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EMBRYONIC STEM CELLS |
aBlood Bank and
dDepartment of Obstetrics and Gynecology, Landspitali-University Hospital, Reykjavík, Iceland;
bInstitute of Immunology, University Hospital, Oslo, Norway;
cBasic Research Program, SAIC-Frederick, Inc., Center for Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, Maryland, USA;
eIceland Genomics Corporation, Reykjavík, Iceland
Key Words. Human • Hematopoietic progenitor cells • Embryonic stem cells • Gene expression
Correspondence: Kristbjorn Orri Gudmundsson, Ph.D., Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute–Frederick, Maryland, USA. Telephone: 301-846-1509; Fax: 301-846-7042; e-mail: orri{at}ncifcrf.gov
Received on September 29, 2006;
accepted for publication on February 13, 2007.
First published online in STEM CELLS EXPRESS February 22, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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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 [4, 5]. These include SCL/tal-1, AML1, Lmo2, HoxB4, PU.1, and Ikaros. Studies in mice show that if these genes are knocked out, either no blood cells are produced or a specific lineage is missing [4, 5]. Identification of genes important for self-renewal has remained elusive, but genes activated through the Wnt [6] and Notch [7] pathways and the homeobox transcription factor HoxB4 [8] have been implicated in this process. Recently it was shown that the basic helix-loop-helix transcription factor HES-1 (Hairy/Enhancer-of-split-1) and the Polycomb group gene Bmi-1 are important for self-renewal of adult and fetal mouse HCSs [9, 10].
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 [11], and are therefore hard to purify to homogeneity. In addition, studying specific differentiation pathways is difficult because of the lack of a direct link between the HSCs and the differentiated cells in vitro. This can be overcome by using genetically modified HSCs, but manipulating the cells in vitro is not without shortcomings, since HSCs tend to differentiate upon prolonged culture [12, 13]. An alternative approach to studying genes affecting HSC self-renewal and differentiation is to use factor-dependent stem/progenitor cell lines that are blocked at various stages of differentiation [14]. Such cell lines have been used to identify genes differentially expressed in mouse myeloid cell differentiation [15–17]. Differential gene expression analysis in such models can then be confirmed in primary HSCs and progenitors.
In this study, we used the human CD34+ acute myeloblastic leukemia cell line KG1 to identify developmentally regulated genes [18–20]. Recently, it was demonstrated that the cell line can be induced to differentiate into dendritic-like cells (DLCs) following stimulation with phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor 
(TNF-) [21]. 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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, 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
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 1–7 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.
Isolation of Various Hematopoietic Cells
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 [24]. The study protocol was approved by the National Bioethics Committee of Iceland (license no. 00/009/AF). The purity of the CD34+ cells was >95% according to FCM analysis after staining with a CD34 antibody. Mesenchymal stem cells were isolated from bone marrow as previously described [25], CD133+, CD34+CD38–, 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.
Differentiation of Umbilical Cord Blood CD34+ Cells
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.
Differentiation of Peripheral Blood CD14+ Cells
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
For growth of different types of colonies (burst-forming unit-erythroid [BFU-E], colony-forming unit-granulocyte, macrophage [CFU-GM], and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte [CFU-GEMM]), 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 (2–5 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
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).
We followed the minimum information about a microarray experiment (MIAME) guidelines for the setup of our experiments and presentation of our data [26]. 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°C 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).
cDNA Synthesis and Subtractive Hybridization
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°C 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.
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 β-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°C for 1 minute; 25–35 cycles 95°C for 30 seconds, 68°C for 1 minute; and final extension at 68°C for 1 minute. PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide. GAPDH and β-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 β-actin was 661 bp.
Q-PCR
Q-PCR was performed as previously described [27]. 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.
Construction of pcDNA4-Dlg7 Vector
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.
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.
Immunoblotting of Dlg7
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).
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 24–48 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.
Apoptosis Detection
Apoptosis in transfected ES cells was detected using the Annexin V-FITC apoptosis detection kit I (Becton Dickinson) as previously described [24]. 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°C in the dark. Then, 400 µl of 1x binding buffer was added, and the samples were analyzed by flow cytometry.
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 <.05. The results are presented as mean ± standard error of the mean.
