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发表于 2009-3-5 00:07 |只看该作者 |倒序浏览 |打印

作者：Fariba Behboda, Wa Xiana, Chad A. Shawb, Susan G. Hilsenbeckc, Anna Tsimelzonc, Jeffrey M. Rosena % t$ ?; Z+ Z6 H" O' p

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      【摘要】! p+ z, e. n; l2 m, G# Y  @
   Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst-effluxing side population cells, mammary gland side population cells (MG-SPs). To better characterize MG-SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (mammary gland non-side population cells ). For analysis, Gene Ontology (GO) that describes genes in terms of biological processes and Ontology Traverser (OT) that performs enrichment analysis were used. OT showed that MG-SP-specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multidrug-resistant transporters, organogenesis, and vasculogenesis. The MG-NSP-upregulated genes were enriched in the GO category of cellular organization and biogenesis, which includes basal epithelial markers, p63, smooth muscle actin, myosin, 6 integrin, cytokeratin (CK) 14, and luminal markers CK8 and CD24. Additional studies showed that a higher percentage of MG-SPs exist in the G1 phase of the cell cycle compared with the MG-NSPs. G1 cell cycle block of MG-SPs may be explained by higher expression of cell cycle-negative regulatory genes such as transforming growth factor-&szlig;2, insulin-like growth factor binding protein-5, P18INK4C, and wingless-5a (Wnt-5a). Accordingly, a smaller percentage of MG-SPs expressed nuclear &szlig;-catenin, possibly as a consequence of the higher expression of Wnt-5a. In conclusion, microarray gene profiling suggests that MG-SPs are a lineage-deficient mammary gland subpopulation expressing key genes involved in cell cycle regulation, development, and angiogenesis.
      【关键词】 Mammary gland side population cells Murine and human Microarray gene profiling4 I0 G; {) i0 I' Y, v* i
   INTRODUCTION3 X1 V& }; V* ^7 s/ N. k* q

Goodell et al. reported almost a decade ago that a population of bone marrow cells was capable of an elevated rate of Hoechst 33342 dye efflux and that flow cytometry (FACS) analysis performed at two different wavelengths permitted the isolation of these cells, termed side population cells (SPs) .

Similar to the bone marrow, the mammary gland in mice and human contains a distinct population of Hoechst-effluxing SPs known as mammary gland SPs (MG-SPs). MG-SPs constitute about 0.25%¨C5% of the epithelial cells .% v9 U) r6 w* P2 P' n

Similar to mice mammary glands, a majority of human breast SPs initially do not express EMA or common acute lymphocytic leukemia antigen (CALLA), markers of human luminal and myoepithelial cells, respectively .

Although these studies have suggested that MG-SPs may be considered candidate stem cells, evidence that they are enriched for functional stem cell activity is still only correlative . With these caveats in mind, and to better characterize the MG-SP population, we have performed comprehensive gene expression profiling studies of these cells using Affymetrix microarrays (Santa Clara, CA, http://www.affymetrix.com). These studies have revealed that MG-SPs differentially express a number of key genes involved in cell cycle control, organogenesis, and angiogenesis and do not express a number of mammary gland differentiation markers. Furthermore, MG-SPs express a number of cell cycle checkpoints and multidrug transporter genes. These gene profiling studies provide an important addition to the stem cell databases and will be useful for future comparisons to other normal mammary gland stem and progenitor cells, as well as an increasing number of cancer models.1 M+ y5 V% y  {- c7 |
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MATERIALS AND METHODS  r* U' f% E$ r, q. Z

Primary Mammary Epithelial Cell Culture and Hoechst Staining1 t* R' {; M$ `+ O% g9 z5 a- ~

The methods for the isolation of mammary glands, preparation of primary mammary epithelial cell cultures, as well as Hoechst staining, have been described previously . For the microarray experiments, primary mammary epithelial cells (PMECs) were prepared from 15 to 20 virgin C57/BL6 mice 4¨C6 weeks of age. PMECs were cultured for 3 days to facilitate the preparation of sufficient numbers of single cells required for flow cytometry analysis and gene profiling. Analysis and sorting were performed on a triple laser MoFlo (Cytomation, Fort Collins, CO, http://www.cytomation.com). The Hoechst dye was excited at 350 nm, and its fluorescence was measured at two wavelengths, 450/20 band pass (Hoechst Blue) and 675 Edge Filler long pass (Hoechst Red).

