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TISSUE-SPECIFIC STEM CELLS

E2F-6 Suppresses Growth-Associated Apoptosis of Human Hematopoietic Progenitor Cells by Counteracting Proapoptotic Activity of E2F-1

^aDivision of Stem Cell Regulation, Center for Molecular Medicine, and
^cDivision of Hematology, Department of Internal Medicine, Jichi Medical School, Shimotsuke, Tochigi, Japan; and
^bCell Regulation Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

Key Words. E2F • Hematopoietic progenitor cell • Apoptosis • Transcription

Correspondence: Yusuke Furukawa, M.D., Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Shimotsuke-City, Tochigi 329-0498, Japan. Telephone: 81-285-58-7400; Fax: 81-285-44-7501; e-mail: furuyu{at}jichi.ac.jp

Received on March 27, 2007; accepted for publication on June 18, 2007.

First published online in STEM CELLS EXPRESS  June 28, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34⁺ human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34^– bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
E2F is a family of transcription factors that control DNA replication, cell cycle progression, DNA repair, and differentiation [1, 2]. Target genes of the E2F family include those encoding enzymes for DNA synthesis (e.g., thymidine kinase and dihydrofolate reductase), regulators of DNA replication (e.g., HsOrc1 and cdc6), and components of cell cycle machinery (e.g., cdc2, cdk2, cyclin A, and c-myc). E2Fs regulate the transcription of these genes either positively or negatively via post-translational modification, such as acetylation and phosphorylation, and binding to pocket proteins (pRB, p107, and p130) and histone deacetylases.

The E2F family comprises at least eight distinct members, E2Fs 1–8, all of which require heterodimerization with a related DRTF1-polypeptide (DP) subunit (DP-1 or DP-2) for full activity [2]. E2Fs are functionally divided into three groups: (a) E2F-1, E2F-2, and E2F-3a, which mainly act as transcriptional activators and facilitate cell cycle progression from G₁ to S phase; (b) E2F-4 and E2F-5, which act as transcriptional repressors upon binding to pocket proteins and play a role in cell cycle arrest and cell differentiation; and (c) E2F-6, E2F-7, and E2F-8, which are considered pocket protein-independent transcriptional repressors because they lack transactivation and pocket protein-binding domains [3–5]. In addition, E2F-1 has the ability to induce apoptosis by upregulating the expression of proapoptotic molecules, such as Apaf-1, ARF, p73, caspase-7, and apoptosis-stimulating kinase 1 (ASK1) [6]. Proapoptotic activity is unique to E2F-1 and is not observed in other members of the E2F family [7].

Accumulating evidence indicates that E2Fs play important roles in hematopoiesis. For example, E2F-1 is expressed in CD34⁺ hematopoietic progenitor cells derived from both bone marrow (BM) and peripheral blood (PB), with higher levels of expression in the former [8]. This difference may reflect the activity of DNA synthesis and the numbers of quiescent CD34⁺ cells between BM and PB. Williams et al. [9] have reported that E2F-4 is the predominant E2F species in human CD34⁺ cells and contributes to maintenance of the quiescent state of CD34⁺ cells with p130. Furthermore, gain-of-function analyses using myeloid cell lines and transgenic megakaryocytes suggest that downregulation of E2F-1 activity is indispensable for terminal differentiation of hematopoietic cells [10, 11]. We have also been studying the biological significance of E2F family proteins in hematopoietic cells [12–16]. In previous studies, we have found that E2F-1 concomitantly promotes cell cycle progression and apoptosis in immature hematopoietic cells and that the major effector of the latter is Apaf-1 but not ARF/p53 nor p73 [15, 16]. The proapoptotic activity of E2F-1 is important for suppressing untoward expansion of proliferating cells and serves as an internal defense mechanism against tumor development in the hematopoietic system. This notion is supported by the high incidence of hematologic malignancies, including malignant lymphoma, in E2F-1-deficient mice [17–19].

