Contribution of the ABC Transporters Bcrp1 and Mdr1a/1b to the Side Population Phenotype in Mammary Gland and Bone Marrow of Mice
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《干细胞学杂志》
a The Netherlands Cancer Institute, Division of Experimental Therapy, Plesmanlaan, Amsterdam, The Netherlands;
b School of Biosciences, Cardiff University, Cardiff, United Kingdom;
c Institute of Cancer Research, Section of Cell and Molecular Biology, London, United Kingdom
Key Words. ATP-binding cassette transporter ? Adult bone marrow stem cells ? Progenitor cells ? Fluoresence-activated cell sorting analysis
Correspondence: Alfred H. Schinkel, Ph.D., Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Telephone: 31-20-5122046; Fax: 31-20-5122050; e-mail: a.schinkel@nki.nl
ABSTRACT
A subpopulation of cells with characteristics of stem cells can be identified by the side population (SP) phenotype, which is based on the efflux of the fluorescent dye Hoechst 33342. This was first shown by Goodell and colleagues, who speculated that the ATP-binding cassette (ABC) transporter MDR1 (P-glycoprotein/ABCB1) was responsible for this phenotype . Recently, however, it has become clear that another ABC transporter, Breast Cancer Resistance Protein (BCRP/ABCG2), is mainly responsible for the SP phenotype, at least in bone marrow . Based on the initial findings in the hematopoietic compartment, SP cells have now been identified in many other tissues including the mammary gland , skeletal muscle, pancreas, lung, retina, liver, testis, heart, and epidermis . In addition to normal tissues, it has further been demonstrated that cancer cell lines and primary tumor cells contain an SP . Those findings led to the proposition that tumors might also contain a minor subpopulation of drug-resistant "cancer" stem cells which might be crucial for their malignancy.
Although the exact physiologic roles of BCRP and MDR1 in stem cells are not yet known, it seems likely that an important function of these transporters is to protect them against the cytotoxic actions of xenotoxins or endogenous compounds. In line with this, it has been suggested that BCRP might protect the stem cell compartment against hypoxic stress by reducing heme or porphyrin accumulation .
Previously, we have identified an SP in human and mouse mammary glands and shown that they constitute an undifferentiated subpopulation that can differentiate into ductal and lobular structures and into myoepithelial and luminal epithelial cell types . To further characterize the respective roles of Bcrp1 and Mdr1a/1b in the SP phenotype, we have generated Bcrp1/Mdr1a/1b triple knockout mice and determined the relative transporter contributions to the SP phenotype in bone marrow and mammary gland.
MATERIALS AND METHODS
Generation and Analysis of Bcrp1–/–/Mdr1a/1b–/– Mice
Previously, we have generated mice with a targeted disruption of the ABC transporters Bcrp1 (Abcg2) or of Mdr1a (Abcb1a) and Mdr1b (Abcb1b) . In this study, we generated triple knockout mice lacking Bcrp1 and both Mdr1a and Mdr1b by crossing of the respective knockout lines. Bcrp1–/–/Mdr1a/1b–/– mice were fertile, had a normal life span, and were born at the expected mendelian ratio, indicating that there was no reduced embryonic viability. Standard plasma clinical chemical analysis and histological analysis revealed no abnormalities except that, similar to the Bcrp1–/– mice , levels of unconjugated bilirubin were slightly increased. Absence of Bcrp1 and Mdr1a/1b together, therefore, appears to be compatible with normal physiologic functioning of mice.
Bcrp1 and Mdr1a/1b Both Contribute to the Mammary SP Phenotype
The relative contributions of Bcrp1 and Mdr1a/1b to the SP phenotype in the mammary gland were determined by comparing mammary preparations from wild-type, Bcrp1–/– and Bcrp1–/–/Mdr1a/1b–/– mice. For determination of SP, we used pools of mammary glands (fourth inguinal pair) from 4–13 mice per analysis. Wild-type mammary glands had levels of epithelial SP cells that were consistent with our previous observations . Glands from Bcrp1–/– mice still had a clear but significantly reduced SP as compared with wild-type mice (0.04% vs. 0.22%, respectively; p = .024; Figs. 1A, 1B, 1D), suggesting that also in the mammary gland Bcrp1 makes a significant contribution to the SP phenotype. Mammary glands from Bcrp1–/–/Mdr1a/1b–/– animals had no detectable SP cells, indicating that together these transporters are fully responsible for the SP phenotype in the mammary gland (Figs. 1C, 1D).