Microarray Studies, C; p. Q# A+ L. b1 \

Total RNA was isolated from the sorted MG-SPs and MG-NSPs using an RNA purification kit (PicoPure RNA Isolation Kit; Arcturus, Mountain View, CA, http://www.arctur.com), followed by mRNA amplification using a T7 global amplification method (Two-Cycle Target Labeling kit; Affymetrix), DNA fragmentation, biotinylation, and hybridization onto Affymetrix 430 2.0 array chips. Normalization and model-based expression analyses were performed using dChip (dChip software; Harvard School of Public Health, Boston, http://www.dchip.org)  as outlined in the experimental design section.; p  l4 K; E; @7 F
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Cell Cycle Analysis

Cell cycle analysis was performed on the sorted MG-SPs, MG-NSPs, and unsorted PMECs according to the protocol described . Briefly, cells were washed in phosphate-buffered saline (PBS) and resuspended in 0.5 ml of PCBS solution (0.1 M phosphate-citrate, 0.15 M NaCl, 5 mM sodium EDTA, 0.5% bovine serum albumin, pH 4.8) containing 0.02% saponin (catalog no. S4521; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and 10 µg/ml 7-aminoactinomycin D (7-AAD; catalog no. A9400; Sigma-Aldrich) followed by incubation for 20 min at room temperature. Cells were then washed one time in PBS and resuspended in 0.5 ml of PCBS containing 10µg/ml 7-AAD and 1 µg/ml pyronin Y (PY) (catalog no. P9172; Sigma-Aldrich). The mixture was kept at 4¡ãC and analyzed the following day using a FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).
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Immunofluorescence
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MG-SPs and MG-NSPs were sorted and plated on collagen-coated coverslips overnight to allow for cell adherence. Cells were then fixed in BD Cytofix/Cytoperm (catalog no. 554722; BD Biosciences, San Diego, http://www.bdbiosciences.com) for 15 min followed by washing and blocking using BD Perm/Wash buffer (catalog no. 554723; BD Biosciences) according to the manufacturer¡¯s guidelines. Immunofluorescence was carried out using nonphosphorylated active ß-catenin (dephosphorylated on Ser37; catalog no. 05-01; Upstate Biotechnology, Lake Placid, NY, http://www.upstate.com) following the standard protocols recommended by the manufacturer. The antibody was used at a dilution of 1:200 at 4¡ãC overnight. Immunofluorescence staining was also performed on cells cytospun onto slides immediately following a sort using another antibody that also recognizes nonphosphorylated active ß-catenin (dephosphorylated on Ser37 or Thr41; catalog no. 05-665; Upstate).; F6 v4 c! n( K% s/ W
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Gene Ontology (GO) and Ontology Traverser, \) ^0 f9 {; Z% M1 V$ ~& T3 w
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Ontology Traverser (OT) is a web-based tool for performing enrichment analysis of microarray gene lists. The Traverser works by mapping gene lists onto the Gene Ontology (GO) data structure and calculating enrichment statistics for each GO node. The enrichment statistics are reported for GO nodes found in any path leading to GO annotations for the probes in the microarray gene list. All GO levels are considered in the analysis, and the entire connectedness of the GO data structure is being used (a directed acyclic graph). Detailed information regarding OT and the formulas used to calculate enrichment statistics have been published.7 p  _8 V" B1 D1 T6 Z( s$ ^

Quantitative Polymerase Chain Reaction

RNA was extracted from MG-SPs and MG-NSPs using an RNA purification kit (PicoPure RNA Isolation Kit; Arcturus). One microgram of RNA per sample was treated with DNase I as recommended by the manufacturer, followed by generation of cDNA using Superscript II (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). One-tenth of the final reaction volume was used in quantitative polymerase chain reactions (Q-PCRs) containing 1 mM MgCl2, platinum Taq (Invitrogen), and 1 x SYBR green (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). PCR was performed using the ABI PRISM 7500 thermocycler (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) with the following cycles: 94¡ãC for 30 seconds, 55¡ãC for 30 seconds, and 72¡ãC for 30 seconds for 40 cycles. Relative quantitation of gene expression was calculated and normalized to glyceraldehyde-3-phosphate dehydrogenase. Q-PCR primers are listed in supplemental online Table 3.