It has been shown that E2F-6 binds to the same consensus sequences within E2F target genes as other E2F family proteins. As it lacks a transactivation domain, E2F-6 is believed to be a dominant-negative repressor for E2F-responsive genes [20]. Gain-of-function analyses of E2F-6 revealed that E2F-6 functions as a repressor of transcription during G₀/G₁ to S phase in nonhematopoietic cells such as U2OS and T98G cell lines and murine fibroblasts [21, 22]. In addition, E2F-6 appears to be a component of large protein complexes, which exert gene silencing in the G₀ phase of the cell cycle by recruiting Polycomb group proteins, including Bmi-1 and EPC1 [23–25]. Although these investigations suggest that E2F-6 is involved in the growth regulation of nonhematopoietic cells by repressing a subset of E2F-target genes, the biological role of E2F-6 in hematopoiesis has not been understood at all. To address this issue, we examined the expression and function of E2F-6 in human CD34⁺ hematopoietic progenitor cells. We have found that E2F-6 is preferentially expressed in CD34⁺ progenitors and suppresses their growth-associated apoptosis by counteracting proapoptotic activity of E2F-1 through competitive binding to an E2F-consensus element of Apaf-1 promoter. Our finding points to a novel and unique role of E2F-6 as a failsafe mechanism against apoptotic loss of human hematopoietic progenitor cells during proliferation.

	MATERIALS AND METHODS

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Preparation and Ex Vivo Culture of Human Hematopoietic Progenitor Cells
CD34⁺ cells were purified by positive selection using an immunomagnetic bead system (magnetic cell sorting; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) from human bone marrow mononuclear cells (BM-MNCs) (purchased from AllCells, Berkeley, CA, http://www.allcells.com) or cord blood mononuclear cells (CB-MNCs) (provided by RIKEN Bioresource Center, Tsukuba, Japan). More than 95% of the enriched cells were shown to be positive for CD34 and negative for lineage markers (Lin^–) by flow cytometry [26]. The rest of cells were used as CD34^– BM-MNCs, which contain myeloid progenitor cells and some terminally differentiated elements, mostly B lymphocytes. CD34⁺/CD38^–/Lin^– and CD34⁺/CD38⁺/Lin^– cells were isolated from CD34⁺ BM-MNCs by fluorescence-activated cell sorting (FACS).

Enriched CD34⁺ cells were cultured in serum-free liquid medium as described previously [27]. Briefly, cells were plated at 5 x 10⁵ cells per milliliter in Iscove's modified Dulbecco's medium supplemented with 1% bovine serum albumin, 10 mg/ml bovine pancreatic insulin, and 200 mg/ml human transferrin (BIT 9500; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) plus 40 mg/ml human low-density lipoproteins (Chemicon, Temecula, CA, http://www.chemicon.com) and 10^–4 M 2-mercaptoethanol. For ex vivo culture of hematopoietic progenitors, three early-acting cytokines (stem cell factor [SCF], thrombopoietin [Tpo], and interleukin-3 [IL-3]) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) were added at 50 ng/ml each according to the method of Ema et al. [28].

Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total cellular RNA was isolated from 1 x 10⁴ cells and reverse-transcribed into cDNA using SuperScript reverse transcriptase and oligo(dT) primers (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) as described previously [27]. Subsequent polymerase chain reaction (PCR) amplification was carried out according to standard methods. Detailed information about primers, including sequences, corresponding nucleotide positions, and PCR product sizes, is shown in supplemental online Table 1. The authenticity of PCR products was ensured by their sizes and confirmed by restriction mapping and direct sequencing in some samples.

Construction and Production of Lentiviral Vectors
We used a lentiviral self-inactivating vector, CSII-cytomegalovirus (CMV)-MCS-internal ribosomal entry site (IRES)-VENUS, which is a polypurine tract-containing plasmid harboring multicloning sites for cDNA insertion followed by the IRES sequence under the control of the CMV promoter (kindly provided by Dr. Hiroyuki Miyoshi, RIKEN Bioresource Center, Ibaraki, Japan), after replacing VENUS with DsRed amplified from pDsRed-expressing vector (Clontech, Mountain View, CA, http://www.clontech.com). The resulting construct was designated the CSII-DsRed vector. We constructed E2F-6 expression vector CSII-E2F-6-DsRed by inserting the coding region of E2F-6 cDNA amplified by reverse transcription (RT)-PCR with the following primers (restriction enzyme-digestion sites are underlined): sense, 5'-TTGGATCCACCATGAGTCAGCA-GCGGCCGGCGAGGAAGT-3'; antisense, 5'-CGAATTCTCAGTT-GCTTACTTCAAGCAATTCTTCA-3'. These vectors were cotransfected into 293FT cells with packaging plasmids (purchased from Invitrogen). Infectious lentiviruses in culture supernatants were harvested, concentrated, and infected as described previously [29, 30]. Transduction efficiencies were monitored by DsRed expression using a flow cytometer.