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RESULTS |
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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).
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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 [21, 30], were also both significantly upregulated, supporting the idea that the KG1 cells were differentiating toward this lineage. Notably, the transcriptional regulator Id2, which has recently been shown to be important for DC differentiation, was also upregulated [31]. The downregulated transcription factors included Meis1, c-Myc, ERF-2, and HoxA9. Meis1, c-Myc, and HoxA9 have all been shown to be important at the HSC level or in HSC differentiation [32–34].
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).
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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).
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).
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.
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Dlg7 Is Not Expressed in Myeloid and Erythroid Colonies or Various Differentiated Blood Cells
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).
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– 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).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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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 [19]. However, St Louis et al. demonstrated that phorbol ester in conjunction with TNF- drives KG1 cells toward the DC lineage [21]. These results were reproduced in our study, supporting the idea that the KG1-DLC differentiation pathway can serve as a model for genetic analysis of DC differentiation. Further indirect support for this claim comes from a study performed by Chomarat et al. in which it was demonstrated that TNF- drives CD34+ cells toward the DC lineage and away from the macrophage lineage [38].
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 [36]. Interestingly, the gene was independently identified as overexpressed in transitional cell carcinoma of the bladder and in hepatocellular carcinoma [35, 39]. The gene has a significant homology to genes encoding proteins of the membrane-associated guanylate kinase family, and SMART analysis (http://smart.embl-heidelberg.de) shows that amino acids 300–600 code for a guanylate kinase-associated protein (GKAP) domain. Dlg7 is highly expressed in fetal liver and shows significant expression in colon, testis, thymus, and bone marrow but is weakly expressed in isolated unfractionated peripheral blood leukocytes [35, 36]. The function of Dlg7 in mammalian cells is currently not known, but its expression has been shown to be tightly regulated during cell cycle progression in HeLa cells and regenerating mouse liver, with expression being low during G1/S transition but gradually increasing during S and G2/M phases and peaking as cells exit from mitosis [35]. In addition, Dlg7 quantity has been shown to be controlled by the Cdk1/cyclin B kinase at the post-translational level [40]. Phosphorylation of Dlg7 by Cdk1-cyclin B promotes its association with SCFFbx7 ubiquitin ligase, leading to Dlg7 ubiquitination and subsequent proteolysis by the proteasome. The importance of the association was further demonstrated by accumulation of intracellular Dlg7 following siRNA inhibition of SCFFbx7 expression [40]. These results raise important questions with regard to the role of Dlg7 and SCFFbx7 in carcinogenesis. Recently, Dlg7 was shown to be phosphorylated by another kinase, the serine/threonine kinase Aurora-A [41], which is known to be frequently overexpressed in human tumors [42, 43]. Dlg7 mutants lacking the Aurora-A phosphorylation sites were unstable and did not allow cell growth at low serum concentrations, indicating a role for Aurora-A as a positive regulator of Dlg7 function [41]. Dlg7 and Aurora-A show similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression. In addition, their expression in a variety of tissues and cell lines is similar [41]. Finally, it has been suggested that Dlg7 has a key role in the assembly of the mitotic spindle [44–46]. According to those studies, Dlg7 might be one of the factors that initiates spindle bipolarity and stabilizes and directs microtubules toward chromosomes.
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 [47]. The high expression of Dlg7 in the bone marrow and in fetal liver further implicate the gene in regulation of HSC cell cycle progression, as these are both very active sites of hematopoiesis. When Dlg7 was overexpressed in ES cells, smaller and significantly fewer EBs were produced compared with control cells. Also, EBs overexpressing Dlg7 expressed higher levels of genes found at the ES cell level, such as Rex1, Bmp4, Nanog, and Oct4 but lower levels of the mesoderm marker Brachyury, which is expressed in differentiating ES cells. This indicates that the gene may be involved in maintaining the stem cell phenotype by reducing the differentiation potential of the cells. In addition, overexpression of Dlg7 rendered the cells more resistant to apoptosis upon serum withdrawal, suggesting that the gene may also provide some sort of survival signal to undifferentiated cells. The ability of the gene to promote survival is in complete agreement with the study by Tsou et al., where overexpression in 293T cells resulted in an enhanced cell growth at low serum levels [35].
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.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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