RESULTS AND DISCUSSION

Experimental Design
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Gene expression profiles were obtained by hybridizing amplified RNA from four replicate MG-SP and MG-NSP samples to Affymetrix 430 2.0 microarray chips. To isolate MG-SP and MG-NSPs, mammary gland cells were stained using the Hoechst dye 33342 and fluorescence displayed at two wavelength emissions, blue and red. The MG-SP and MG-NSP regions are indicated by trapezoids on the left (R1) and right (R2), respectively (Fig. 1). We and others have previously shown that the R1 region is composed of side population cells since verapamil markedly inhibits their appearance . Selected quality assessment parameters are shown in supplemental online Table 1. One of the replicate sets was eliminated from further analysis due to the high percentage of probe sets called an array outlier after model-based expression analysis by dChip. The genes were filtered to eliminate those with very low expression values in most samples. From 45,000 probe sets, 16,744 probe sets were retained and used for further analysis. For normalization purposes and to allow for data comparison with other centers, universal mouse reference RNA (Stratagene, La Jolla, CA, http://www.stratagene.com) was hybridized to two chips. All other arrays were normalized to one of the universal mouse reference RNA arrays expressing average intensity closest to the median intensity of all chips. For comparison, a paired t test was performed with a false discovery rate (FDR) by permutation of class labels set at
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Figure 1. A representative mammary gland side population cell (MG-SP) profile. Mammary gland cells were stained using the Hoechst dye 33342 and fluorescence displayed at two wavelength emissions, blue (FL7) and red (FL8). MG-SP and mammary gland non-side population cell (MG-NSP) regions are indicated by trapezoids on the left (R1) and right (R2), respectively. As indicated by the flow cytometry profile, MG-SP represent approximately 2% and MG-NSPs approximately 16.33% of the mammary gland cells. The remaining 81.67% are dead cells and cellular debris.

Figure 2. Differentially expressed number of genes by the mammary gland side population cells (MG-SPs) and non-side population cells (MG-NSPs). Each bar represents the number of genes differentially expressed by MG-SPs and MG-NSPs based on a paired t test comparison plotted against the selected criteria, p value, fold change, and false discovery rate (FDR). a, 1.2-fold change, FDR 9.4%, p value  .05; b, 1.5-fold, FDR 3.4%, p value  .05; c, twofold change, FDR 0%, p value  .05; d, twofold change, FDR 0%, p value  .01.
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Gene Ontology Grouping of MG-SP- and MG-NSP-Specific Genes

To examine the significant biological processes represented by MG-SP and MG-NSP-specific genes, we used GO annotations (http://www.geneontology.org) that describe genes in terms of their associated biological processes and OT that performs enrichment analysis of microarray gene lists. Gene lists that were differentially expressed by MG-SPs and MG-NSPs (771 genes at 1.5-fold difference, p value  .05%, FDR
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Figure 3. Ontology Traverser analysis of mammary gland side population cell (MG-SP) and non-side population cell (MG-NSP)-specific genes. Significant Gene Oncology categories represented by MG-SP-specific (A) and MG-NSP-specific (B) genes. The group of genes used for this analysis included 771 differentially expressed genes at a 1.5-fold difference in expression value, FDR = 3.4%, and a p value  .05.
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MG-SP Gene Profiles

Cell Cycle Regulation and Cell Cycle Checkpoints. One of the significant findings in this analysis was that MG-SP-specific genes were mapped to the enriched GO categories of cell cycle, cell cycle regulation, and cell cycle checkpoints (Fig. 3A). The genes in these categories included those encoding G2/M cell cycle proteins such as cyclin A2; cyclin B1; cyclin B2; Ki67; ubiquitin-conjugated enzyme e2c; kinesin family members 11, 22, and 2c; kinetochore-associated protein 2; and cdc 20 (supplemental online Table 4A). Cell cycle analysis of the MG-SPs and MG-NSPs showed that approximately 83% of the MG-SPs existed in G1 phase of the cell cycle compared with 61% of the MG-NSPs and 43% of the unsorted PMECs (Table 1; Fig. 4). The mechanism responsible for the accumulation of a higher percentage of MG-SPs in G1 phase of the cell cycle requires further studies. However, we speculate that the MG-SP G1 phase cell cycle block may be explained by the increased expression of negative cell cycle regulatory proteins such as p18INK4C (an early G1 phase cell cycle inhibitor); checkpoint kinase Chk1; aurora kinases A and B; transducer of ERBB2, 2 (TOB-2); insulin-like growth factor binding protein-5 (IGFBP-5); and ligands such as transforming growth factor ß-2 (TGF-ß2) and Wnt-5a, a noncanonical Wnt ligand.+ r8 i. T# a& ^2 K8 a: c# H* e

Table 1. Cell cycle analysis of MG-SP, MG-NSP, and unsorted PMEC cells
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Figure 4. Flow cytometry analysis of MG-SP and MG-NSP cell cycle status. MG-SPs and MG-NSPs were sorted and stained with PY and 7-AAD for the quantification of RNA and DNA, respectively, as described in Materials and Methods. A higher percentage of MG-SPs (83.6%) exist in G1 phase of the cell cycle compared with MG-NSPs (61%). Abbreviations: 7-AAD, 7-aminoactinomycin D; MG-NSP, mammary gland non-side population cell; MG-SP, mammary gland side population cell; PY, pyronin Y.