Immunoblotting
Immunoblotting was carried out according to the standard method using the following antibodies: anti-E2F-1 (C-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-E2F-6 (TFE61; Active Motif, Carlsbad, CA, http://www.activmotif.com), anti-c-myc (N-262; Santa Cruz Biotechnology), anti-p73 (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com), anti-ASK1 (Cell Signaling Technology, Denver, http://www.cellsignal.com), anti-ARF (C-18; Santa Cruz Biotechnology), anti-cdc2 (Santa Cruz Biotechnology), anti-dihydrofolate reductase (anti-DHFR) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), anti-Apaf-1 (NT; Millennium Biotechnology, Ramona, CA, http://www.mlnm.com), and anti-β-actin (Ab-1; Oncogene Science, Uniondale, NY, http://www.oncogene.com).

Flow Cytometric Analysis and FACS
Flow cytometric analysis and FACS were carried out by FACSAria (Becton Dickinson) as previously described [30, 31]. CD34 positivity was determined by reactivity with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 antibody (Becton Dickinson). Apoptosis was determined by reactivity with FITC-conjugated Annexin-V (MBL International Corp., Nagoya, Japan, http://www.mblintl.com).

Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Upstate, Lake Placid, NY, http://www.upstate.com) according to the manufacturer's instructions. DsRed-positive Kpef-1 cells were harvested by cell sorting and fixed with 1% formaldehyde at 37°C for 5 minutes. Cells were then washed with phosphate-buffered saline, resuspended in lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) in the presence of protease inhibitors, sonicated, and centrifuged to obtain supernatants. The supernatants were incubated with either anti-E2F-1 or anti-E2F-6 antibody at 4°C overnight. The mixture was incubated with protein A-agarose beads at 4°C for 1 hour and centrifuged to collect the beads. DNA fragments bound on the beads were purified with washing and subjected to PCR to detect promoter regions containing E2F-binding sites. The sequences of oligonucleotide primers are as follows (corresponding nucleotide positions are shown in parentheses): Apaf-1 promoter, sense, 5'-GGGGCTTGGGGTGTGTTTATTTGCATA-3' (–96 to –70), and antisense, 5'-GTCTTCCCGGCCTGTGGCGCCCTTCC-3' (+159 to +184) [16]; cdc2 promoter, sense, 5'-TGCGCTCGCACTCAGTTGGCGCCC-3' (–186 to –163), and antisense, 5'-GAAGCCAAGTGCGAGCAGTTTC A-3' (–11 to +12) [12].

Ex Vivo Culture and Colony Assays of Transduced CD34⁺ Cells
Enriched CD34⁺ cells were cultured in serum-free medium for 24 hours as described above. Then, lentiviruses were added to cell suspension in the presence of 8 µg/ml polybrene and transduced for 24 hours [29]. After transduction, the cells were resuspended in fresh medium and further cultured for 9 days. The cultured cells were harvested at the indicated time points and subjected to flow cytometric analysis to detect apoptosis by staining with annexin-V. For colony assays, DsRed-positive cells were harvested by FACS after lentiviral transduction and seeded in methylcellulose medium (Stem Cell Technologies). The colony numbers were counted after 14 days [32].

	RESULTS

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Endogenous Expression of E2F-Family Transcription Factors in Human Bone Marrow-Derived Hematopoietic Progenitor Cells
We first examined the endogenous expression of E2F-family genes, E2F-1–E2F-6, DP-1, and DP-2, in CD34⁺ and CD34^– BM-MNCs from healthy volunteers using RT-PCR. Functionally, CD34⁺ and CD34^– BM-MNCs correspond to hematopoietic progenitor cells and their differentiated offspring, respectively. As shown in Figure 1A, there was a striking difference in the expression of E2F transcripts between the two populations. CD34⁺ cells moderately expressed E2F-1, E2F-6, and DP-1 and faintly expressed E2F-2, E2F-4, and DP-2. E2F-1, E2F-2, E2F-4, and DPs were markedly upregulated in CD34^– BM-MNCs, whereas only E2F-6 was downregulated to an almost undetectable level. Notably, E2F-3 and E2F-5 were below the detection limit in both populations.

