Gene Expression Analysis of Hematopoietic Progenitor Cells Identifies Dlg7 as a - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:53 |只看该作者 |倒序浏览 |打印

作者：Kristbjorn Orri Gudmundssona, Leifur Thorsteinssona, Olafur E. Sigurjonssona,b, Jonathan R. Kellerc, Karl Olafssond, Torstein Egelandb, Sveinn Gudmundssona, Thorunn Rafnare


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      【摘要】: A/ s- A+ ?$ d" A- C5 i; `) R
   Inducible hematopoietic stem/progenitor cell lines represent a model for studying genes involved in self-renewal and differentiation. Here, gene expression was studied in the inducible human CD34  acute myelogenous leukemia cell line KG1 using oligonucleotide arrays and suppression subtractive cloning. Using this approach, we identified Dlg7, the homolog of the Drosophila Dlg1 tumor suppressor gene, as downregulated at the early stages of KG1 differentiation. Similarly, Dlg7 was expressed in normal purified umbilical cord blood CD34 CD38¨C progenitors but not in the more committed CD34 CD38  population. Dlg7 expression was not detected in differentiated cells obtained from hematopoietic colonies, nor was expression detected in purified T-cells, B-cells, and monocytes. When analyzed in different types of stem cells, Dlg7 expression was detected in purified human bone marrow-derived CD133  progenitor cells, human mesenchymal stem cells, and mouse embryonic stem (ES) cells. Overexpression of Dlg7 in mouse ES cells increased their growth rate and reduced the number of EBs emerging upon differentiation. In addition, the EBs were significantly smaller, indicating an inhibition in differentiation. This inhibition was further supported by higher expression of Bmp4, Oct4, Rex1, and Nanog in EBs overexpressing Dlg7 and lower expression of Brachyury. Finally, the Dlg7 protein was detected in liver and colon carcinoma tumors but not in normal adjacent tissues, suggesting a role for the gene in carcinogenesis. In conclusion, our results suggest that Dlg7 has a role in stem cell survival, in maintaining stem cell properties, and in carcinogenesis.1 M8 F1 k$ d/ t/ j& n2 y8 V
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Human Hematopoietic progenitor cells Embryonic stem cells Gene expression9 X$ L( t  }( W: k$ B" N7 t
   INTRODUCTION3 e5 D3 |9 [& B& q) H

All cell lineages of the blood are derived from a small number of hematopoietic stem cells (HSCs) ./ @% Q! p" ^3 O

The genetic program controlling stem cell self-renewal and differentiation is yet to be elucidated, although significant contributions have been made. To date, many lineage- and stage-specific genes and transcription factors have been identified .6 W5 m4 @) [; {6 w/ }
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Genetic studies of human HSCs are complicated by the fact that they are a small, heterogeneous population, comprising less than 0.01% of nucleated bone marrow cells . Differential gene expression analysis in such models can then be confirmed in primary HSCs and progenitors.
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In this study, we used the human CD34  acute myeloblastic leukemia cell line KG1 to identify developmentally regulated genes . Using this culture model, we identified a number of genes that showed a significant change in expression upon differentiation and could play a role in HSC self-renewal and differentiation. In this report, we focus on one of these genes, Dlg7 (KIAA0008; Hurp), the homolog of the Drosophila Dlg1 tumor suppressor gene, which we identified as downregulated early in the differentiation process.
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MATERIALS AND METHODS! P8 o4 C; j) m7 n+ }

Cell Lines and Culture Reagents

The human CD34  myeloblastic cell line KG1 was purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org). The CCE embryonic stem (ES) cell line, originally derived by Drs. Gordon Keller and Elisabeth Robertson , was kindly provided by Stem Cell Technologies (London, http://www.stemcell.com) and maintained according to their recommendations. TNF-, interleukin-4 (IL-4), and granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from R&D Systems (Abingdon, U.K., http://www.rndsystems.com). PMA was purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).

