Contribution of the ABC Transporters Bcrp1 and Mdr1a/1b to the Side Population P - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

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) x9 |& M6 P. ?* G# o/ S& L

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.nl9 _8 W1 p4 l- E% o+ s: N7 \
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ABSTRACT# u3 W, c9 e3 j. L# a  A

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.1 s, `* ~4 i! x7 N7 R( W" o
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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 .* y; h$ c/ n/ A$ \: C
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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.
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MATERIALS AND METHODS/ i4 ~; b! O* G1 Q
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Generation and Analysis of Bcrp1–/–/Mdr1a/1b–/– Mice2 g; o! z5 b. M' Q% q, d9 F

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.. m. r) z6 i- G+ |/ Q9 @

Bcrp1 and Mdr1a/1b Both Contribute to the Mammary SP Phenotype* w: ^0 y, n2 E, t9 w$ G
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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).
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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.) T, y: x2 z6 f. I
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Loss of Mdr1a/1b Frequently Results in an Increased SP Phenotype in Bone Marrow, Which Is Reversible by Ko143/ u- L  B* l! T
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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

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' v3 m' O( ~3 Y4 P6 O# o0 w
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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.+ B6 j, _8 s! P4 Q4 K1 v! E. J

DISCUSSION
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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." N* K3 e1 ]& u% N% P

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