View larger version (34K): [in this window] [in a new window]   Figure 1. Expression of endogenous E2F-family transcription factors in CD34+ and CD34– bone marrow mononuclear cells (BM-MNCs). (A): The expression of E2F and DP subunits was detected by semiquantitative reverse transcription (RT)-PCR in CD34+ and CD34– BM-MNCs. PCR amplification was carried out with 1 µl of cDNA solution (corresponding to 500 cells) in a 50-µl reaction mixture containing 5 units of Taq polymerase, 10 mM Tris-HCl (pH 8.5), 50 mM KCl, 1.5 mM MgCl2, 100 mM dNTPs, and 74 kBq of [32P]dCTP in the presence of specific primer pairs (200 nM each) (supplemental online Table 1). PCR products were resolved on 5% native polyacrylamide gels and visualized by autoradiography. The results of 16, 18, 20, 22, 24, and 26 amplification cycles are shown. (B): We isolated CD34+/CD38– and CD34+/CD38+ cells from CD34+ BM-MNCs and CD34–/CD33+ cells from CD34– BM-MNCs by fluorescence-activated cell sorting and examined the expression of E2F-6 by RT-PCR. PCR products were resolved on 2% agarose gels and visualized by staining with ethidium bromide. Because PCR was carried out without [32P]dCTP, the amplification cycles between 30 and 40 were in a linear range. GAPDH mRNA was simultaneously amplified to normalize the amounts of RNA in each sample. (C): The signal intensities of E2F-6 were quantified with a densitometer, and their means are shown as the ratio against those of GAPDH in corresponding samples. Abbreviations: DP, DRTF1-polypeptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Next, we investigated the changes in the expression of E2F-6 during the proliferation and differentiation of CD34⁺ progenitor cells. To this end, we cultured CD34⁺ BM-MNCs with a combination of early-acting cytokines (SCF, Tpo, and IL-3), which mainly supports the proliferation of progenitors, and serially monitored the expression of E2F-6 mRNA. E2F-6 expression was readily detected in unstimulated CD34⁺ cells, increased during culture with a maximal level at day 4, and then decreased at day 12 (Fig. 2A, 2B). Cell cycle analysis revealed that CD34⁺ cells entered the cell cycle after 2 days of cytokine stimulation and continued to proliferate until day 10 of culture (Fig. 2C). CD34 positivity was more than 80% until 4 days but declined to less than 10% after 10 days of culture (Fig. 2D). Overall, E2F-6 was moderately expressed in CD34⁺ quiescent progenitor cells, upregulated during proliferation, and downregulated after differentiation into CD34^– committed progenitors. This pattern suggests that E2F-6 functions during proliferation of human hematopoietic progenitor cells.

View larger version (24K): [in this window] [in a new window]   Figure 2. Upregulation of E2F-6 expression during culture of CD34+ cells. (A): We cultured CD34+ bone marrow mononuclear cells in the presence of stem cell factor, thrombopoietin, and interleukin-3 and determined the expression of E2F-6 mRNA at the indicated time points by semiquantitative reverse transcription-PCR. The results of 30, 35, and 40 amplification cycles are shown. GAPDH was used as an internal control. (B): The signal intensities of E2F-6 were quantified with densitometer, and their means are shown as the ratio against those of GAPDH in corresponding samples. (C): Cells were harvested at day 0 (before culture) and day 2 (after culture) of culture and stained with propidium iodide to obtain DNA histograms. The percentages of cells in sub-G1, G0/G1, and S+G2/M fractions were calculated with the ModFitLT 2.0 program (BD Biosciences, San Diego, http://www.bdbiosciences.com). (D): CD34 positivity was analyzed by reactivity with a fluorescein isothiocyanate-conjugated anti-CD34 antibody on flow cytometry during culture of CD34+ cells. The means ± SD (bars) of three independent experiments are shown. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction.

View larger version (39K): [in this window] [in a new window]   Figure 3. Overexpression of E2F-6 by the lentiviral transduction system. (A): Schematic representation of lentiviral expression vectors CSII-DsRed and CSII-E2F-6-DsRed; indicates a packaging signal. Details of construction are given in Materials and Methods. (B): Kpef-1 cells transduced with lentivirus carrying CSII-DsRed and CSII-E2F-6-DsRed vectors were designated Kpef-1-mock and Kpef-1-E2F-6, respectively. DsRed positivity was measured after 2 days of transduction by flow cytometry. (C): P2 were collected by fluorescence-activated cell sorting and subjected to immunoblot analysis for E2F-6 and β-actin (loading control) expression. Abbreviations: CMV, cytomegalovirus promoter; cPPT, central polypurine tract; CTS, central termination sequence; IRES, internal ribosomal entry site; LTR, long terminal repeat; P2, DsRed-positive cells; PRE, woodchuck hepatitis virus post-transcriptional regulatory element; RRE, rev-responsive element, SSC-A, side scatter channel-A.

