TY - JOUR
AU - Feuer, Gerold
AB - Abstract Human T-cell lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus and the etiologic agent of adult T-cell leukemia (ATL), an aggressive CD4+ malignancy. HTLV-2 is highly homologous to HTLV-1; however, infection with HTLV-2 has not been associated with lymphoproliferative diseases. Although HTLV-1 infection of CD4+ lymphocytes induces cellular replication and transformation, infection of CD34+ human hematopoietic progenitor cells (HPCs) strikingly results in G0/G1 cell cycle arrest and suppression of in vitro clonogenic colony formation by induction of expression of the cdk inhibitor p21cip1/waf1 (p21) and concurrent repression of survivin. Immature CD34+/CD38− hematopoietic stem cells (HSCs) were more susceptible to alterations of p21 and survivin expression as a result of HTLV-1 infection, in contrast to more mature CD34+/CD38+ HPCs. Knockdown of p21 expression in HTLV-1-infected CD34+ HPCs partially abrogated cell cycle arrest. Notably, HTLV-2, an HTLV strain that is not associated with leukemogenesis, does not significantly modulate p21 and survivin expression and does not suppress hematopoiesis from CD34+ HPCs in vitro. We speculate that the remarkable differences in the activities displayed by CD34+ HPCs following infection with HTLV-1 or HTLV-2 suggest that HTLV-1 uniquely exploits cell cycle arrest mechanisms to establish a latent infection in hematopoietic progenitor/hematopoietic stem cells and initiates preleukemic events in these cells, which eventually results in the manifestation of ATL. Disclosure of potential conflicts of interest is found at the end of this article. Cell cycle heterogeneity, Leukemia, Lentiviral vectors, Stem/progenitor cell, Viral persistence Introduction CD34+ hematopoietic progenitor cells (HPCs) are a heterogeneous pluripotent cell population that includes hematopoietic stem cells (HSCs) and HPCs are capable of differentiating into various hematopoietic lineages. CD34+ hematopoietic progenitor/hematopoietic stem cells (HP/HSCs) have previously been shown to be cellular targets for viral infection, including cytomegalovirus (CMV), human herpesvirus (HHV) type 6, HHV8, measles virus, and Moloney murine leukemia virus [1, 2, 3, 4, 5, 6, 7–8]. Cell cycle progression and differentiation is highly regulated in CD34+ HPCs, which demonstrate unique expression patterns of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors. The cdk inhibitors p21cip1/waf1 (p21) and p27kip1 (p27), in particular, have been shown to be key contributors in restricting cell cycle entry from G0 and maintaining quiescence in hematopoietic stem cells [9, 10–11]. Survivin, originally identified as a member of the inhibitor of apoptosis protein family, has recently been implicated in regulating hematopoiesis, cell cycle control, and transformation [12, 13, 14–15]. Survivin is expressed in normal adult bone marrow and in CD34+ HPCs, and expression is upregulated by hematopoietic growth factors [15]. Notably, survivin has been shown to be a key mediator of early cell cycle entry in CD34+ HP/HSCs and regulates progenitor cell proliferation through p21-dependent and -independent pathways [16]. Survivin is functionally associated with p21, releasing p21 from the cdk4/p21 complexes and facilitating cell cycle progression [17, 18, 19–20]. Survivin interacts with the cdk4/cyclin D complex and enhances RB phosphorylation, and the targeted overexpression of survivin enhances cell cycle progression [20, 21]. Cumulatively, these data implicate survivin as an integral cellular factor that regulates multiple aspects of hematopoiesis. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+ leukemia [22]. Although HTLV-1 shares 70% homology with HTLV-2, infection with HTLV-2 has not been definitively linked with development of neoplasia. HTLV-1 can transform mature T lymphocytes in vitro, and the viral Tax oncoprotein (Tax1) is intimately linked to cellular transformation and viral pathogenesis in vivo. Tax1 is a transcriptional transactivator protein that promiscuously induces cellular gene expression by activation of transcription factors such as nuclear factor κB (NF-κB) and cyclic AMP response element-binding protein/activating transcription factor (CREB/ATF) [23]. Tax1 has also been shown to trans-repress transcription of certain cellular genes, including bax [24], human β-polymerase [25], cyclin A [26], lck [27], MyoD [28], INK4 [29], and p53 [30]. We have previously shown that overexpression of Tax1 in CD34+ HPCs, using a lentivirus vector (LV), induces G0/G1 cell cycle arrest and suppresses multilineage hematopoiesis in vitro by transcriptional upregulation of the cdk inhibitors p21 and p27 [31]. Here, we demonstrate that HTLV-1 infection of CD34+ HPCs suppresses hematopoiesis as a result of induction of G0/G1 cell cycle arrest by modulation of p21 and survivin gene expression. Notably, CD34+/CD38− HSCs demonstrate exquisite sensitivity to cell-cycle arrest following HTLV-1 infection, suggesting that HTLV-1 may efficiently establish a latent infection in these cell types in vivo. HTLV-2 does not display these properties following infection of CD34+ HPCs, demonstrating distinct biological differences between these two virus strains with respect to regulation of cellular processes in HP/HSCs. Materials and Methods Isolation, Infection, and Purification of CD34+ HPCs Human CD34+ HPCs were prepared from human fetal liver (ABR, Oakland, California) as previously described [32, 33]. Purified CD34+ HPCs (3 × 106) were infected with HTLV-1 or HTLV-2 by cocultivation with lethally irradiated (103 rads) SLB-1 (HTLV-1-infected), 729/ACH (HTLV-1-infected), and 729/pH6Neo (HTLV-2-infected) donor cells at a ratio of 1:5, as previously described [31, 34]. We routinely and reproducibly saw relatively high percentages of CD34+/CD38− cells in fetal liver-derived stem cell preparations (>75%), as previously reported [35, 36]. After 2 days cells were stained with phycoerythrin (PE)-conjugated anti-CD34 antibody and a mouse monoclonal anti-HTLV envelope antibody (gp46env) (Zeptometrix, Buffalo, NY, http://www.zeptometrix.com) (3 μl per sample) followed by a secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse monoclonal IgG (Dako, Glostrup, Denmark, http://www.dako.com), and infected cells coexpressing both CD34 and gp46env were purified by fluorescence-activated cell sorting (FACS). Mock-infected cells and CD34+ HPCs that were cocultivated with HTLV-infected donor cell lines but were not infected (CD34+/gp46env−) were also purified by FACS. Clonogenic Colony Forming Assays Clonogenic colony forming assays were performed as previously described [8, 33]. FACS-purified CD34+/HTLV gp46env+, CD34+/HTLV gp46env−, and mock-infected cells were plated in 2 ml of MethoCult H4433 medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) at 2,000 cells per 35 × 10 mm plate, in triplicate, and incubated at 37°C in a humidified atmosphere with 5% CO2. Total clonogenic colony forming granulocyte-macrophage (CFU-GM), erythroid burst (BFU-E), and high proliferative pluripotent (HPP) colonies were identified by morphology at 10–15 days postplating and counted under an inverted fluorescent microscope (DMIL; Leica, Heerbrugg, Switzerland, http://www.leica.com) [37]. Cell Cycle Analysis of CD34+ HPCs CD34+ HPCs were stained with Hoechst 33342 (Hst) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) and Pyronin Y (Polysciences Inc., Warrington, PA, http://www.polysciences.com) using a protocol modified from the one previously described [31, 38, 39]. Briefly, cells (2 × 106) were stained with 1 μg/ml Hst followed by 0.2 μg/ml Pyronin Y (PY) in 50 μM verapamil solution in Hst buffer. Cells were permeabilized/fixed (Cytofix/Cytoperm Solution; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) and stained with a mouse monoclonal antibody (mAB) that recognizes both HTLV-1 and HTLV-2 capsid (p19gag) protein (Zeptometrix) (3 μl per sample), a modification from previously published protocols. Gates for cycling CD34+ cell subpopulations were established as follows: CD34+ HPCs were serum-starved (to arrest cells in G0) and were treated with roscovitine (20 μM) (Calbiochem, San Diego, http://www.emdbiosciences.com), a cdk inhibitor that arrests cells in G1, or were transduced with a lentiviral vector encoding the human immunodeficiency virus 1 (HIV-1) Vpr gene, which arrests cells in G2 [33, 40, 41–42]. Cells that displayed increased RNA (as determined by PY staining) were defined as