Figure 1. Bcrp1 and Mdr1a/1b are required for the mammary side population (SP) phenotype. (A, B, C): Flow-cytometric SP analysis of mammary epithelial samples from (A) wild-type, (B) Bcrp1–/–, and (C) Bcrp1/Mdr1a/1b–/– mice. Percentages of SP cells within the gated regions of the fluorescence-activated cell sorting (FACS) traces are shown in each panel. (D): Distribution of percentages of SP cells from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1–/–/Mdr1a/1b–/– mice. Numbers of mice used to generate the data were 28 (wild-type), 40 (Bcrp1–/–), 12 (Mdr1a/1b–/–), and 17 (Bcrp1/Mdr1a/1b–/–). Each separate analysis is represented by a single point. The bar indicates the mean.
Loss of Mdr1a/1b Frequently Results in an Increased SP Phenotype in Bone Marrow, Which Is Reversible by Ko143
We next determined the relative contributions of Bcrp1 and Mdr1a/1b to the SP phenotype in bone marrow by comparing wild-type, Bcrp1–/–, Mdr1a/1b–/– and Bcrp1–/–/Mdr1a/1b–/– mice (Fig. 2). A clear but significantly reduced SP was detected in bone marrow of Bcrp1–/– mice as compared with wild-type mice (0.05% ± 0.07% versus 0.18% ± 0.20%, respectively; p < .01). In contrast to the mammary gland, however, bone marrow of Bcrp1–/–/Mdr1a/1b–/– mice still had a detectable SP (0.05% ± 0.08%; p < .01 as compared with wild-type), showing that the SP cells remaining in Bcrp1–/– bone marrow may not exclusively depend on Mdr1a/1b for efflux of Hoechst 33342 (Fig. 2B). Our data showing that Bcrp1 is responsible for the majority of the SP phenotype in hematopoietic cells are consistent with a previous suggestion that Bcrp1 is exclusively responsible for the SP phenotype in hematopoietic cells . Surprisingly, the percentage of SP cells detected in the bone marrow of Mdr1a/b–/– mice was frequently higher than wild-type controls, but this did not reach statistical significance due to high interindividual variation (mean 0.35% ± 0.36% vs. 0.18% ± 0.20%, respectively; p = .17). To further characterize this unexpected increase in SP in Mdr1a/1b–/– mice, we measured SPs in individual mice rather than in pooled samples. It should be noted that in other studies, hematopoietic SP was determined in pools of mice, masking possible interindividual differences . To compensate for the inherent variability in the measurement of percentage of SP, a larger number of samples was analyzed from each genotype (Fig. 2B). In total, between 28 and 46 individual marrows (using either one, or pools of two or four marrows per experiment) were analyzed for the genotypes shown. We found that the SP in the Mdr1a/1b–/– mice was highly variable and that some mice had exceptionally high SPs. The low variability of the SP in the Bcrp1–/–/Mdr1a/1b–/– mice shows that the variation in the Mdr1a/1b–/– mice is not due to the technical procedure. It should be noted that some variation was also observed in the wild-type and Bcrp1–/– mice which might be due to variable expression of the respective transporters. Treatment of the cells with the specific Bcrp1-inhibitor Ko143 strongly reduced the SP phenotype in both wild-type and Mdr1a/1b–/– animals (0.02% ± 0.02%; p < .01 and 0.05% ± 0.06%; p < .01 as compared with untreated wild-type, respectively), suggesting that Bcrp1 is primarily responsible for the SP phenotype in Mdr1a/1b–/– mice. In cases in which a significant SP fraction was detected in the Ko143-treated Mdr1a/1b–/– samples, the corresponding untreated half of the sample was found to have higher-than-normal SP levels, suggesting the involvement of a third transporter compensating for the loss of Mdr1a/1b.