Differentiation of KG1 Cells
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KG1 cells (5 x 105 cells per milliliter) were differentiated into DLCs by culturing in 175-cm2 culture flasks (Nunc, Roskilde, Denmark, http://www.nuncbrand.com) for 1¨C7 days in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal calf serum (FCS) (both from Invitrogen, Paisley, U.K., http://www.invitrogen.com), PMA (20 ng/ml), and TNF- (20 ng/ml). Differentiation of cultured cells was monitored on an inverted microscope (Leitz, Wetzlar, Germany, http://www.leica-microsystems.com) and by flow cytometric (FCM) analysis using a FACSCalibur flow cytometer (Becton, Dickinson and Company, San Diego, http://www.bd.com). The antibodies used in the FCM analysis, CD14 FITC, CD45 FITC, CD13 PE, CD33 PE, CD34 PE, CD80 PE, CD83 PE, CD86 PE, and HLA-DR PE, were all purchased from Becton Dickinson.8 i- Q" t  O% R2 y; O& C
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Isolation of Various Hematopoietic Cells
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CD34  cells were isolated from umbilical cord blood using the Dynal CD34 Progenitor Cell Selection System (Dynal, Oslo, Norway, http://www.invitrogen.com) as previously described , CD133 , CD34 CD38¨C, and CD34 CD38  cells were isolated from bone marrow aspirates using CD133 isolation kit and Direct CD34 progenitor cell isolation kit (both from Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), followed by further enrichment by cell sorting on a FACSVantage-DIVA cell sorter (Becton Dickinson). CD14  cells (monocytes) were isolated from buffy coats of healthy adult blood donors using EasySep immunomagnetic selection (Stem Cell Technologies). CD4 , CD8 , and CD19  cells (T- and B-cells) were isolated from buffy coats of healthy adult blood donors using positive selection by Dynabeads (Dynal). Purity of cells was assessed by FCM analysis.& x' o9 C2 y7 H: [0 ^

Differentiation of Umbilical Cord Blood CD34  Cells
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CD34  cells were differentiated toward the dendritic cell (DC) lineage using three different cytokine cocktails: (a) PMA and TNF- (20 ng/ml each); (b) GM-CSF, TNF-, and IL-4 (20 ng/ml each); (c) GM-CSF, TNF-, and SCF (20 ng/ml each). The CD34  cells were cultured for 18 days in IMDM supplemented with 20% FCS and the cytokine cocktails. Differentiation of cultured cells was monitored on an inverted microscope and by FCM analysis using the same markers as with the KG1 cells.: t  z; r- I+ H

Differentiation of Peripheral Blood CD14  Cells- c/ v8 K: b, k# O# k" F7 L. F; q$ B( T
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CD14  cells were differentiated toward DCs by culturing for 10 days in RPMI 1640 containing 10% normal human serum and TNF-, IL-4, and GM-CSF (10 ng/ml each). Differentiation of cultured cells was monitored on an inverted microscope and by FCM analysis using the same markers as were used for the KG1 cells.

Clonogenic Cultures
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For growth of different types of colonies (burst-forming unit-erythroid ), isolated cord blood CD34  cells were cultured in MethoCult GF  methylcellulose (Stem Cell Technologies) for 14 days. Individual colonies were picked and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (Q-PCR) analysis.

Culture and Differentiation of Mouse ES Cells

The CCE ES cells were cultured in gelatin-coated culture flasks or plates in ES cell media containing LIF and passaged every second day or when confluence was between 50% and 70%. For differentiation into EBs, ES cells (2¨C5 x 102 cells per 35-mm dish) were cultured for 7 days in basic methylcellulose medium containing IMDM, 15% FCS, 2 mM L-glutamine (all from Stem Cell Technologies), and 150 µM monothioglycerol (Sigma-Aldrich) and subsequently quantitated.

RNA Isolation and Oligonucleotide Array Analysis* X* P9 e7 x. w  q% r! d% F( _8 s
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Total RNA was isolated from KG1 cells and DLCs using Trizol reagent (Invitrogen) or the RNAqueous 4PCR kit (Ambion, Austin, TX, http://www.ambion.com). For the oligonucleotide array analysis, RNA was isolated by Trizol, followed by a second cleanup with RNeasy Total RNA isolation kit (Qiagen, Valencia, CA, http://www1.qiagen.com).8 s4 ?8 n2 _* V