View larger version (37K): [in this window] [in a new window]   Figure 4. E2F-6 rescues Kpef-1 cells from apoptotic cell death induced by forced expression of E2F-1. Kpef-1-mock and Kpef-1-E2F-6 cells were cultured in the absence (IPTG[-]) or presence (IPTG[+]) of IPTG to induce E2F-1 overexpression. (A): Total numbers of DsRed-positive cells were calculated by flow cytometry at the indicated time points. The means ± SD (bars) of three independent experiments are shown. The p value was calculated by Student's t test. (B): DsRed-positive cells were harvested by fluorescence-activated cell sorting at day 6 and subjected to Wright-Giemsa staining for morphological examination. The arrowheads indicate apoptotic cells. Representative photomicrographs at day 6 are shown (original magnification, x1,000). (C): After 6 days of culture, cells were subjected to cell cycle analysis. The percentages of cells in sub-G1 fraction (M1) were calculated with the ModFitLT 2.0 program. (D): After 6 days of culture, cells were stained with FITC-conjugated annexin-V in preparation for flow cytometry. Representative histogram plots of DsRed+ fractions are shown. Annexin-V positivity is indicated as a percentage in each histogram. Abbreviations: FITC, fluorescein isothiocyanate; IPTG, isopropyl-β-D-thiogalactoside.

Finally, we asked whether E2F-6 could suppress other types of apoptosis, such as those associated with cell differentiation and anticancer drug treatment. Previously, we have shown that K562 cells undergo apoptosis after differentiation induced by phorbol esters and sodium butyrate and by treatment with various antineoplastic agents, including cytosine arabinoside and FK228 [33, 34]. E2F-6 was not able to block the apoptosis-promoting effects of these agents in K562 cells (data not shown). Taken together, these results suggest that E2F-6 is not a general inhibitor of apoptosis but rather specifically counteracts the proapoptotic activity of E2F-1.

E2F-6 Dominant-Negatively Suppresses Transactivation of Apaf-1, a Pivotal Mediator of E2F-1-Dependent Apoptosis
To determine the pathway of antiapoptotic signal transduction by E2F-6, we screened for the expression of E2F-1-dependent apoptosis regulators Apaf-1, ARF, p73, c-myc, and ASK1 in Kpef-1 cells. The expressions of cdc2 and DHFR, as controls, were detected simultaneously, as they are primarily involved in cell cycle regulation as E2F-target molecules. The abundance of Apaf-1 protein was dramatically increased in parallel with an increase in E2F-1 levels in IPTG-treated Kpef-1-mock cells (Fig. 5A). E2F-6 reduced the E2F-1-induced increase of Apaf-1 expression to an extent comparable to the decrease of apoptosis (nearly 50%; Fig. 4). In contrast, we failed to detect obvious differences in the expression of other E2F-1-dependent apoptosis regulators (data not shown), as well as cdc2 and DHFR, between IPTG-treated Kpef-1-mock and Kpef-1-E2F-6 cells (Fig. 5A). This is in line with previous reports in which Apaf-1 is a major effector of E2F-1-dependent apoptosis [15, 35] and further suggests that the antiapoptotic effect of E2F-6 is mediated by the suppression of Apaf-1 expression.

View larger version (45K): [in this window] [in a new window]   Figure 5. E2F-6 inhibits upregulation of Apaf-1 by E2F-1 via competitive binding to the E2F-consensus site of Apaf-1 promoter. (A): Kpef-1-mock and Kpef-1-E2F-6 cells were cultured in the presence of IPTG for up to 3 days. DsRed+ cells were harvested by fluorescence-activated cell sorting (FACS) at the indicated time points for immunoblot analysis for the expression of E2F-1, E2F-6, Apaf-1, cdc2, DHFR, and β-actin (a loading control). (B): Kpef-1-mock and Kpef-1-E2F-6 cells were cultured in the absence (–) or presence (+) of IPTG for 2 days. One million DsRed+ cells were harvested by FACS and subjected to chromatin immunoprecipitation assay. Chromatin suspensions were immunoprecipitated with rabbit anti-E2F-1 antibody, mouse anti-E2F-6 antibody, control rabbit IgG, and control mouse IgG. The resulting precipitants were subjected to polymerase chain reaction (PCR) to amplify the promoter regions containing E2F-binding sites of the Apaf-1 and cdc2 genes. The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis. Representative data of 50 cycles are shown. "No DNA" and "input" indicate that PCR was performed without DNA templates and with genomic DNA, respectively. Abbreviations: DHFR, dihydrofolate reductase; IPTG, isopropyl-β-D-thiogalactoside.