TGF-ß2 and IGFBP-5 expression levels were also increased in the LRCs in the skin bulge, an epidermal stem cell niche microenvironment, and have been proposed to mediate the quiescent state of these cells . These results suggest that the self-renewal and engraftment of the MG-SPs may be similarly affected by the negative regulation imposed by these proteins.
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Wnt-5a is a Wnt ligand that activates the noncanonical Wnt signaling pathway in many biological systems, including Xenopus embryos and mammalian cells . Similar to the skin bulge, we speculate that the block of MG-SPs in the G1 phase of the cell cycle may be due to an inactive canonical Wnt signaling. Whether Wnt-5a contributes to the inactivation of canonical Wnt signaling and the G1 phase block of the MG-SPs requires further investigation.2 @5 m0 |" z; O7 _1 G
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Figure 5. Immunofluorescence staining for active ß-catenin. (A): Representative MG-SP, MG-NSP, and Wnt hyperplasia tumor cells stained for active ß-catenin. Most MG-NSP and Wnt hyperplasia tumor cells express a distinct nuclear active ß-catenin staining in the heterochromatin regions. (B): Bar graph indicates the percentage of unsorted PMECs (1% ¡À 0.2%), MG-SPs (11.3% ¡À 6%), and MG-NSPs (41% ¡À 3.2%) that stained positive for the nuclear active ß-catenin. A total of 651 unsorted PMECs, 354 MG-SPs, and 568 MG-NSPs were counted. Abbreviations: MG-NSP, mammary gland non-side population cell; MG-SP, mammary gland side population cell; PMEC, primary epithelial cell.

A number of cell cycle checkpoint genes, such as those encoding aurora kinases A and B, Gadd45g, Chk1, and cyclin D type binding protein 1, displayed significantly higher expression in the MG-SPs compared with MG-NSPs. These genes are normally upregulated in response to genotoxic agents to prevent cell cycle entry, allowing time for DNA repair. These data suggest that MG-SPs might have an active DNA damage response to ensure chromosomal repair and genomic stability. As mentioned previously, isolation of MG-SPs requires use of the DNA vital dye, Hoechst 33342. Hoechst 33342 is toxic, which may cause cell death or the induction of a DNA damage response. However, a DNA damage response is usually very rapid and involves post-translational processing, relocalization, and conformational change or stability of pre-existing proteins . Up-regulation of 8 of the 11 genes was associated with poor prognosis, and 4 of those 8 genes were also upregulated in the MG-SPs, including those encoding Ki67, cyclin B1, BUB1, and kinetochore-associated 2 (KNTC2). Three of the four genes (those encoding Ki67, cyclin B1, and KNTC2) were included in the Ontology Traverser-enriched GO category of cell cycle regulation (supplemental online Table 4A).

Multidrug-Resistant Transporters. MG-SPs, compared with MG-NSPs, express increased levels of MDRTPs such as ATP-binding cassette, subfamily B (MDR/TAP), member 1A (mdr 1a); ATP-binding cassette, subfamily B (MDR/TAP), member 1B (mdr 1b); ATP-binding cassette, subfamily C (CFTR/MRP), member 4; ATP-binding cassette, subfamily C (CFTR/MRP), member 9; and ATP-binding cassette, subfamily G (WHITE), member 2 (Bcrp1 or ABCG2). The expression of these transporters by the MG-SPs suggested that the SP phenotype may be contributed by a number of ATP-binding cassette transporters in the mammary gland, not just Bcrp1 as seen in the hematopoietic stem cells. These results have been confirmed by the analysis of the SP population in Bcrp1/Mdr 1a/1b triple knockout mice ., k* u) j) N3 o- s; `$ _" O

Cells that express MDRTPs, such as SPs, are protected from chemotoxic agents as well as from hypoxic conditions. MDRTPs are responsible for the efflux of agents such as mitoxantrone, topotecan, doxorubicin, daunorubicin, and methotrexate . These results, coupled with higher expression of DNA damage repair and checkpoint genes by the MG-SPs, suggest that several mechanisms may be in place to protect MG-SPs against cellular insult and to ensure the maintenance of chromosomal integrity specifically in these cells.$ y" H* q" ?, b0 A0 V' a