We followed the minimum information about a microarray experiment (MIAME) guidelines for the setup of our experiments and presentation of our data . The array analysis was performed on KG1 cells and KG1-derived DLCs in two separate differentiation experiments using two sets of RNA samples, which were tested on individual chips. Double-stranded cDNA was synthesized from KG1 RNA and day 4 DLC RNA using the SMART cDNA synthesis kit (Clontech, Palo Alto, CA, http://www.clontech.com). Biotinylated cRNA was generated using the MessageAmp amplification kit (Ambion), and 10 µg of cRNA was hybridized to Affymetrix human HG-U95A array at 45¡ãC for 16 hours (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). The DNA chips were stained, washed, and scanned according to the manufacturer's protocol. Scanned GeneChip DAT files were analyzed by the Gene Chip Analysis Suite Software (Affymetrix). Fold changes were defined using signal log ratio. The complete microarray data were deposited into the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress; accession no. E-MEXP-345).
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cDNA Synthesis and Subtractive Hybridization5 F* a/ ~& V+ J

Double-stranded cDNA (ds-cDNA) was synthesized from KG1 and DLC RNA using the SMART cDNA synthesis kit (Clontech). ds-cDNA was purified using CHROMA SPIN-1000 columns (Clontech) followed by RsaI digestion at 37¡ãC for 3 hours to produce shorter, blunt-ended molecules. Digested cDNA was purified further using NucleoTrap PCR kit (Clontech). Differentially expressed genes were identified using the PCR-Select cDNA subtraction kit (Clontech) according to the manufacturer's recommendations.
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RT-PCR Analysis

Single stranded-cDNA was generated from 200 ng of total RNA using the Superscript first-strand synthesis system (Invitrogen). PCRs using gene-specific primers for human Osteopontin, JunB, Id2, Dlg7, GAPDH, and ¦Â-actin were performed using the Advantage 2 PCR kit (Clontech). Primers used are listed in supplemental online Table 1. Cycle conditions were as follows: 95¡ãC for 1 minute; 25¨C35 cycles 95¡ãC for 30 seconds, 68¡ãC for 1 minute; and final extension at 68¡ãC for 1 minute. PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide. GAPDH and ¦Â-actin control primers were purchased from Stratagene (La Jolla, CA, http://www.stratagene.com). The expected size of the GAPDH product was 600 base pairs (bp), and that of ¦Â-actin was 661 bp.
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Q-PCR
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Q-PCR was performed as previously described . cDNA for Q-PCR was generated using the Superscript first-strand synthesis system (Invitrogen). The Q-PCRs were performed using the DNA Engine Opticon continuous fluorescence detection system (MJ Research, Inc. Waltham, MA, http://www.bio-rad.com) and the SYBR Green fluorescent dye (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) or TaqMan probes. TaqMan probes were labeled with FAM and BHQ-1. Relative amounts of Dlg7 transcripts were normalized against GAPDH in each cDNA sample. Standard curves were generated using cloned Dlg7. Primers and TaqMan probes for Dlg7 were as follows: Dlg7 sense, 5'-TGATGTTCGAGCAATCCGACCTG-3'; Dlg7 antisense, 5'-TGGGCATTACAGGCTGCACAACT-3'; Dlg7 TaqMan probe, 5'-TCCAAGACAAACTTCTGAAAAGAA-3'. All other primers used are listed in supplemental online Table 1.
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Construction of pcDNA4-Dlg7 Vector  J) W: E( Z# G0 D9 ]1 P, v
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The coding sequence of mouse Dlg7 was amplified from mouse CCE ES cell cDNA using the following primers: forward primer, 5'-ATGCTGGTGTCACGTTTTGCCAGTC-3'; reverse primer, 5'-TCATAGTGGTGAGAAGAGAATGAGG-3'. The PCR product was gel-purified and cloned into a pcDNA4/HisMax TOPO vector (Invitrogen), and plasmids with correct inserts were identified by restriction analysis and sequencing.
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Antibody Generation

