells. Blood 2001;97:1888–1891.[Abstract/Free Full Text]

  47. Hesdorffer C, Ayello J, Ward M et al. Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation. J Clin Oncol 1998;16:165–172.[Abstract/Free Full Text]

  48. Abonour R, Williams DA, Einhorn L et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000;6:652–658.[CrossRef][Medline]

  49. Johnstone RW, Ruefli AA, Smyth MJ. Multiple physiological functions for multidrug transporter P-glycoprotein? Trends Biochem Sci 2000;25:1–6.[CrossRef][Medline]

  50. Bezombes C, Maestre N, Laurent G et al. Restoration of TNF-alpha-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833. FASEB J 1998;12:101–109.[Abstract/Free Full Text]

  51. Toti F, Schindler V, Riou JF et al. Another link between phospholipid transmembrane migration and ABC transporter gene family, inferred from a rare inherited disorder of phosphatidylserine externalization. Biochem Biophys Res Commun 1997;241:548–552.[CrossRef][Medline]

  52. Pallis M, Russell N. P-glycoprotein expression is associated with resistance to spontaneous in vitro apoptosis in AML. Leukemia 1999;13:1468–1469.[CrossRef][Medline]

  53. Pallis M, Russell N. P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin–ceramide apoptotic pathway. Blood 2000;95:2897–2904.[Abstract/Free Full Text]

  54. Maguer-Satta V, Oostendorp R, Reid D et al. Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates. Blood 2000;96:4118–4123.[Abstract/Free Full Text]

  55. Kuwata T, Wang IM, Tamura T et al. Vitamin A deficiency in mice causes a systemic expansion of myeloid cells. Blood 2000;95:3349–3356.[Abstract/Free Full Text]

  56. Matsushita H, Kizaki M, Kobayashi H et al. Restoration of retinoid sensitivity by MDR1 ribozymes in retinoic acid-resistant myeloid leukemic cells. Blood 1998;91:2452–2458.[Abstract/Free Full Text]

  57. Germann UA, Chambers TC, Ambudkar SV et al. Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells. J Biol Chem 1996;271:1708–1716.[Abstract/Free Full Text]

  58. Ramachandran C, Kunikane H, You W et al. Phorbol ester-induced P-glycoprotein phosphorylation and functionality in the HTB-123 human breast cancer cell line. Biochem Pharmacol 1998;56:709–718.[CrossRef][Medline]

Received September 7, 2001; accepted for publication October 22, 2001.



This article has been cited by other articles:


Home page
 Circ. Res.Home page
O. Pfister and R. Liao
Pump to Survive: Novel Cytoprotective Strategies for Cardiac Progenitor Cells
Circ. Res., May 9, 2008; 102(9): 998 - 1001.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
C. M. Martin, A. Ferdous, T. Gallardo, C. Humphries, H. Sadek, A. Caprioli, J. A. Garcia, L. I. Szweda, M. G. Garry, and D. J. Garry
Hypoxia-Inducible Factor-2{alpha} Transactivates Abcg2 and Promotes Cytoprotection in Cardiac Side Population Cells
Circ. Res., May 9, 2008; 102(9): 1075 - 1081.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Engelmann, H. Shen, and O. J. Finn
MCF7 Side Population Cells with Characteristics of Cancer Stem/Progenitor Cells Express the Tumor Antigen MUC1
Cancer Res., April 1, 2008; 68(7): 2419 - 2426.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
U. D. Lichtenauer, I. Shapiro, K. Geiger, M. Quinkler, M. Fassnacht, R. Nitschke, K.-D. Ruckauer, and F. Beuschlein
Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R
Endocrinology, March 1, 2008; 149(3): 1314 - 1322.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. Yin, P. Castagnino, and R. K. Assoian
ABCG2 Expression and Side Population Abundance Regulated by a Transforming Growth Factor {beta}-Directed Epithelial-Mesenchymal Transition
Cancer Res., February 1, 2008; 68(3): 800 - 807.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
P. Sundaram, W. Han, N. Cohen, B. Echalier, J. Albin, and L. Timmons
Caenorhabditis elegans ABCRNAi Transporters Interact Genetically With rde-2 and mut-7
Genetics, February 1, 2008; 178(2): 801 - 814.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Wu, Q. Wei, V. Utomo, P. Nadesan, H. Whetstone, R. Kandel, J. S. Wunder, and B. A. Alman
Side Population Cells Isolated from Mesenchymal Neoplasms Have Tumor Initiating Potential
Cancer Res., September 1, 2007; 67(17): 8216 - 8222.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. Lan, D. Cui, K. Nowka, and M. Derwahl
Stem Cells Derived from Goiters in Adults Form Spheres in Response to Intense Growth Stimulation and Require Thyrotropin for Differentiation into Thyrocytes
J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3681 - 3688.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Bhattacharya, A. Das, K. Mallya, and I. Ahmad
Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling
J. Cell Sci., August 1, 2007; 120(15): 2652 - 2662.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. M. Ho, A. V. Ng, S. Lam, and J. Y. Hung
Side Population in Human Lung Cancer Cell Lines and Tumors Is Enriched with Stem-like Cancer Cells
Cancer Res., May 15, 2007; 67(10): 4827 - 4833.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Wang, L.-P. Guo, L.-Z. Chen, Y.-X. Zeng, and S. H. Lu
Identification of Cancer Stem Cell-Like Side Population Cells in Human Nasopharyngeal Carcinoma Cell Line
Cancer Res., April 15, 2007; 67(8): 3716 - 3724.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
G. Huper and J. R. Marks
Isogenic Normal Basal and Luminal Mammary Epithelial Isolated by a Novel Method Show a Differential Response to Ionizing Radiation
Cancer Res., April 1, 2007; 67(7): 2990 - 3001.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
E. Savary, J. P. Hugnot, Y. Chassigneux, C. Travo, C. Duperray, T. Van De Water, and A. Zine
Distinct Population of Hair Cell Progenitors Can Be Isolated from the Postnatal Mouse Cochlea Using Side Population Analysis
Stem Cells, February 1, 2007; 25(2): 332 - 339.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
D. Dingli and F. Michor
Successful Therapy Must Eradicate Cancer Stem Cells
Stem Cells, December 1, 2006; 24(12): 2603 - 2610.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. A. Weinreich, I. Lintmaer, L. Wang, H. D. Liggitt, M. A. Harkey, and C. A. Blau
Growth factor receptors as regulators of hematopoiesis
Blood, December 1, 2006; 108(12): 3713 - 3721.
[Abstract] [Full Text] [PDF]