Figure 2. Bcrp1, but not Mdr1a/1b, is required for the bone marrow side population (SP) phenotype. (A): Flow-cytometric SP analysis of bone marrow samples from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1/Mdr1a/1b–/– mice. Percentages of SP cells within the gated regions of the fluorescence-activated cell sorting (FACS) traces are shown in each panel. (B): Distribution of percentages of SP cells from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1/Mdr1a/1b–/– mice. Numbers of individual marrows used to generate the data were 46 (wild-type), 34 (Bcrp1–/–), 28 (Mdr1a/1b–/–), 28 (Bcrp1/Mdr1a/1b–/–), six (wild-type/Ko143 treated), and six (Mdr1a/1b–/–/Ko143 treated). Each separate analysis is represented by a single point. The bar indicates the mean.
Immunohistochemical Localization of Bcrp1 in Mammary Gland
Finally, we were interested whether Bcrp1 could be used as a marker to immunohistochemically detect cells in the virgin mammary gland representing the SP. The low background of staining demonstrated using Bcrp1 null tissues, allowed the identification of a small number of positive cells in wild-type virging lands (Figs. 3A–3D, 3F). As might be expected from the low proportion of SP cells in the virgin gland (normally less than 1%), very few positive cells were detected (Figs. 3A–3C). The few cells detected were closely associated with mammary ducts, were often single, and were almost always basal to smooth muscle actin–positive cells (Fig. 3F) as assessed by immunohistochemical staining. No positive cells were ever detected in the luminal cells of mid- and large-sized ducts. As there are major alterations in cellular proliferation and apoptosis throughout the estric cycle, staged glands were stained for Bcrp1, for a marker of proliferation (proliferating cell nuclear antigen ), and for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining). No major changes in the pattern of Bcrp1 expression were noted during the cycle (data not shown). In addition, no obvious relationship was detected between Bcrp1-expressing cells and those staining for PCNA or TUNEL (data not shown).
Figure 3. Localization of Bcrp1 in the virgin mouse mammary gland. Immunohistochemical detection of Bcrp1 in virgin mouse mammary glands: (A–C) wild-type, (D, E) Bcrp1–/–. (F): Smooth muscle actin staining in virgin wild-type mammary gland. Arrows indicate positively staining cells. Bars = 100 μM.
Although Bcrp1-expressing cells in the virgin gland are clearly basal to myoepithelial cells, they are intimately associated with mammary ducts and could thus be represented within the primary SP. However, we cannot formally exclude the possibility that cells intimately associated with the epithelium express levels of Bcrp1 below the histological detection limit and are capable of exporting sufficient Hoechst 33342 dye to be represented within the SP cell population. The niche that contains mammary epithelial stem cells remains to be identified, and the Bcrp1-expressing cells (as determined histologically and phenotypically) must thus be considered as candidate stem cells.
DISCUSSION
Our data show that the relative contributions of the ABC transporters Bcrp1 and Mdr1a/1b to the SP phenotype in different tissues, in this case bone marrow and mammary gland, can vary. As a consequence, the sensitivity of tissues to cytotoxic drugs that are substrates of these ABC transporters will also vary, which in turn might affect the chemotherapeutical treatment of patients. Our data also show that loss of expression of Mdr1a/1b in bone marrow resulted in an increased SP and suggest that this is due to compensatory upregulation of Bcrp1 and possibly a third transporter.
ACKNOWLEDGMENTS
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.
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.
Zhou S, Morris JJ, Barnes Y et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 2002;99:12339–12344.
Zhou S, Zong Y, Lu T et al. Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone. Biotechniques 2003;35:1248–1252.
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.
Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.
Alvi AJ, Clayton H, Joshi C et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 2002;5:R1–R8.
Welm BE, Tepera SB, Venezia T et al. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 2002;245:42–56.
Clayton H, Titley I, Vivanco M. Growth and differentiation of progenitor/stem cells derived from the human mammary gland. Exp Cell Res 2004;297:444–460.
Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.
Lechner A, Leech CA, Abraham EJ et al. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun 2002;293:670–674.
Summer R, Kotton DN, Sun X et al. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97–104.
Bhattacharya S, Jackson JD, Das AV et al. Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest Ophthalmol Vis Sci 2003;44:2764–2773.