Polyclonal rabbit antibodies were generated against Dlg7 using two different peptides from the protein. The synthetic peptides were purified by HPLC, verified by mass spectrometry, and used for immunization conjugated to KLH. The peptide sequences were as follows: CANENEPEGKVPSKGRPAKNV (anti-Dlg7-A), ISFGGNLITFSPLQPGEF (anti-Dlg7-B). These peptide sequences were found to be unique for Dlg7 according to protein BLAST analysis. Peptides and antibodies were produced and affinity-purified by Bethyl Laboratories (Montgomery, TX, http://www.bethyl.com). The specificity of each antibody was analyzed by ELISA using plates coated with the peptides and by immunoblotting using dot blots of the peptides.
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Immunoblotting of Dlg78 Y8 p$ C2 x& r8 i! ?. f/ W, t5 |/ @
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Various cell lysates were analyzed by immunoblotting for their expression of the Dlg7 protein. Ten to 20 µg of proteins were separated on 10% NuPAGE Novex Bis-Tris gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were probed with anti-Dlg7 and visualized by ECL (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). For quantitation of proteins, exposed films were scanned and protein bands analyzed with the GeneTools Image Analysis software (Syngene, Cambridge, U.K., http://www.syngene.com).3 d; g/ x/ E) E' h
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Transfection of ES Cells

CCE ES cells were plated in six-well plates (Falcon) in ES cell medium without antibiotics 1 day before transfection. Seventy percent to 80% confluent cells were transfected with 2 µg of pcDNA4 (control) or pcDNA4-Dlg7 plasmids using Lipofectamine 2000 (Invitrogen) and assayed for transgene expression 24¨C48 hours post-transfection by probing ES cell immunoblots with anti-His antibody (Invitrogen). To establish stable transfectants, transfected ES cells were split 1:10 post-transfection and cultured in complete medium containing 0.4 mg/ml Zeocin (Invitrogen). Stable clones were maintained in medium containing 0.4 mg/ml Zeocin.7 j% a) @( w& t+ g8 Y8 @  g
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Apoptosis Detection  t: ]- N2 J2 d( _1 ^0 e
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Apoptosis in transfected ES cells was detected using the Annexin V-FITC apoptosis detection kit I (Becton Dickinson) as previously described . Briefly, 1 x 105 cells were stained with 5 µl of Annexin V-FITC and 5 µl of propidium iodide and incubated for 15 minutes at 22¡ãC in the dark. Then, 400 µl of 1x binding buffer was added, and the samples were analyzed by flow cytometry.2 j2 E8 K, M) ^& G! ?

Statistical Analysis

Statistical calculations were made using GraphPad Prism version 3.00 for Windows (GraphPad Software, Inc., San Diego, http://www.graphpad.com). Significance tests were performed using unpaired Student's t-tests. The difference was considered significant if p
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RESULTS
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The CD34  KG1 Progenitor Cell Line Differentiates into DLCs

We initiated experiments to identify novel genes that regulate self-renewal and differentiation using the KG1 progenitor cell line. This cell line has previously been reported to differentiate into DLCs .

KG1 cell differentiation turned out to be most efficient using a combination of PMA and TNF- (Fig. 1), the differentiation being essentially complete within 4 days. Going beyond day 7 was not beneficial since the cells terminally differentiated without proliferating and cell death became pronounced in the cultures. In the 4 days of stimulation, the cells became strongly adherent and acquired DC morphology (Fig. 1A, 1B). Upregulation of DC-related molecules was evident by flow cytometric analysis (Fig. 1C); the upregulated molecules included CD80 (B7.1), CD83, CD86 (B7.2), and HLA-DR. In addition, the myeloid cell-specific marker CD13 was upregulated, whereas the stem/progenitor cell marker CD34 and the myeloid progenitor marker CD33 were downregulated (data not shown).# @& O! ^) z5 c- _7 ?/ b7 z' {
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Figure 1. Morphological and flow cytometric analysis of dendritic-like cell (DLC) differentiation of KG1 cells. Unstimulated KG1 cells (A) showed characteristic changes in morphology following phorbol 12-myristate 13-acetate and tumor necrosis factor- stimulation for 4 d (B). Microscopic magnification, x200. (C): Flow cytometric analysis of viable cells showed upregulation of the dendritic cell-related cell surface molecules CD80, CD83, CD86, and HLA-DR. Black open lines are isotype-specific controls, black filled lines are unstimulated KG1 cells, and gray open lines are d4 DLCs. Mean fluorescence values for d0 and d4 are shown inside the graphs. Abbreviations: d, day; HLA-DR, human leukocyte antigen-DR; PE, phycoerythrin; PerCP, peridin-chlorophyll.
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Gene Expression Profiling of KG1 Cell Differentiation  |' q8 Z8 Y& c' B" M) h# }