Angiogenesis. Another enriched GO category for MG-SPs was angiogenesis and vascular development (Fig. 3A; supplemental online Table 4A). The genes included in this category were known endothelial-specific genes such as Tie-2 (a receptor for angiopoietin 1 and 2), enos (endothelial nitric-oxide synthase), angiopoietin-2, Flt-1 (also known as vascular endothelial growth factor receptor-1), and FLK-1 (also known as vascular endothelial growth factor receptor-2). Others that belong to this category but were not endothelial-specific genes were plexin D1, neuropillin 1, endoglin, EGF-like domain 7 (Egfl7), and cysteine rich protein 61 (CYR61). CYR61 and Egfl7 are known major inducers of angiogenesis. CYR61 is a member of the cysteine-rich CYR61 family of secreted matricellular proteins, which are key modulators of cell-matrix interactions ) by flow cytometry. As seen in Figure 6, a higher percentage of MG-SPs express CD31 compared with MG-NSPs (18.6% ¡À 7.8% vs. 2.17% ¡À 0.8%).5 L' |& L# b  B' |  ^, e! D
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Figure 6. Expression of CD31 in MG-SPs versus MG-NSPs. Primary epithelial cells were stained with Hoechst 3342 as described in Materials and Methods, followed by staining with the anti-CD31 antibody. MG-SPs and MG-NSPs were gated and analyzed for the expression of CD31. A significantly higher percentage of MG-SPs expresses CD31 compared with MG-NSPs (18.6% ¡À 7.8% vs. 2.17% ¡À 0.8%, respectively). Bar graph represents accumulated data from four independent experiments. Abbreviations: MG-NSP, mammary gland non-side population cell; MG-SP, mammary gland side population cell.

We speculate that MG-SPs may be an important component of the mammary gland stem cell niche, contributing to mammary gland development, remodeling, and neovascularization. Neovascularization involves the incorporation of endothelial precursor cells derived mainly from the bone marrow or blood. However, solid tissue, such as muscle-derived SPs, may also contain endothelial precursor cells. It has been shown that muscle-derived SPs expressed endothelial markers Tie-2 and CD31 and engrafted into endothelium during vascular regeneration in response to injury .

The present study indicates that about 18% of the MG-SPs expressed the endothelial surface marker CD31, compared with approximately 2% of the MG-NSPs. Previous studies have shown that MG-SPs can give rise to differentiated epithelial structures in vitro and to mammary gland outgrowths in vivo . Whether this subpopulation of MG-SPs belongs to the endothelial and/or epithelial lineage requires a comprehensive analysis using other endothelial and/or epithelial markers and functional in vivo transplantation studies.# R9 c% A, ~" m! q/ Z: G1 ]
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Organogenesis. OT showed enrichment of genes involved in organogenesis, pattern formation, and development in the MG-SPs. These genes included transcription factors such as the homeobox A3 (Hoxa3), homeobox A5 (Hoxa5), Tbx18, Hesr-1 (a homeobox and notch target gene), Gli3 (a hedgehog transcription factor), Msx-1, and Msx-2. Hedgehog signaling and Notch pathways are known to play essential roles in the self-renewal of stem cells in neural, hematopoietic, intestinal, and mammary gland tissue .2 J' @7 d$ U! J' q
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Homeobox genes Msx-1 and Msx-2 play essential roles in mammary gland development . Therefore, these results suggest that Msx-1 and Msx-2 genes may regulate the development and fate determination of the MG-SPs. It is likely that MG-SPs in the periductal areas may mediate epithelial-stromal interactions and thus may regulate mammary gland development.% ^  B6 |  r- t9 l5 p
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Homeobox A5 (Hoxa5) has been shown to be downregulated in a majority of human breast tumors by promoter methylation . However, a role for Hoxa3 in the mammary gland has not been reported.& @$ o  V+ j! G" E4 ~

Hairy/enhancer of split-related (Hesr) proteins, such as Hesr-1/Hey1, are basic helix-loop-helix transcription factors implicated in cell fate decisions. Hesr-1 is a direct transcriptional target of the Notch signaling pathway. The Notch signal transduction pathway has also been implicated in the self-renewal of stem cells in hematopoietic, neural, and germ cells. Using Notch receptor activating ligands as well as blocking antibodies, Dontu et al. have shown a role for the Notch pathway in self-renewal and fate determination of mammary gland stem cells .' ~. v5 e2 a* _0 J
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A role for Gli3 in mammary gland development has not been reported, except for the possible involvement of Gli3 in the development of mammary placode induction . Together, these data indicate that Gli3 may mediate MG-SPs self-renewal and/or fate determination.