Shimano K, Satake M, Okaya A et al. Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 2003;163:3–9.
Lassalle B, Bastos H, Louis JP et al. "Side population" cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 2003;131:479–487.
Martin CM, Meeson AP, Robertson SM et al. Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev Biol 2004;265:262–275.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A 2004;101:781–786.
Setoguchi T, Taga T, Kondo T. Cancer stem cells persist in many cancer cell lines. Cell Cycle 2004;3:414–415.
Hirschmann-Jax C, Foster AE, Wulf GG et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A 2004;101:14228–14233.
Krishnamurthy P, Ross DD, Nakanishi T et al. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J Biol Chem 2004;279:24218–24225.
Jonker JW, Buitelaar M, Wagenaar E et al. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci U S A 2002;99:15649–15654.
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.
Smalley MJ, Titley J, O’Hare MJ. Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting. In Vitro Cell Dev Biol Anim 1998;34:711–721.
Naylor S, Smalley MJ, Robertson D et al. Retroviral expression of Wnt-1 and Wnt-7b produces different effects in mouse mammary epithelium. J Cell Sci 2000;113:2129–2138.
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 U S A 1997;94:4028–4033.
Allen JD, Van Loevezijn A, Lakhai JM et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 2002;1:417–425.
Welm B, Behbod F, Goodell MA et al. Isolation and characterization of functional mammary gland stem cells. Cell Prolif 2003;36(suppl 1):17–32.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Montanaro F, Liadaki K, Schienda J et al. Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 2004;298:144–154.
Jonker JW, Merino G, Musters S et al. The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat Med 2005;11:127–129.
Petersen OW, Gudjonsson T, Villadsen R et al. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia. Cell Prolif 2003;36(suppl 1):33–44.
Clarke RB, Smith GH. Stem cells and tissue homeostasis in mammary glands. J Mammary Gland Biol Neoplasia 2005;10:1–3.
Morroni M, Giordano A, Zingaretti MC et al. Reversible transdifferentiation of secretory epithelial cells into adipocytes in the mammary gland. Proc Natl Acad Sci U S A 2004;101:16801–16806.(Johan W. Jonkera, Jamie F)
b School of Biosciences, Cardiff University, Cardiff, United Kingdom;
c Institute of Cancer Research, Section of Cell and Molecular Biology, London, United Kingdom
Key Words. ATP-binding cassette transporter ? Adult bone marrow stem cells ? Progenitor cells ? Fluoresence-activated cell sorting analysis
Correspondence: Alfred H. Schinkel, Ph.D., Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Telephone: 31-20-5122046; Fax: 31-20-5122050; e-mail: a.schinkel@nki.nl
ABSTRACT
A subpopulation of cells with characteristics of stem cells can be identified by the side population (SP) phenotype, which is based on the efflux of the fluorescent dye Hoechst 33342. This was first shown by Goodell and colleagues, who speculated that the ATP-binding cassette (ABC) transporter MDR1 (P-glycoprotein/ABCB1) was responsible for this phenotype . Recently, however, it has become clear that another ABC transporter, Breast Cancer Resistance Protein (BCRP/ABCG2), is mainly responsible for the SP phenotype, at least in bone marrow . Based on the initial findings in the hematopoietic compartment, SP cells have now been identified in many other tissues including the mammary gland , skeletal muscle, pancreas, lung, retina, liver, testis, heart, and epidermis . In addition to normal tissues, it has further been demonstrated that cancer cell lines and primary tumor cells contain an SP . Those findings led to the proposition that tumors might also contain a minor subpopulation of drug-resistant "cancer" stem cells which might be crucial for their malignancy.
Although the exact physiologic roles of BCRP and MDR1 in stem cells are not yet known, it seems likely that an important function of these transporters is to protect them against the cytotoxic actions of xenotoxins or endogenous compounds. In line with this, it has been suggested that BCRP might protect the stem cell compartment against hypoxic stress by reducing heme or porphyrin accumulation .