To identify genes either upregulated or downregulated in KG1 differentiation, gene expression profiling was performed on oligonucleotide arrays using undifferentiated KG1 cells and day 4 DLCs. This time point was chosen because the morphology and expression of cell surface markers confirmed that the KG1 cells were differentiating toward the DLCs. Of the 12,000 genes represented on the chip, approximately 700 (5.8%) were detected as either upregulated or downregulated during differentiation, using 2.0-fold change as a cutoff level (deposited into ArrayExpress). The difference in gene expression was more dramatic when genes were upregulated than when they were downregulated. For instance, the gene for osteopontin, a secreted phosphoprotein known to be produced by DCs and to induce DC migration to lymph nodes .' r& b' G1 Z! x6 d

A number of transcription factors and transcriptional regulators were significantly upregulated during KG1 differentiation. These included JunB, ISGF-3, Egr3, ERF-1, MafF, and Musculin/ABF1, none of which have previously been implicated in DC differentiation. The nuclear factor (NF)-B gene family members p50-NF-kappaB homolog and RelB, which are thought to be important regulators of DC differentiation .# b, |6 {- S9 i4 k

In addition to the oligonucleotide arrays, we also performed suppression subtractive hybridization on KG1 cells and day 4 DLCs. This was done to detect differentially expressed genes not represented on the array and also to complement the array results. The subtractive hybridization was used for detecting both downregulated and upregulated genes. Two hundred clones from each reaction were sequenced and compared with the NCBI sequence database. Thirty clones were identified as downregulated (supplemental online Table 2), whereas 21 clones were found to be upregulated (supplemental online Table 3). For some of the genes (e.g., osteopontin), multiple clones were detected, presumably reflecting the high expression level. As predicted, some of the same genes were detected as either up- or downregulated on both the array and by subtractive cloning. In addition to osteopontin, these genes included IL-8, inhibitor of apoptosis-1, leupaxin, IL1RN, and Dlg7.

Taken together, the array and subtractive hybridization data show gene expression patterns consistent with published data on the DC differentiation pathway. More important, upregulation or downregulation of novel genes with a potential role in self-renewal and differentiation was also detected.

Confirmation of Array and Subtractive Hybridization Results by Kinetic RT-PCR Analysis

To confirm the results obtained from the arrays and the subtractive hybridization, we looked at three upregulated genes by RT-PCR, osteopontin, JunB, and Id2. According to both array and subtractive hybridization data, osteopontin is highly upregulated during DLC differentiation of KG1 cells. This was confirmed by RT-PCR analysis of osteopontin at four time points during differentiation (Fig. 2A). RNA transcripts were undetectable in unstimulated cells but expressed at day 4, and expression was maintained at day 7. Expression of JunB and Id2 was also in complete agreement with the array data: both were nearly undetectable in unstimulated cells but upregulated at days 1 through 7 (Fig. 2B).) G3 _+ N5 o7 P2 o8 x, o

Figure 2. Osteopontin, JunB, Id2, and Dlg7 expression in DLC differentiation of KG1 cells. (A): KG1 cells initiated expression of the osteopontin gene after PMA and TNF (20 ng/ml each) stimulation. (B): Expression of the transcription factor JunB and the transcriptional regulator Id2 was also upregulated. (C): Quantitative reverse transcription-polymerase chain reaction analysis of Dlg7 expression showed a gradual downregulation upon differentiation. (D): The Dlg7 protein was detected in KG1 cells (lane 1). On days 3 and 7, the protein was detected in lower amounts in DLCs (lanes 2 and 3) compared with the KG1 cells. GAPDH detection was used as a loading control. Abbreviations: d, day; DLC, dendritic-like cell; PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor .  B* p- W# c4 T4 |
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We chose the gene Dlg7, which encodes a putative tumor suppressor protein, for further study. Both the array and subtractive hybridization experiments showed this gene to be downregulated once differentiation was initiated, suggesting that its expression was linked to the progenitor phenotype. In addition, previous studies had indicated its presence in bone marrow but not in peripheral blood mononuclear cells and also in the regulation of the cell cycle, which is very relevant when studying stem cell fate. Q-PCR analysis confirmed downregulation of Dlg7 (Fig. 2C).
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Furthermore, immunoblot analysis of lysates from KG1 cells, day 3 DLCs, and day 7 DLCs demonstrated that the Dlg7 protein is detected in KG1 cells but is reduced during differentiation (Fig. 2D).
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Gene Expression During DC Differentiation of Umbilical Cord Blood CD34  Cells Compared with DLC Differentiation of KG1 Cells