MG-NSP-Specific Gene Profiles: Cellular Organization and Biogenesis5 d6 O: C3 E( z' f7 G. C

MG-NSPs express a number of genes belonging to the enriched GO category of cellular organization and biogenesis (Fig 3.; supplemental online Table 4B). These genes include basal-specific mammary epithelial cell markers such as p63, smooth muscle actin, myosin, 6 integrin (CD49f), and CK5/14, as well as the luminal epithelial markers CK8 and CD24. We also compared our data to the tumor database reported by Perou et al. .( Z8 u" m# g" ~+ {* k2 I
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CONCLUSION! q, o$ w: }4 D5 U5 s( T
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We have performed gene profiling on a subpopulation of mammary gland cells known as mammary gland side population cells. The stem cell potential of these cells has been demonstrated by several in vitro studies; however, direct in vivo evidence is still lacking. Therefore, the exact identity of the MG-SPs is still not known. These gene profiling studies have attempted to clarify SP identity by revealing unique genes, signaling pathways, and markers expressed in this population. In summary, our gene profiling using OT has allowed us to build several hypotheses that require further testing and functional validation. Specifically, MG-SPs lacked the expression of several lineage-specific mammary gland markers, such as myoepithelial and/or luminal markers. Therefore, MG-SPs appear to represent a heterogeneous stem/progenitor cell population lacking mammary gland cell differentiation markers. It is also possible that MG-SPs may serve as a component of the mammary gland stem cell niche, providing support to the mammary gland stem cells. MG-SPs displayed reduced expression of CK6, a putative mammary gland stem/progenitor cell marker. Thus, CK6 expression may be higher in the more committed progenitors. In addition, MG-SPs may have mechanisms in place to protect them from DNA damage, hypoxic conditions, and chemotoxic insults since they express DNA damage response genes as well as MDRTPs. Why in particular these cells as compared to other mammary epithelial cells require such protective mechanisms is not clear and requires further investigation. The higher expression of endothelial-specific genes, angiogenic factors, and genes involved in development by MG-SPs suggests that these cells may be an important component of the stem cell niche and as such may play an essential role in mammary gland development. Additionally, gene profiling showed that MG-SPs are in a state of readiness to enter the cell cycle, since they express a number of G2/M cell cycle regulatory genes and they exist mainly in the G1 phase of the cell cycle. The blockade of MG-SPs in G1 may be due to an inactive canonical Wnt signaling pathway. It is also intriguing to speculate that MG-SPs may be involved in tumorigenesis, since these cells express several of the MDRTPs, as well as 4 out of the 11 cancer stem cell signature genes. In contrast, MG-NSPs express a number of mammary gland luminal and myoepithelial markers, suggesting that MG-NSPs may include differentiated or more committed progenitor cells.$ p+ B8 ^+ P" ^& V0 ?
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ACKNOWLEDGMENTS
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We thank Drs. Daniel Medina, Margaret A. Goodell, Yi Li, Kaylee Schwertfeger, Tracy Vargo Gogola, and Heather L. Lamarca for helpful comments. We thank Dr. Charles M. Perou for the comparison of microarray data to the reported human tumor database and for helpful comments. In addition, we thank Mike Cubbage, Chris Threeton, and Tatiana Goltsova for excellent flow cytometry assistance and Lisa While and Hilary N. Lewis at the Baylor Microarray Core Facility. This work was supported by grants F32 CA101560-03 (F.B.) and UO1 CA84243 (J.M.R.) from the National Cancer Institute.
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AUTHORS¡¯ NOTE) L1 V- Z6 a) z/ l( [0 a
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Recently, MG-SPs have been analyzed for the expression of endothelial specific adhesion molecule-1 (ESAM-1) by FACS. ESAM-1 is an adhesion molecule shown to be upregulated by long-term HSCs . The majority of MG-SPs expressed ESAM compared to MG-NSPs (83.4 ¡À 6.4 vs. 4.2 ¡À 0.5).

DISCLOSURES  Y: r( Y5 _2 P' \( F# y
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The authors indicate no potential conflicts of interest.: F2 R- J1 E, V& r' M5 p
      【参考文献】

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¨C1806.