Previously, we have identified an SP in human and mouse mammary glands and shown that they constitute an undifferentiated subpopulation that can differentiate into ductal and lobular structures and into myoepithelial and luminal epithelial cell types . To further characterize the respective roles of Bcrp1 and Mdr1a/1b in the SP phenotype, we have generated Bcrp1/Mdr1a/1b triple knockout mice and determined the relative transporter contributions to the SP phenotype in bone marrow and mammary gland.
MATERIALS AND METHODS
Generation and Analysis of Bcrp1–/–/Mdr1a/1b–/– Mice
Previously, we have generated mice with a targeted disruption of the ABC transporters Bcrp1 (Abcg2) or of Mdr1a (Abcb1a) and Mdr1b (Abcb1b) . In this study, we generated triple knockout mice lacking Bcrp1 and both Mdr1a and Mdr1b by crossing of the respective knockout lines. Bcrp1–/–/Mdr1a/1b–/– mice were fertile, had a normal life span, and were born at the expected mendelian ratio, indicating that there was no reduced embryonic viability. Standard plasma clinical chemical analysis and histological analysis revealed no abnormalities except that, similar to the Bcrp1–/– mice , levels of unconjugated bilirubin were slightly increased. Absence of Bcrp1 and Mdr1a/1b together, therefore, appears to be compatible with normal physiologic functioning of mice.
Bcrp1 and Mdr1a/1b Both Contribute to the Mammary SP Phenotype
The relative contributions of Bcrp1 and Mdr1a/1b to the SP phenotype in the mammary gland were determined by comparing mammary preparations from wild-type, Bcrp1–/– and Bcrp1–/–/Mdr1a/1b–/– mice. For determination of SP, we used pools of mammary glands (fourth inguinal pair) from 4–13 mice per analysis. Wild-type mammary glands had levels of epithelial SP cells that were consistent with our previous observations . Glands from Bcrp1–/– mice still had a clear but significantly reduced SP as compared with wild-type mice (0.04% vs. 0.22%, respectively; p = .024; Figs. 1A, 1B, 1D), suggesting that also in the mammary gland Bcrp1 makes a significant contribution to the SP phenotype. Mammary glands from Bcrp1–/–/Mdr1a/1b–/– animals had no detectable SP cells, indicating that together these transporters are fully responsible for the SP phenotype in the mammary gland (Figs. 1C, 1D).
Figure 1. Bcrp1 and Mdr1a/1b are required for the mammary side population (SP) phenotype. (A, B, C): Flow-cytometric SP analysis of mammary epithelial samples from (A) wild-type, (B) Bcrp1–/–, and (C) Bcrp1/Mdr1a/1b–/– mice. Percentages of SP cells within the gated regions of the fluorescence-activated cell sorting (FACS) traces are shown in each panel. (D): Distribution of percentages of SP cells from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1–/–/Mdr1a/1b–/– mice. Numbers of mice used to generate the data were 28 (wild-type), 40 (Bcrp1–/–), 12 (Mdr1a/1b–/–), and 17 (Bcrp1/Mdr1a/1b–/–). Each separate analysis is represented by a single point. The bar indicates the mean.