S-phase, whereas increased Hst staining levels determined G2/M gates. Cell cycle arrest was determined by comparison of cell numbers in S+G2/M in relation to cell numbers in the G1 and G0 gates. Cell cycle analysis was performed on an LSRII flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR, http://www.flojo.com). Statistical significance was analyzed using the Student t test and single-tail analysis of variance (p < .05) using mock-infected cells as controls. Flow Cytometry CD34+ HPCs were stained with 5 μl each of PerCP-conjugated anti-human CD34 antibody (Becton Dickinson) and an APC-conjugated anti-human CD38 antibody (Becton Dickinson) cells were then permeabilized/fixed and stained with mouse mAB, which recognizes both HTLV-1 and HTLV-2 capsid (p19gag) (Zeptometrix) (3 μl per sample), and with either a rabbit mAB against human survivin (5 μl per sample) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) or a rabbit mAB against human p21cip1/waf1 (5 μl per sample) (Santa Cruz Biotechnology) followed by FITC-conjugated goat anti-mouse IgG (1 μl per sample) (Dako) and PE-conjugated goat anti-rabbit IgG (2 μl per sample) (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Samples were analyzed using FlowJo software. Infection of CD34+ HPCs and Primary CD4+ T Cells with Vesicular Stomatitis Protein G Pseudotyped Lentiviral Vectors Vesicular stomatitis protein G-pseudotyped lentivirus vector (LVs) stocks were generated, and CD34+ HPCs and primary CD4+ T cells were transduced with lentivirus vectors (multiplicity of infection = 5) as previously described [31, 33, 34, 43, 44]. Cells were analyzed for green fluorescent protein (GFP) expression 24–48 hours post-transduction by flow cytometry. Transient Transfection Assays 293T cells were cotransfected with survivin promoter-luciferase constructs and LV constructs expressing Tax1, Tax1(−), or Tax2 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's protocol. Briefly, cells (105) were transfected with 2 μg of LV constructs or 2 μg of pUC19 (mock) and 2 μg of either pLuc-2870 or pLuc-178 [19]. Survivin promoter constructs and the number of nucleotides of upstream sequences and key transcription factor binding sites are described as follows: pLuc-2870, 2.8 kilobases (kb) of the promoter sequence containing nine E-box sequences, two CREB binding sequences, and three p300 binding sequences; pLuc-178, 178 base pairs (bp) of the promoter sequence containing one p300 binding sequence. The survivin promoter sequence was analyzed for transcription factor binding sites using the MAPPER database [45]. For dose-response transient transfections, 293T cells (105) were cotransfected with increasing amount of Tax1 vector (in micrograms, as indicated) and 4 μg of pLuc-178. pUC-19 DNA was used to standardize total amount of DNA (8 μg) in each transfection. Cotransfection of pLuc-2870 (2 μg) and increasing amounts of either LV-Tax1, LV-Tax1(−), or pc53-SN (a p53 expression vector) was performed in 293T cells. Luciferase assays were normalized to protein concentration levels and performed in triplicate using a plate-reading luminometer (Luminoskan RS; Labsystems, Needham Heights, MA, http://www.thermo.com) and the Luciferase Assay System (Promega, Madison, WI, http://www.promega.com). Statistical analysis was performed with Tax1(−) lysates as a negative control, using the Student t test (p < .05). Transfection of Short Interfering RNA and Propidium Iodide Cell Cycle Staining ON-TARGETplus SMARTpool p21 short interfering RNA (siRNA) and siCONTROL Non-Targeting siRNA (Dharmacon, Lafayette, CO, http://www.dharmacon.com) were obtained and resuspended per the manufacturer's protocol (Dharmacon). Briefly, 5 μl of 20 μM siRNA stock solution of both p21 siRNA and nontargeting siRNA was used to transfect 2 × 106 CD34+ HPCs using the Human CD34 cell Nucleofector kit and Nucleofector machine (Amaxa, Gaithersburg, MD, http://www.amaxa.com) according to the manufacturer's recommendation and previously published reports [46]. The efficiency of transfection was monitored using siGLO lamin A/C siRNA (Dharmacon). Propidium iodide cell cycle staining was performed as described previously [47]. Western Blot Analysis Western blot analysis for survivin expression in transfected 