E2F-6 Suppresses Growth-Associated Apoptosis and Enhances Clonogenic Growth of Human Hematopoietic Progenitor Cells
Finally, we attempted to confirm the antiapoptotic function of E2F-6 in normal hematopoiesis. For this purpose, we performed gain-of-function analysis using primary human CD34⁺ cells derived from cord blood after confirming that the pattern of E2F-6 expression was almost identical to that of CD34⁺ BM-MNCs (supplemental online Fig. 2). CD34⁺ CB-MNCs were lentivirally transduced with E2F-6 or mock-transduced and then cultured in serum-free liquid medium supplemented with three early-acting cytokines (SCF, Tpo, and IL-3) for 8 days. On the basis of the results of our experiments with Kpef-1 cells, we first examined the effects of E2F-6 on apoptosis of CD34⁺ cells in culture. As shown in Figure 6A, the proportion of annexin-V-positive cells was significantly reduced by E2F-6 overexpression at day 4. However, there was no difference in annexin-V reactivity between mock- and E2F-6-transfected cells at day 8 of culture. As CD34-positive cells differentiate at day 8 (Fig. 2D), these results suggest that E2F-6 specifically inhibits cell growth-associated but not differentiation-related apoptosis.

View larger version (25K): [in this window] [in a new window]   Figure 6. E2F-6 inhibits growth-associated apoptosis of human primary CD34+ cells by suppressing Apaf-1 expression. CD34+ cells were isolated from human cord blood and cultured at 1 x 105 cells per milliliter in serum-free medium overnight as described in Materials and Methods. Then, cells were either transduced with E2F-6 or mock-transduced overnight after the addition of concentrated lentivirus suspensions. The next day, cells were washed twice with fresh medium and cultured in the presence of stem cell factor (SCF), thrombopoietin, and interleukin-3 (IL-3). (A): Cells were harvested after 4 and 8 days of transduction and subjected to flow cytometric analysis for annexin-V reactivity. Data shown are the means ± SD (bars) of annexin-V-positive cells in DsRed+ fractions (n = 3). The p value was calculated by Student's t test. (B): Total cellular RNA was isolated from DsRed+ cells for semiquantitative reverse transcription-PCR analysis to detect the expression of E2F-1, E2F-6, Apaf-1, and GAPDH (an internal control). Data shown are the representative results of one series of three independent experiments. (C): One thousand lentivirus-transduced cord blood CD34+ cells were seeded in methylcellulose medium supplemented with SCF, IL-3, IL-6, erythropoietin, granulocyte macrophage-colony stimulating factor, and G-CSF (Methocult GF+ H4435; Stem Cell Technologies) and cultured for 14 days. Data show the average colony numbers of three independent experiments. The p values were calculated by Student's t test. Abbreviations: BFU-E, burst-forming unit-erythroid; CFU-G, colony-forming unit-granulocyte; CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte; CFU-GM, colony-forming unit-granulocyte and macrophage; CFU-M, colony-forming unit-macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Next, we investigated whether the suppression of Apaf-1 was also important for antiapoptotic action of E2F-6 in primary CD34⁺ cells. DsRed-positive cells were harvested by FACS at day 4 of transfection with either CSII-DsRed (mock) or CSII-E2F-6-DsRed (E2F-6) and subjected to RT-PCR analysis for Apaf-1 expression. We found that E2F-6 markedly decreased the expression of Apaf-1 in CD34⁺ cells without affecting the abundance of E2F-1, ARF, c-myc, and p73 transcripts (Fig. 6B; data not shown). This is fully compatible with the results obtained with the Kpef-1 cell line and suggests that E2F-6 suppresses cell growth-associated apoptosis of hematopoietic progenitors by repressing E2F-1-induced Apaf-1 transactivation in a dominant-negative manner.