Cell lines serve as useful models for differential gene expression analysis; however, since KG1 cells are de facto immortalized tumor cells, care should be taken to verify the results in normal HSCs. To this end, we analyzed the expression of osteopontin, JunB, Id2, and Dlg7 in DC differentiation of cord blood CD34  progenitor cells. The CD34  cells were cultured with three different combinations of cytokines, all thought to drive the cells toward the DC lineage. In all combinations, the cultured cells acquired the morphology of DCs and flow cytometric analysis showed upregulation of the cell surface markers CD80, CD83, CD86, and HLA-DR (data not shown).
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JunB was detected at low levels but osteopontin and Id2 were not detected at all in unstimulated CD34  cells. Using a combination of GM-CSF, TNF-, and IL-4 to differentiate the cells, expression of all three genes was upregulated and stayed relatively constant through days 12 and 18 (Fig. 3A). Analogous to the KG1 model, Dlg7 is expressed in undifferentiated CD34  cells, but expression is undetectable after differentiation is initiated (Fig. 3A). Taken together, the KG1-DLC differentiation model is highly comparable to DC differentiation of normal umbilical cord blood CD34  cells for all the genes tested.

Figure 3. Gene expression during dendritic cell differentiation of cord blood CD34  hematopoietic progenitor cells and Dlg7 expression in purified CD34 CD38¨C and CD34 CD38  bone marrow hematopoietic progenitor cells. (A): Cord blood CD34  progenitor cells, differentiated for 18 days with granulocyte macrophage colony-stimulating factor tumor necrosis factor interleukin 4 (20 ng/ml each cytokine), showed upregulation of Osteopontin, JunB, and Id2 expression and downregulation of Dlg7 expression. (B): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Dlg7 expression in purified CD34 CD38¨C and CD34 CD38  hematopoietic progenitor cells showed predominant expression in the CD34 CD38¨C fraction. (C): Quantitative PCR analysis of Dlg7 expression in purified CD34 CD38¨C and CD34 CD38  cells confirmed the RT-PCR analysis. Abbreviation: -RT, reverse transcriptase.0 o4 }" o& o$ y: n

Dlg7 Is Expressed in Primitive CD34 CD38¨C Progenitor Cells but Not in CD34 CD38  Cells9 y' B& _% w$ M" s
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The downregulation of Dlg7 in CD34  cells suggested that its expression needs to be turned off for differentiation to proceed. Therefore, we looked at the expression of Dlg7 in sorted bone marrow CD34 CD38¨C and CD34 CD38  cells by RT-PCR. The CD34 CD38¨C fraction corresponds to the most primitive hematopoietic progenitors, whereas the CD34 CD38  cells are considered more committed. RT-PCR (Fig. 3B) and Q-PCR (Fig. 3C) analysis demonstrated that Dlg7 is only detected in CD34 CD38¨C cells but not in CD34 CD38  cells.

Dlg7 Is Not Expressed in Myeloid and Erythroid Colonies or Various Differentiated Blood Cells, K3 B' a' a8 L5 i: s
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Next, we looked at the expression of Dlg7 in clonogenic cultures. Gene expression was analyzed by RT-PCR and Q-PCR in various types of colonies generated from cord blood CD34  cells. After 14 days of culture, Dlg7 was not detected in differentiated cells from any type of colony (data not shown).. }" y8 [- i& a" h+ m  _

In addition, we analyzed the expression of Dlg7 by RT-PCR in T-helper cells (CD4 ), cytotoxic T-cells (CD8 ), monocytes (CD14 ), and B-cells (CD19 ) isolated from peripheral blood. Dlg7 was not detected in any cell type (data not shown). Thus, Dlg7 is exclusively found in CD34 CD38¨C progenitor cells.