293FT cells were performed as previously described [48, 49]. Briefly, 293T cells (2 × 105) were transfected with LV-Tax1 or Tax(−) or were mock-transfected (4 μg of DNA) with Lipofectamine 2000 (Invitrogen), and protein was extracted at 48 hours post-transfection. Proteins were then separated on a 4%–12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and transferred to membrane, blocked in 5% nonfat dry milk in 1× Tris-buffered saline, washed, and incubated with monoclonal antibody against survivin ([D-8] sc-17779; Santa Cruz Biotechnology; dilution, 1:100) for 48 hours followed by a secondary goat anti-mouse horseradish peroxidase-conjugated antibody for 2 hours (Santa Cruz Biotechnology; dilution, 1:500). Membranes were then stained with a monoclonal antibody against human β-actin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to standardize for protein loading. Results HTLV-1 Infection Suppresses Clonogenic Colony Formation in CD34+ HPCs We have previously shown that HTLV-1 can infect CD34+ HPCs and that proviral sequences are maintained during cellular differentiation in vitro and in vivo [32]. To quantitatively establish whether HTLV infection affects hematopoiesis, CD34+ HPCs were infected with HTLV-1 or HTLV-2 by cocultivation with lethally irradiated SLB-1 (HTLV-1-infected cell line) or 729/pH6Neo (HTLV-2-infected cell line) donor cells [34, 50]. HTLV does not efficiently infect cells as a cell-free virus, requiring cocultivation of uninfected target cells with lethally irradiated persistently infected donor cell lines. CD34+ HPCs expressing HTLV-1 envelope proteins (CD34+/gp46env+ cells) were viably purified by FACS, and more than 98% of the FACS-purified cells coexpressed CD34 and HTLV gp46env. Both HTLV-1- and HTLV-2-infected CD34+ HPCs express similar levels of Tax oncoprotein 2 days postinfection, as visualized by flow cytometry using a polyclonal antibody that detects both Tax1 and Tax2 (Santa Cruz Biotechnology) (Fig. 1A). Purified CD34+/gp46env+ cells were plated into a semisolid medium (MethoCult H4433) that allows for the development of clonogenic hematopoietic colonies, including CFU-GM, erythroid precursors (BFU-E), and high proliferative pluripotent (HPP). HTLV-1-infected CD34+ HPCs displayed a marked suppression of multilineage hematopoiesis in contrast to HTLV-2-infected or mock-infected CD34+ HPCs (approximately twofold) (Fig. 1B). Clonogenic colonies that matured from HTLV-1- and HTLV-2-infected CD34+ HPCs had similar levels of proviral sequences as detected by RQ-polymerase chain reaction (PCR) (average number of HTLV proviral copies per cell in clonogenic colonies, 1.3 [SD = 0.23] and 1.2 [SD = 0.54] for HTLV-1- and HTLV-2-infected HPCs, respectively) (data not shown). The relative ratios of CFU-GM, BFU-E, and HPP colonies that developed from HTLV-1-infected CD34+ HPCs were similar to the ratios that developed from either mock-infected or HTLV-2-infected CD34+ HPCs. Figure 1. Open in new tabDownload slide HTLV-1 productively infects CD34+ hematopoietic progenitor cells (HPCs), and infection suppresses clonogenic colony formation in vitro. Human fetal liver-derived CD34+ HPCs were purified and infected with HTLV-1 or HTLV-2 by cocultivation with SLB-1 (HTLV-1-infected), 729/ACH (HTLV-1-infected), or 729/pH6Neo (HTLV-2-infected) donor cells, respectively. Two days postinfection, CD34+/gp46env+ and CD34+/gp46env− cells were fluorescence-activated cell sorting (FACS)-purified and plated into MethoCult. (A): Relative levels of Tax expression in FACS-purified HTLV-1 (blue line) and HTLV-2 (pink line)-infected CD34+ HPCs. Red line represents cells stained with secondary antibody only. (B): Enumeration of clonogenic colony forming activity. Colonies were visually scored and enumerated 14 days after plating. The experiments were performed four times, and error bars represent SD. CD34+ used in these experiments was purified from four different donor tissues. (C): Clonogenic colony forming activity of CD34+ HPCs infected with HTLV-1 (SLB-1 or 729/ACH cells). Uninfected cells represent FACS-purified CD34+ HPCs from SLB-1 or 729/ACH cocultures that did not express gp46env. Colonies were visually scored and enumerated 10 days after plating. This experiment was performed