Loss of Mdr1a/1b Frequently Results in an Increased SP Phenotype in Bone Marrow, Which Is Reversible by Ko143
We next determined the relative contributions of Bcrp1 and Mdr1a/1b to the SP phenotype in bone marrow by comparing wild-type, Bcrp1–/–, Mdr1a/1b–/– and Bcrp1–/–/Mdr1a/1b–/– mice (Fig. 2). A clear but significantly reduced SP was detected in bone marrow of Bcrp1–/– mice as compared with wild-type mice (0.05% ± 0.07% versus 0.18% ± 0.20%, respectively; p < .01). In contrast to the mammary gland, however, bone marrow of Bcrp1–/–/Mdr1a/1b–/– mice still had a detectable SP (0.05% ± 0.08%; p < .01 as compared with wild-type), showing that the SP cells remaining in Bcrp1–/– bone marrow may not exclusively depend on Mdr1a/1b for efflux of Hoechst 33342 (Fig. 2B). Our data showing that Bcrp1 is responsible for the majority of the SP phenotype in hematopoietic cells are consistent with a previous suggestion that Bcrp1 is exclusively responsible for the SP phenotype in hematopoietic cells . Surprisingly, the percentage of SP cells detected in the bone marrow of Mdr1a/b–/– mice was frequently higher than wild-type controls, but this did not reach statistical significance due to high interindividual variation (mean 0.35% ± 0.36% vs. 0.18% ± 0.20%, respectively; p = .17). To further characterize this unexpected increase in SP in Mdr1a/1b–/– mice, we measured SPs in individual mice rather than in pooled samples. It should be noted that in other studies, hematopoietic SP was determined in pools of mice, masking possible interindividual differences . To compensate for the inherent variability in the measurement of percentage of SP, a larger number of samples was analyzed from each genotype (Fig. 2B). In total, between 28 and 46 individual marrows (using either one, or pools of two or four marrows per experiment) were analyzed for the genotypes shown. We found that the SP in the Mdr1a/1b–/– mice was highly variable and that some mice had exceptionally high SPs. The low variability of the SP in the Bcrp1–/–/Mdr1a/1b–/– mice shows that the variation in the Mdr1a/1b–/– mice is not due to the technical procedure. It should be noted that some variation was also observed in the wild-type and Bcrp1–/– mice which might be due to variable expression of the respective transporters. Treatment of the cells with the specific Bcrp1-inhibitor Ko143 strongly reduced the SP phenotype in both wild-type and Mdr1a/1b–/– animals (0.02% ± 0.02%; p < .01 and 0.05% ± 0.06%; p < .01 as compared with untreated wild-type, respectively), suggesting that Bcrp1 is primarily responsible for the SP phenotype in Mdr1a/1b–/– mice. In cases in which a significant SP fraction was detected in the Ko143-treated Mdr1a/1b–/– samples, the corresponding untreated half of the sample was found to have higher-than-normal SP levels, suggesting the involvement of a third transporter compensating for the loss of Mdr1a/1b.
Figure 2. Bcrp1, but not Mdr1a/1b, is required for the bone marrow side population (SP) phenotype. (A): Flow-cytometric SP analysis of bone marrow samples from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1/Mdr1a/1b–/– mice. Percentages of SP cells within the gated regions of the fluorescence-activated cell sorting (FACS) traces are shown in each panel. (B): Distribution of percentages of SP cells from wild-type, Bcrp1–/–, Mdr1a/1b–/–, and Bcrp1/Mdr1a/1b–/– mice. Numbers of individual marrows used to generate the data were 46 (wild-type), 34 (Bcrp1–/–), 28 (Mdr1a/1b–/–), 28 (Bcrp1/Mdr1a/1b–/–), six (wild-type/Ko143 treated), and six (Mdr1a/1b–/–/Ko143 treated). Each separate analysis is represented by a single point. The bar indicates the mean.
Immunohistochemical Localization of Bcrp1 in Mammary Gland
Finally, we were interested whether Bcrp1 could be used as a marker to immunohistochemically detect cells in the virgin mammary gland representing the SP. The low background of staining demonstrated using Bcrp1 null tissues, allowed the identification of a small number of positive cells in wild-type virging lands (Figs. 3A–3D, 3F). As might be expected from the low proportion of SP cells in the virgin gland (normally less than 1%), very few positive cells were detected (Figs. 3A–3C). The few cells detected were closely associated with mammary ducts, were often single, and were almost always basal to smooth muscle actin–positive cells (Fig. 3F) as assessed by immunohistochemical staining. No positive cells were ever detected in the luminal cells of mid- and large-sized ducts. As there are major alterations in cellular proliferation and apoptosis throughout the estric cycle, staged glands were stained for Bcrp1, for a marker of proliferation (proliferating cell nuclear antigen ), and for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining). No major changes in the pattern of Bcrp1 expression were noted during the cycle (data not shown). In addition, no obvious relationship was detected between Bcrp1-expressing cells and those staining for PCNA or TUNEL (data not shown).
Figure 3. Localization of Bcrp1 in the virgin mouse mammary gland. Immunohistochemical detection of Bcrp1 in virgin mouse mammary glands: (A–C) wild-type, (D, E) Bcrp1–/–. (F): Smooth muscle actin staining in virgin wild-type mammary gland. Arrows indicate positively staining cells. Bars = 100 μM.