If the above notion is correct, E2F-6 could enhance the growth of CD34⁺ progenitor cells induced by early-acting cytokines, because E2F-1 is implicated in the suppression of untoward expansion of hematopoietic cells at this stage [15]. To address this question, we examined the effects of E2F-6 on the clonogenic growth of CD34⁺ CB-MNCs. After lentiviral transduction, DsRed-positive cells were purified by FACS and seeded in methylcellulose medium containing SCF, IL-3, IL-6, erythropoietin, granulocyte macrophage-colony stimulating factor (GM-CSF), and granulocyte-colony stimulating factor (G-CSF) to induce both proliferation and differentiation. As shown in Figure 6C, E2F-6 significantly increased colony numbers after 2 weeks of culture. It is of note that there was a 1.5-fold increase in the numbers of colony-forming units (CFU)-granulocyte, erythroid, macrophage, and megakaryocyte, which correspond to immature progenitors, whereas no difference was observed in more differentiated offspring of CD34⁺ progenitors, such as burst-forming units-erythroid and colony-forming units-granulocyte. These results suggest that E2F-6 acts in favor of the suppression of apoptosis of proliferative progenitor cells.

Finally, we detected the endogenous expression of Apaf-1 and E2F-1 during ex vivo culture of CD34⁺ cells. As shown in supplemental online Figure 4, the kinetics of Apaf-1 expression correlated well with that of E2F-1, suggesting that Apaf-1 is also under the regulation of E2F-1 in primary CD34⁺ cells. The loss of E2F-1-dependent expression of Apaf-1 explains the absence of the antiapoptotic effect of E2F-6 at later stages of culture of CD34⁺ cells. Taken together, these results indicate that E2F-6 specifically inhibits Apaf-1-mediated apoptosis of cycling CD34⁺ cells but not their differentiated offspring.

	DISCUSSION

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It is widely accepted that E2F family transcription factors regulate various important cellular events, including DNA replication, cell cycle progression, DNA repair, and differentiation. Activating E2Fs (E2F-1, E2F-2, and E2F-3a) are considered to be essential for cell proliferation [36]. Previously, Leone et al. [37] pointed to a specific role of E2F-3 in the regulation of transcription in cycling epithelial cells. Although both E2F-1 and E2F-3 are important for initial cell cycle entry, only E2F-3 is required once cells are cycled in epithelial cells and is essential for their proliferation [37, 38]. In this study, however, we could not detect the expression of E2F-3 in hematopoietic cells. This finding is in line with the fact that hematopoiesis is intact in E2F-3 knockout mice [38] and suggests that E2F-3 is dispensable for hematopoiesis. Among activating E2Fs, E2F-1 and E2F-2 were present in hematopoietic progenitor cells, and their expression increased along with cell proliferation and differentiation into mature cells. Loss-of-function studies using knockout mice revealed that hematopoietic abnormalities were associated with the loss of E2F-2 but not E2F-1, although E2F-1-deficient mice frequently develop hematologic malignancies [39]. These findings suggest that E2F-2 is the major activating E2F in hematopoietic cells.

Among repressive E2Fs, we found that E2F-4 and E2F-6 were expressed in hematopoietic cells. E2F-6 was specifically expressed in CD34⁺ progenitors, and E2F-4 expression was dominant in CD34^– bone marrow mononuclear cells. It has been demonstrated that E2F-6 exerts transcriptional silencing of genes in the G₀ phase of the cell cycle [23]. Furthermore, functional studies have revealed that E2F-6 inhibits the cell cycle entry of quiescent NIH3T3 cells into the S phase and delays the exit of cycling U2OS cells from the S phase [21, 22]. As hematopoietic stem and progenitor cells are quiescent or slowly cycling in bone marrow, we initially speculated that E2F-6 is involved in the cell cycle regulation of progenitor cells. However, we found that E2F-6 expression was relatively weak in CD34⁺ cells, including quiescent CD34⁺/CD38^– stem cells, and was upregulated by cytokine stimulation. In addition, overexpression of E2F-6 did not suppress the cell growth of a K562 subline and colony formation of CD34⁺ cells in culture. In support of our observations, Gaubatz et al. [21] demonstrated that ectopic expression of E2F-6 did not affect the cell cycle profile or proliferative capacity of asynchronously growing Saos-2 and NIH3T3 cells. Movassagh et al. [40] also demonstrated that overexpression of E2F-6 did not have a significant effect on cardiac myocyte proliferation. On the other hand, Williams et al. [9] have shown that E2F-4 is the predominant E2F protein in both quiescent and proliferating CD34⁺ cells and forms a complex with p130 and p107 in G₀/G₁ and S phases of the cell cycle, respectively. Expression analysis of murine hematopoietic stem cells (HSCs) revealed that the abundance of p130 was higher in long-term repopulating HSCs than short-term repopulating HSCs. In contrast, the expression of p107 was higher in the latter than in the former, and E2F-4 and E2F-6 were constitutively expressed in both populations [41]. These findings suggest that E2F-4/p130 maintains HSCs in the G₀/G₁ phase by repressing E2F target genes involved in S-phase progression. A switch from the E2F-4/p130 complex to p107-associated E2F complexes recruits HSCs into the cell cycle. When these data are taken together, it is unlikely that E2F-6 is actively involved in cell cycle regulation of hematopoietic progenitor cells.