We used the anti-Dlg7 antibody to assess the presence of the Dlg7 protein in isolated CD34  cells, CD14  cells, and DCs differentiated from the CD14  cells. The Dlg7 protein was only present in CD34  cells, not in CD14  cells or during DC differentiation (Fig. 4A).

Figure 4. Immunoblot analysis of Dlg7 in CD34  progenitor cells, CD14  cells, and DCs and Dlg7 gene expression analysis in AC133  cells, mesenchymal stem cells, and mouse ES cells. (A): The Dlg7 protein was detected in CD34  progenitor cells (lane 1) but not in CD14  cells or during DC differentiation (lanes 2¨C5). The blot was exposed to the film for 1¨C2 minutes to reveal the Dlg7 band but for 15 seconds for GAPDH detection. (B): The Dlg7 gene was expressed in AC133  cells (lane 1), bone marrow mesenchymal stem cells (lane 2), and mouse ES cells (lane 3). (C): Dlg7 was expressed in ES cells but was gradually downregulated upon EB differentiation. Abbreviations: d, day; DC, dendritic cell; ES, embryonic stem.
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Since Dlg7 was only detected in CD34  progenitor cells, we decided to analyze different types of stem cells to find out whether the expression was stem cell-restricted in any way. RT-PCR analysis of purified human AC133  cells and mesenchymal stem cells revealed that the gene was expressed in these cell types (Fig. 4B). Table 1 summarizes Dlg7 expression in various cell types.6 ^, B8 A4 Y1 s4 p% A4 H1 x6 Z
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Table 1. Summary of Dlg7 expression in various stem cells, progenitors, and differentiated cells
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Overexpression of Dlg7 in Mouse ES Cells Decreases Their EB Differentiation Potential and Promotes Survival
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To start to dissect the potential functional role of Dlg7, we made several attempts to overexpress the gene in KG1 cells to see what effect this would have on the differentiation process. However, KG1 cells are notoriously resistant to transfection, and it was not possible to use this model to assay the effect of Dlg7 overexpression in these cells. Therefore, we decided to analyze the effects of overexpressing the gene in mouse ES cells. These cells can be differentiated into EBs, which contain cells corresponding to all three germ layers, and subsequently into blood cells using colony cultures. Dlg7 was expressed in ES cells; it was significantly downregulated in EBs at day 7 and showed little or no expression in EBs at day 10 (Fig. 4C). Dlg7 was not detected in blood cells derived from EBs (data not shown). We transfected ES cells with pcDNA4 or pcDNA4-Dlg7 constructs and selected cells stably expressing Dlg7 (Fig. 5A, 5B). Cells overexpressing Dlg7 showed a higher growth rate than control cells, although the difference was not statistically significant (Fig. 5C). No difference was detected in the viability of ES cells transfected with the two constructs (data not shown). These cells were used to initiate EB differentiation cultures, and the effects of Dlg7 overexpression on EB formation were analyzed on day 7. Cells stably overexpressing Dlg7 produced smaller and significantly fewer EBs than control cells (p

Figure 5. Effects of Dlg7 overexpression in mouse ES cells on EB differentiation and immunoblot analysis of various T and adjacent N lysates. (A): Dlg7 expression in transduced ES cells. ES cells were stably transfected with pcDNA4-control or pcDNA4-Dlg7 plasmids using Zeocin selection. Quantitative reverse transcription-polymerase chain reaction showed more than 50-fold higher Dlg7 expression in ES cells transduced with pcDNA4-Dlg7. (B): The Dlg7 protein was detected in transduced cells by immunoblotting and anti-His staining. (C): ES cells overexpressing Dlg7 showed increased growth rate compared with control cells. Control or Dlg7-expressing ES cells (1 x 105) were cultured for 48 hours in media, harvested, and counted. The figure is representative of three independent experiments. Microscopic magnification, x125. (D): EBs differentiated from ES cells overexpressing Dlg7 were significantly smaller than control EBs. Microscopic magnification, x31.25 and x125. (E): ES cells overexpressing Dlg7 produced significantly fewer EBs than control cells (*, p 6 O& g/ D7 s+ V. O. V$ Z  w. y; z- y