Although Bcrp1-expressing cells in the virgin gland are clearly basal to myoepithelial cells, they are intimately associated with mammary ducts and could thus be represented within the primary SP. However, we cannot formally exclude the possibility that cells intimately associated with the epithelium express levels of Bcrp1 below the histological detection limit and are capable of exporting sufficient Hoechst 33342 dye to be represented within the SP cell population. The niche that contains mammary epithelial stem cells remains to be identified, and the Bcrp1-expressing cells (as determined histologically and phenotypically) must thus be considered as candidate stem cells.
DISCUSSION
Our data show that the relative contributions of the ABC transporters Bcrp1 and Mdr1a/1b to the SP phenotype in different tissues, in this case bone marrow and mammary gland, can vary. As a consequence, the sensitivity of tissues to cytotoxic drugs that are substrates of these ABC transporters will also vary, which in turn might affect the chemotherapeutical treatment of patients. Our data also show that loss of expression of Mdr1a/1b in bone marrow resulted in an increased SP and suggest that this is due to compensatory upregulation of Bcrp1 and possibly a third transporter.
ACKNOWLEDGMENTS
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.
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.
Zhou S, Morris JJ, Barnes Y et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 2002;99:12339–12344.
Zhou S, Zong Y, Lu T et al. Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone. Biotechniques 2003;35:1248–1252.
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.
Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.
Alvi AJ, Clayton H, Joshi C et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 2002;5:R1–R8.
Welm BE, Tepera SB, Venezia T et al. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 2002;245:42–56.
Clayton H, Titley I, Vivanco M. Growth and differentiation of progenitor/stem cells derived from the human mammary gland. Exp Cell Res 2004;297:444–460.
Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.
Lechner A, Leech CA, Abraham EJ et al. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun 2002;293:670–674.
Summer R, Kotton DN, Sun X et al. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97–104.
Bhattacharya S, Jackson JD, Das AV et al. Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest Ophthalmol Vis Sci 2003;44:2764–2773.
Shimano K, Satake M, Okaya A et al. Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 2003;163:3–9.
Lassalle B, Bastos H, Louis JP et al. "Side population" cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 2003;131:479–487.
Martin CM, Meeson AP, Robertson SM et al. Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Dev Biol 2004;265:262–275.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci U S A 2004;101:781–786.
Setoguchi T, Taga T, Kondo T. Cancer stem cells persist in many cancer cell lines. Cell Cycle 2004;3:414–415.
Hirschmann-Jax C, Foster AE, Wulf GG et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A 2004;101:14228–14233.
Krishnamurthy P, Ross DD, Nakanishi T et al. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J Biol Chem 2004;279:24218–24225.
Jonker JW, Buitelaar M, Wagenaar E et al. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci U S A 2002;99:15649–15654.
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.
Smalley MJ, Titley J, O’Hare MJ. Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting. In Vitro Cell Dev Biol Anim 1998;34:711–721.
Naylor S, Smalley MJ, Robertson D et al. Retroviral expression of Wnt-1 and Wnt-7b produces different effects in mouse mammary epithelium. J Cell Sci 2000;113:2129–2138.
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 U S A 1997;94:4028–4033.
Allen JD, Van Loevezijn A, Lakhai JM et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 2002;1:417–425.
Welm B, Behbod F, Goodell MA et al. Isolation and characterization of functional mammary gland stem cells. Cell Prolif 2003;36(suppl 1):17–32.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Montanaro F, Liadaki K, Schienda J et al. Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 2004;298:144–154.
Jonker JW, Merino G, Musters S et al. The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat Med 2005;11:127–129.
Petersen OW, Gudjonsson T, Villadsen R et al. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia. Cell Prolif 2003;36(suppl 1):33–44.
Clarke RB, Smith GH. Stem cells and tissue homeostasis in mammary glands. J Mammary Gland Biol Neoplasia 2005;10:1–3.
Morroni M, Giordano A, Zingaretti MC et al. Reversible transdifferentiation of secretory epithelial cells into adipocytes in the mammary gland. Proc Natl Acad Sci U S A 2004;101:16801–16806.(Johan W. Jonkera, Jamie F)