We obtained evidence indicating that E2F-6 regulates apoptosis instead of cell cycling in human hematopoietic cells. E2F-6 is strongly expressed in hematopoietic progenitors during cytokine-induced proliferation and suppresses their growth-associated apoptosis by counteracting the proapoptotic activity of E2F-1 through competitive binding to Apaf-1 promoter. This is contradictory to previous observations in nonhematopoietic cells, in which E2F-6 arrested cells in the S phase by repressing E2F-dependent genes that regulate G₁/S transition [22, 42]. However, Movassagh et al. [40] showed that E2F-6 depletion resulted in apoptotic cell death but not growth alterations in cardiac myocytes. They speculated that transactivation of E2F-1-dependent proapoptotic genes occurs by displacement of E2F-6 with E2F-1 in promoter regions. Therefore, the biological functions of E2F-6 may vary depending on cell type. Regarding the mechanisms of E2F-6 action, we demonstrated that E2F-6 actually competed with E2F-1 for Apaf-1 promoter binding. In support of our observation, Oberley et al. [43] proposed a similar mechanism in transcriptional regulation of the BRCA1 promoter. They demonstrated that BRCA1 transcription was controlled by the level of occupancy of either E2F-1 or E2F-6 on the E2F site.

The structural analysis of human E2F-6 promoter revealed the presence of two putative E2F binding regions [44]. E2F-1 can bind to the regions to transactivate the E2F-6 gene. In the present study, the expression of E2F-6 correlated well with that of E2F-1 in CB-CD34⁺ cells, suggesting that E2F-6 transcription is governed by E2F-1 in CD34⁺ progenitor cells. E2F-1 is induced in quiescent cells in response to mitogenic stimuli and promotes both cell cycle progression and apoptosis [15, 45]. The apoptosis-inducing ability of E2F-1 is important for suppressing untoward expansion of proliferating cells and thus provides an internal defense mechanism against tumor development. In contrast, deregulated activation of E2F-1 may increase apoptotic loss of hematopoietic progenitor cells. In fact, deregulated activation of E2F-1 is observed in the bone marrow of myelodysplastic syndrome, in which apoptosis of hematopoietic progenitors is accelerated [46]. It is interesting to investigate the role of E2F-6 in this process as a therapeutic target.

Through this study, we realized that the function of E2F-6 in hematopoietic cells is quite similar to that of Bmi-1. Previous studies have demonstrated that Bmi-1 acts as an antiapoptotic factor during self-renewal of HSCs, and its overexpression increased colony formation of primitive HSCs [47, 48]. Bmi-1 binds to E2F-6 and forms a mammalian Polycomb complex, which is a strong transcriptional repressor. Recruitment of the complex to promoter regions results in strong gene silencing [23–25]. In addition, Ink4a and ARF, which mediate cellular senescence and apoptosis, are defined as major targets of Bmi-1 [32, 47]. It is possible that E2F-6 regulates the fate of hematopoietic progenitor cells in cooperation with Bmi-1. An investigation is currently under way in our laboratory to test this hypothesis.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We are grateful to Drs. Seiji Madoiwa, Jun Mimuro, Yoichi Sakata, Katsutoshi Ozaki, Masaaki Takatoku, and Masanori Niimura (Jichi Medical School) for helpful discussions and technical assistance. We also thank Prof. Atsushi Iwama (Department of Cellular and Molecular Medicine, Chiba University) for critical reading of the manuscript. This work was supported in part by the High-Tech Research Center Project for Private Universities Matching Fund Subsidy from MEXT 2002–2006 and by grants from the Vehicle Racing Commemorative Foundation and Sankyo Foundation of Life Science (to Y.F.). J.K. is a winner of the Jichi Medical School Young Investigators Award.

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