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¨C1034.  Q6 i3 r- S- q7 P
, T( p1 N! \- K  t5 t
Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123¨C134.

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¨C2773.4 e& |9 H, A! Q' f" C. |: i) H' x

He DN, Qin H, Liao L et al. Small intestinal organoid-derived SP cells contribute to repair of irradiation-induced skin injury. Stem Cells Dev 2005;14:285¨C291.

Jonker JW, Freeman J, Bolscher E et al. Contribution of the ABC-transporters Bcrp1 and Mdr1a/1b to the side population phenotype in mammary gland. STEM CELLS 2005;23:1059¨C1065.
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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 2004;131:479¨C487.2 q, w' v. \9 N3 X( b
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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¨C674.
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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¨C275.% J6 a. H0 t9 @: c6 n5 D! u
/ k% ~& U7 I. l2 R+ X0 D* R; M
Nadin BM, Goodell MA, Hirschi KK. Phenotype and hematopoietic potential of side population cells throughout embryonic development. Blood 2003;102:2436¨C2443.! T' F$ Z( L  O+ h' l9 U

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¨C9.. }5 C6 {' c; u

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¨CL104.

Yano S, Ito Y, Fujimoto M et al. Characterization and localization of side population cells in mouse skin. Stem Cells 2005;23:834¨C841.( s9 i5 U& e2 @
0 V6 c% D# ^0 d- Y+ M8 O8 S0 \
Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005;5:275¨C284." `8 ?, j! r# k' ^( c  ]

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¨C14233.% E2 g# B, z0 h; G! \
8 R6 J% Q  @+ N1 d7 Y1 n
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¨C24225.
& a: V# ?! L) L) m
Patrawala L, Calhoun T, Schneider-Broussard R et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2  and ABCG2¨C cancer cells are similarly tumorigenic. Cancer Res 2005;65: 6207¨C6219.

Smalley MJ, Clarke RB. The mammary gland "side population": A putative stem/progenitor cell marker? J Mammary Gland Biol Neoplasia 2005;10:37¨C47.* @( J2 {4 w$ p8 |$ O$ l7 N$ H. i

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¨C56.

Alvi AJ, Clayton H, Joshi C et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 2003;5:R1¨CR8.
* ^' G7 g. m/ G) y
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¨C460.
! D; I3 x  }  |" n! t
Clarke RB, Spence K, Anderson E et al. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol 2005;277:443¨C456.2 ?- G& \, _9 _

Dontu G, Abdallah WM, Foley JM et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003;17:1253¨C1270.

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¨C1345.

Li C, Wong WH. Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection. Proc Natl Acad Sci U S A 2001;98:31¨C36.5 r0 S7 |1 J/ ~1 m. U, g

Schmid I, Cole SW, Korin YD et al. Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence. Cytometry 2000;39:108¨C116.
) r: X9 \- r9 s: _
Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359¨C363.

Xia C, Bao Z, Tabassam F et al. GCIP, a novel human grap2 and cyclin D interacting protein, regulates E2F-mediated transcriptional activity. J Biol Chem 2000;275:20942¨C20948.
( P7 ~( f7 m3 O$ U/ F7 k8 _( c
Topol L, Jiang X, Choi H et al. Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol 2003;162:899¨C908.5 p5 {" s9 Z, i" c0 }

Ishitani T, Kishida S, Hyodo-Miura J et al. The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2 ) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol 2003;23: 131¨C139.0 U7 n+ f6 [; X# H, F3 T

Veeman MT, Axelrod JD, Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 2003; 5:367¨C377.2 }& K: l0 j; L9 \, f6 v3 a3 ?

Wong GT, Gavin BJ, McMahon AP. Differential transformation of mammary epithelial cells by Wnt genes. Mol Cell Biol 1994;14:6278¨C6286.
: C9 }* C, O4 Q
Olson DJ, Gibo DM. Antisense wnt-5a mimics wnt-1-mediated C57MG mammary epithelial cell transformation. Exp Cell Res 1998;241:134¨C141.  Z& C: o6 D( v: {# ^
% B2 `# |; X5 k( P9 z( s
Liu BY, McDermott SP, Khwaja SS et al. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A 2004;101:4158¨C4163.
! Z) s6 o+ P) y+ k! ~# p, L7 `/ q
Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004;432:324¨C331.

DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 1999;126:4557¨C4568.: |6 D+ G6 j; d) W, B) _

Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004; 432:316¨C323.

Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest 2005;115:1503¨C1521.

Ee PL, Kamalakaran S, Tonetti D et al. Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. Cancer Res 2004;64:1247¨C1251.

Bornstein P. Diversity of function is inherent in matricellular proteins: An appraisal of thrombospondin 1. J Cell Biol 1995;130:503¨C506.& e! h3 \$ f, t) P/ {, m2 Q. I

Yang Z, Kyriakides TR, Bornstein P. Matricellular proteins as modulators of cell-matrix interactions: Adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2. Mol Biol Cell 2000;11:3353¨C3364.

Mo FE, Muntean AG, Chen CC et al. CYR61 (CCN1) is essential for placental development and vascular integrity. Mol Cell Biol 2002;22: 8709¨C8720.: y# i! K- _! a1 o: w0 Q
* A  K* r/ \: G
Parker LH, Schmidt M, Jin SW et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 2004; 428:754¨C758.

Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003;9:702¨C712.# r6 E& |/ w9 f/ f) b  I

Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA, Hirschi KK. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. J Clin Invest 2003;111:71¨C79." I" y% ~: I& u! R8 Y+ P$ W

Cleaver O, Melton DA. Endothelial signaling during development. Nat Med 2003;9:661¨C668.$ v" @5 i% }" y( F1 i
/ c1 I- C3 x' z" Q
Shen Q, Goderie SK, Jin L et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science 2004;304:1338¨C1340.

Wurmser AE, Nakashima K, Summers RG et al. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature 2004;430:350¨C356.  k, a4 R2 _7 I) Z  B+ F
/ D" T( w' g! P7 X3 P! H6 u
Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109¨C1121.. l1 x5 `- c: |$ E' d

Sakamoto M, Hirata H, Ohtsuka T et al. The basic helix-loop-helix genes Hesr1/Hey1 and Hesr2/Hey2 regulate maintenance of neural precursor cells in the brain. J Biol Chem 2003;278:44808¨C44815.

Dontu G, Jackson KW, McNicholas E et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 2004;6:R605¨CR615.
4 Z: Z5 h# A9 S5 U# n
Satokata I, Ma L, Ohshima H et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet 2000;24:391¨C395.0 Q" A2 J4 h6 E  d. Y5 z
" a0 l! g5 j. p1 s8 h2 |: W
Satokata I, Maas R. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 1994;6:348¨C356.
$ S' T* ^/ L" O. i
Friedmann Y, Daniel CW. Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions. Dev Biol 1996; 177:347¨C355.6 ^) F6 N$ Z1 H6 @- C- e
& @( @% t$ P8 |2 s$ ^) R
Phippard DJ, Weber-Hall SJ, Sharpe PT et al. Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development. Development 1996;122:2729¨C2737.

Raman V, Martensen SA, Reisman D et al. Compromised HOXA5 function can limit p53 expression in human breast tumours. Nature 2000;405:974¨C978.0 |4 b- h: x' m0 u4 q

Raman V, Tamori A, Vali M et al. HOXA5 regulates expression of the progesterone receptor. J Biol Chem 2000;275:26551¨C26555.' }& m7 k& f0 O" y

Kameda Y, Nishimaki T, Takeichi M et al. Homeobox gene hoxa3 is essential for the formation of the carotid body in the mouse embryos. Dev Biol 2002;247:197¨C209.: b* s. z, L7 Y- W) w
( \! A* u* k$ B2 N
Lewis MT, Veltmaat JM. Next stop, the twilight zone: Hedgehog network regulation of mammary gland development. J Mammary Gland Biol Neoplasia 2004;9:165¨C181.
( A1 e5 M3 |, y8 F( t
Lei Q, Zelman AK, Kuang E et al. Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord. Development 2004;131:3593¨C3604.
0 r# t5 w) j# x( D  c' p9 n% S4 q
McDermott A, Gustafsson M, Elsam T et al. Gli2 and Gli3 have redundant and context-dependent function in skeletal muscle formation. Development 2005;132:345¨C357.$ k, L- {/ `7 E6 l

Perou CM, Sorlie T, Eisen MB et al. Molecular portraits of human breast tumours. Nature 2000;406:747¨C752.

Li Y, Welm B, Podsypanina K et al. Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells. Proc Natl Acad Sci U S A 2003; 100:15853¨C15858.
# w) V2 z3 C" ]& a: [
Forsberg EC, Prohaska SS, Katzman S et al. Differential expression of novel potential regulators in hematopoietic stem cells. PLOS Genetics 2005;1:3 e28.

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