The Dlg7 Protein Is Detected in Large Amounts in Colon and Liver Tumors1 V) [% M; a4 Z4 [0 o
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Previous studies using PCR-based methods or in situ hybridization indicated that Dlg7 is expressed in colon, breast, bladder, and hepatocellular carcinoma . These observations are very interesting in light of our results, which indicate that Dlg7 expression is tightly regulated at the stem cell level. Tumor cells have been shown to acquire certain stem cell properties such as dedifferentiation and increased proliferation potential. To follow up on this, we looked for the Dlg7 protein in lysates prepared from various human tumor tissues and adjacent normal tissue. Immunoblotting analysis clearly demonstrated that Dlg7 is detected in large amounts in colon and liver tumors but is completely absent from adjacent normal tissues, indicating a role for the protein in these tumors (Fig. 5F). In contrast, the protein was not detected in breast, bladder, kidney, or lung tumors.
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DISCUSSION

In this study, we used the progenitor cell line KG1 to identify genes that are potentially important in stem cell self-renewal and differentiation. By using a well-defined model, it is possible to bypass the problem of obtaining stem cells in sufficient numbers and purity, which is of paramount importance when comparing gene expression in different cell populations. However, the cell line models are not without shortcomings. They are tumor cells, often propagated for years, accumulating changes that do not necessarily reflect the real stem cells in all respects. Therefore, we also validated our results in normal CD34  progenitor cells.
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KG1, the cell line used in this study, was originally believed to differentiate to the monocyte/macrophage lineage when stimulated with phorbol esters .
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Genes that are downregulated during differentiation may provide important clues as to how differentiation is prevented and the stem cell phenotype maintained. In our model, a putative tumor suppressor gene (Dlg7) was identified as downregulated on the arrays and in the subtractive hybridization experiments, making it a candidate stem cell gene. The gene is not well-characterized, but it contains interesting features, which prompted us to study it further. Dlg7 is a novel-cell-cycle regulated gene, recently shown to be a homolog of the Drosophila melanogaster discs large-1 (dlg1) tumor suppressor gene . According to those studies, Dlg7 might be one of the factors that initiates spindle bipolarity and stabilizes and directs microtubules toward chromosomes.& F; i+ m5 \4 L  o; J% W. h
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According to our results, Dlg7 is expressed mainly at the hematopoietic progenitor cell level, but expression is completely turned off upon differentiation. This indicates a role for this family of proteins in the regulation of stem cell growth and differentiation. This is further supported by the recent cloning of the GKAP gene, mars, which was shown to be strongly expressed in Drosophila premeiotic germ cells but not in somatic or postmeiotic cells .3 ^  F: {7 N, k+ v6 _: G( p

In conclusion, our results indicate that the putative tumor suppressor gene Dlg7 has a role in the maintenance of stem cell properties. In addition, high levels of the Dlg7 protein in colon and liver tumors indicate a role for Dlg7 in the etiology of these cancers.5 ~# r% i1 `0 l) |8 M% q+ V, P

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest." R- }" ^# H6 C, [% J4 ^2 |. ^
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ACKNOWLEDGMENTS/ w. Z  z8 n% v# Q. O0 h% ^
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We thank Dr. Kelvin P. Lee for valuable information regarding the cell line model; Dr. Sigridur Valgeirsdottir and Dr. Kristin Bergsteinsdottir at Iceland Genomics Corporation, Reykjavik, Iceland, for the array analysis; and Prof. Eirikur Steingrimsson and his staff at the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland for valuable information and assistance in cloning and sequencing. We also thank Stefan Jonsson at the Institute for Experimental Pathology at Keldur for the quantitative RT-PCR analysis and Dr. Kimberly Klarmann at the National Cancer Institute, Frederick, MD, for critically reading the manuscript. This work was supported by grants from the Icelandic Centre for Research (no. 021940002), the Landspitali-University Hospital Research Fund, and the Norwegian Research Council. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This project was funded in whole or in part by the National Cancer Institute, NIH, under contract NO1-CO12400. K.O.G. is currently affiliated with the Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute¨CFrederick, Maryland.
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