once, with cells being plated in triplicate, and error bars represent SD. Statistical analysis was performed using single-tail analysis of variance and Student t tests using mock-infected CD34+ cells as control; *, p < .05. Abbreviation: HTLV, human T-cell lymphotropic virus. Figure 1. Open in new tabDownload slide HTLV-1 productively infects CD34+ hematopoietic progenitor cells (HPCs), and infection suppresses clonogenic colony formation in vitro. Human fetal liver-derived CD34+ HPCs were purified and infected with HTLV-1 or HTLV-2 by cocultivation with SLB-1 (HTLV-1-infected), 729/ACH (HTLV-1-infected), or 729/pH6Neo (HTLV-2-infected) donor cells, respectively. Two days postinfection, CD34+/gp46env+ and CD34+/gp46env− cells were fluorescence-activated cell sorting (FACS)-purified and plated into MethoCult. (A): Relative levels of Tax expression in FACS-purified HTLV-1 (blue line) and HTLV-2 (pink line)-infected CD34+ HPCs. Red line represents cells stained with secondary antibody only. (B): Enumeration of clonogenic colony forming activity. Colonies were visually scored and enumerated 14 days after plating. The experiments were performed four times, and error bars represent SD. CD34+ used in these experiments was purified from four different donor tissues. (C): Clonogenic colony forming activity of CD34+ HPCs infected with HTLV-1 (SLB-1 or 729/ACH cells). Uninfected cells represent FACS-purified CD34+ HPCs from SLB-1 or 729/ACH cocultures that did not express gp46env. Colonies were visually scored and enumerated 10 days after plating. This experiment was performed once, with cells being plated in triplicate, and error bars represent SD. Statistical analysis was performed using single-tail analysis of variance and Student t tests using mock-infected CD34+ cells as control; *, p < .05. Abbreviation: HTLV, human T-cell lymphotropic virus. SLB-1 is an HTLV-1-transformed T-cell line established from an ATL patient. 729/ACH and 729/pH6-Neo are B-lymphoid cell lines transfected with infectious molecular clones of HTLV-1 and HTLV-2, respectively. To verify that the differences observed following infection of CD34+ HPCs with HTLV-1 and HTLV-2 were not simply due to the differences in the cellular source of the virus, we compared and analyzed the colony forming potential of CD34+ HPCs infected with either SLB-1 or 729/ACH cells (Fig. 1C). Infection with either 729/ACH or SLB-1 cells resulted in similar reductions of CFU activity in vitro. We also examined the ability of uninfected CD34+ HPCs purified from the cocultured cells to form clonogenic colonies and found that these cells showed clonogenic colony forming activity similar to that of mock-infected cells. This directly demonstrates that suppression of hematopoiesis requires infection of the CD34+ HPCs by HTLV-1. HTLV-1 infection of CD34+ HPCs also did not alter the relative differentiation profiles and was reflective of the suppression of hematopoiesis following transduction of CD34+ HPCs with HTLV-1 Tax (Tax1) [33]. HTLV-1 Induces G0/G1 Cell Cycle Arrest in CD34+ HPCs To investigate whether suppression of hematopoiesis in HTLV-1-infected CD34+ HPCs was a result of cell cycle perturbation, CD34+ HPCs were infected with HTLV-1 or HTLV-2, and CD34+/HTLV p19gag+ cells were stained with Hst and PY [38, 39]. Staining with Hst and PY distinguishes cells in distinct phases of the cell cycle on the basis of their relative amount of DNA and RNA content, respectively. CD34+ HP/HSCs reside in G0, display a low RNA content and are generally in quiescence, whereas cells transitioning from G1 to S display increased RNA levels and concurrently elevated PY and Hst staining. As a control CD34+ HPCs were cultured in the absence of cytokine growth factors (IL-3, IL-6, stem cell factor, and Flt-3L) to induce arrest in G0. Cells were also treated with roscovitine K, a drug that induces G1 arrest, or were transduced with a lentiviral vector encoding HIV-1 Vpr, which has been previously shown to arrest cells in G2 (Fig. 2C) [33, 40, 41–42]. The serum-starved, roscovitine-treated, and Vpr-transduced CD34+ HPCs were then used in combination with PY and Hst staining patterns to establish individual gates for cycling cell subpopulations (Fig. 2A–2C). HTLV-1 infection of CD34+ HPCs resulted in significant G0/G1 cell cycle arrest at 48 and 72 hours postinfection (Fig. 2F, 2J), in contrast to HTLV-2-infected or mock-infected HPCs. The percentage of cells that accumulated in G0/G1 following HTLV-1 infection was reflective of the cell cycle arrest pattern demonstrated by treating cells with roscovitine K. Transduction of CD34+ cells with HIV-1 Vpr resulted in a significant accumulation of cells in G2, as previously shown [31]. No arrest of HTLV-1-infected CD34+ HPCs was detected at 24 hours, suggesting that proviral integration and viral gene expression was required for the manifestation of this phenotype (data not shown). Notably, uninfected stem cells (CD34+/ HTLV-1p19gag−) from the SLB-1 coculture failed to show accumulation of cells in G0/G1, in contrast to CD34+/p19gag+ cells (45% vs. 80%, respectively), demonstrating that HTLV-1 infection was necessary for cell cycle arrest in CD34+ HPCs (Fig. 2F, 2H). These data show that HTLV-1 infection of CD34+ HPCs induces cell cycle arrest in G0/G1 and that HTLV-2 fails to alter patterns of cell cycle progression following infection. Figure 2. Open in new tabDownload slide HTLV-1 infection arrests CD34+ hematopoietic progenitor cells (HPCs) in G0/G1. (A–C): Representative histograms of Hoechst 33342 (Hst)/Pyronin Y (PY) analysis of serum-starved (A), roscovitine-treated (B), and human immunodeficiency virus 1 Vpr (C)-transduced CD34+ HPCs. CD34+ cell subpopulations arrested under each treatment condition shown in (A–C) were used to establish G0, G1, and G2 gates for flow cytometric analysis (red boxes), as previously reported [31, 40, 41]. (D): Representative histogram of Hst/PY analysis of mock-infected CD34+ HPCs at 48 hrs postinfection. (E): Histogram of p19gag expression on HTLV-1-infected CD34+ HPCs. The histogram bisector population represents the selection gate used to analyze the cell-cycle profile of CD34+/HTLV p19gag+ and CD34+/HTLV p19gag− cells. (F–I): Representative dot plot of Hst/PY flow cytometric profiles of CD34+/HTLV-1 p19gag+ cells (F), CD34+/HTLV-2 p19gag+ cells (G), CD34+/HTLV-1 p19gag− cells (H), and CD34+/HTLV-2 p19gag− cells (I). Numbers represent the percentage of cells in each quadrant. All cells were stained with Hst/PY and were subsequently permeabilized and fixed prior to flow cytometric analysis. (J): Quantification of the percentage of cells in G0/G1 at 48 hrs (solid bar) and 72 hrs (hatched bar) postinfection from four experiments. The majorities of serum-starved CD34+ cells were not viable at 48 hrs, underwent apoptosis, and were not analyzed. The experiments were performed four times, and error bars represent the SD. Statistical analysis was performed using single-tail analysis of variance and Student t tests using mock-infected cells as control; *, p < .05. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. Figure 2. Open in new tabDownload slide HTLV-1 infection arrests CD34+ hematopoietic progenitor cells (HPCs) in G0/G1. (A–C): Representative histograms of Hoechst 33342 (Hst)/Pyronin Y (PY) analysis of serum-starved (A), roscovitine-treated (B), and human immunodeficiency virus 1 Vpr (C)-transduced CD34+ HPCs. CD34+ cell subpopulations arrested under each treatment condition shown in (A–C) were used to establish G0, G1, and G2 gates for flow cytometric analysis (red boxes), as previously reported [31, 40, 41]. (D): Representative histogram of Hst/PY analysis of mock-infected CD34+ HPCs at 48 hrs postinfection. (E): Histogram of p19gag expression on HTLV-1-infected CD34+ HPCs. The histogram bisector population represents the selection gate used to analyze the cell-cycle profile of CD34+/HTLV p19gag+ and CD34+/HTLV p19gag− cells. (F–I): Representative dot plot of Hst/PY flow cytometric profiles of CD34+/HTLV-1 p19gag+ cells (F), CD34+/HTLV-2 p19gag+ cells (G), CD34+/HTLV-1 p19gag− cells (H), and CD34+/HTLV-2 p19gag− cells (I). Numbers represent the percentage of cells in each quadrant. All cells were stained with Hst/PY and were subsequently permeabilized and fixed prior to flow cytometric analysis. (J): Quantification of the percentage of cells in G0/G1 at 48 hrs (solid bar) and 72 hrs (hatched bar) postinfection from four experiments. The majorities of serum-starved CD34+ cells were not viable at 48 hrs, underwent apoptosis, and were not analyzed. The experiments were performed four times, and error bars represent the SD. Statistical analysis was performed using single-tail analysis of variance and Student t tests using mock-infected cells as control; *, p < .05. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. HTLV-1 Infection Modulates the Expression of p21cip1/waf1 in CD34+/CD38− HSCs CD34+/CD38− cells represent an immature subpopulation consisting of HSCs that predominantly reside in quiescence and retain the capacity to divide as well as differentiate into multiple hematopoietic lineage cells [51, 52, 53, 54, 55–56]. p21cip1/waf1 (p21) is a CDK inhibitor that is involved in the modulation of cell cycle progression of HSCs and has been shown to sustain stem cells in quiescence [10, 57]. We have previously shown that HTLV-1 infection transcriptionally upregulates p21 expression in CD34+ HPCs and that the transcriptional upregulation of p21 was more robust in HTLV-1-infected CD34+/CD38− in comparison with HTLV-1-infected CD34+/CD38+ cells [31]. To verify that intracellular p21 protein levels were reflective of the transcriptional modulation, CD34+ HPCs were infected with HTLV-1 or HTLV-2, and CD34+/CD38−/p19gag+ and CD34+/CD38+/p19gag+ cells were analyzed for intracellular expression of p21cip1/waf1 (Fig. 3). HTLV-1-infected CD34+/CD38− cells showed significantly higher intracellular levels of p21 protein at 48 and 72 hours postinfection in comparison with HTLV-2-infected or mock-infected CD34+/CD38− cells. Conversely, the levels of p21 in HTLV-1-infected and HTLV-2-infected CD34+/CD38+ cells were modestly elevated at 48 and 72 hours postinfection. No significant difference in p21 expression levels was detected at 24 hours postinfection, indicating that modulation of p21 expression occurs concurrently with cell cycle arrest/progression (data not shown). These data suggest that HTLV-1 robustly induces p21 expression in immature CD34+/CD38− HSCs, in contrast to HTLV-2. Figure 3. Open in new tabDownload slide HTLV-1 induces p21cip1/waf1 expression in CD34+ hematopoietic progenitor cells (HPCs)/hematopoietic stem cells. (A): HTLV-1-infected CD34+ HPCs stained with an isotype control antibody. (B, C): Representative scatter plots demonstrating HTLV-1 p19gag+ and CD34 expression on mock-infected (B) and HTLV-1-infected (C) CD34+ HPCs. Region I (red square) represents the selection gate used to analyze CD34+/HTLV p19gag+ cells. (D): Representative scatter plot showing the CD38 expression on positively selected CD34+/HTLV p19gag+ cells. Regions II and III (red squares) represent gates used to analyze CD34+/CD38−/HTLV p19gag+ or CD34+/CD38+/HTLV p19gag+ cells, respectively. (E, F): Representative histograms of intracellular p21 expression in CD34+/CD38− HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (E) and 72 (F) hrs postinfection. (G, H): Representative histograms showing intracellular p21 expression in CD34+/CD38+ HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (G) and 72 (H) hrs postinfection. Red lines in (E–H) represent cells stained with secondary antibody only. (I, J): Representative scatter plot showing the CD38 expression on mock-infected (I) and CD34+/HTLVp19gag− (J) HPCs. Data are representative of four experiments using purified CD34+ HPCs from three different donors. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. Figure 3. Open in new tabDownload slide HTLV-1 induces p21cip1/waf1 expression in CD34+ hematopoietic progenitor cells (HPCs)/hematopoietic stem cells. (A): HTLV-1-infected CD34+ HPCs stained with an isotype control antibody. (B, C): Representative scatter plots demonstrating HTLV-1 p19gag+ and CD34 expression on mock-infected (B) and HTLV-1-infected (C) CD34+ HPCs. Region I (red square) represents the selection gate used to analyze CD34+/HTLV p19gag+ cells. (D): Representative scatter plot showing the CD38 expression on positively selected CD34+/HTLV p19gag+ cells. Regions II and III (red squares) represent gates used to analyze CD34+/CD38−/HTLV p19gag+ or CD34+/CD38+/HTLV p19gag+ cells, respectively. (E, F): Representative histograms of intracellular p21 expression in CD34+/CD38− HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (E) and 72 (F) hrs postinfection. (G, H): Representative histograms showing intracellular p21 expression in CD34+/CD38+ HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (G) and 72 (H) hrs postinfection. Red lines in (E–H) represent cells stained with secondary antibody only. (I, J): Representative scatter plot showing the CD38 expression on mock-infected (I) and CD34+/HTLVp19gag− (J) HPCs. Data are representative of four experiments using purified CD34+ HPCs from three different donors. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. Knockdown of p21 Releases HTLV-1-Infected CD34+ HPCs from Cell Cycle Arrest To evaluate whether silencing p21 expression results in the reversal of cell cycle arrest in HTLV-1-infected CD34+ HPCs, HTLV-1-infected CD34+ HPCs were transfected with p21 siRNA [58, 59]. p21 siRNA expression resulted in a significant downmodulation of intracellular p21 protein levels in HTLV-1-infected CD34+ HPCs at 48–72 hours post-transfection (Fig. 4B, 4C). Downmodulation of p21 expression in HTLV-1-infected CD34+ HPCs resulted in a concurrent two- to threefold increase in the percentage of infected CD34+ HPCs that accumulate in the S+G2/M subpopulation in comparison with control siRNA-transfected cells (Fig. 4E, 4F). These data demonstrate that p21 substantially contributes to cell cycle arrest in HTLV-1-infected CD34+ HP/HSCs. Figure 4. Open in new tabDownload slide Knockdown of p21 abrogates cell-cycle arrest in HTLV-1-infected CD34+ hematopoietic progenitor cells (HPCs). (A–C): Representative histogram showing p21 expression in CD34+/HTLV p19gag+ cells transfected with p21 siRNA (blue lines) and control siRNA (pink lines) at 24 (A), 48 (B), and 72 (C) hrs post-transduction. (D–F): Representative histograms of propidium iodide cell cycle profile of CD34+/HTLV p19gag+ cells transfected with p21 siRNA (red lines) and control siRNA (blue lines) 24 (D), 48 (E), and 72 (F) hrs post-transduction. Red lines represent cells stained with secondary antibody only. Data are representative of two separate experiments using purified CD34+ HPCs from two different donors. Abbreviations: hrs, hours; siRNA, short interfering RNA. Figure 4. Open in new tabDownload slide Knockdown of p21 abrogates cell-cycle arrest in HTLV-1-infected CD34+ hematopoietic progenitor cells (HPCs). (A–C): Representative histogram showing p21 expression in CD34+/HTLV p19gag+ cells transfected with p21 siRNA (blue lines) and control siRNA (pink lines) at 24 (A), 48 (B), and 72 (C) hrs post-transduction. (D–F): Representative histograms of propidium iodide cell cycle profile of CD34+/HTLV p19gag+ cells transfected with p21 siRNA (red lines) and control siRNA (blue lines) 24 (D), 48 (E), and 72 (F) hrs post-transduction. Red lines represent cells stained with secondary antibody only. Data are representative of two separate experiments using purified CD34+ HPCs from two different donors. Abbreviations: hrs, hours; siRNA, short interfering RNA. HTLV-1 Infection Downmodulates Survivin in CD34+ HPCs Survivin plays a pivotal role in initiating cell cycle entry, progression, and maturation of CD34+ HP/HSCs and is required for egress of CD34+ HPCs from quiescence [13, 15, 17, 38, 60]. To establish whether HTLV-1 infection modulates the expression of endogenous survivin in CD34+ HPCs, CD34+/CD38−/p19gag+ and CD34+/CD38+/p19gag+ cell subpopulations were stained with rabbit mAB against human survivin (Santa Cruz Biotechnology) and analyzed for intracellular survivin expression (Fig. 5). HTLV-1-infected CD34+/CD38− HSCs showed significant downmodulation of survivin at 48 and 72 hours postinfection, in contrast to HTLV-2-infected and mock-infected cells. Both HTLV-1 and HTLV-2 repressed survivin expression at modest levels in more mature CD34+/CD38+ cells at 72 hours postinfection. This demonstrates that suppression of survivin is most acute in HTLV-1-infected CD34+/CD38− HSCs. Figure 5. Open in new tabDownload slide HTLV-1 downmodulates survivin expression in CD34+/CD38− hematopoietic stem cells and CD34+/CD38+ hematopoietic progenitor cells (HPCs). (A): HTLV-1-infected cells stained with isotype control. (B, C): Representative histograms demonstrating intracellular expression of HTLV-1 p19gag and CD34 on mock-infected (B) and HTLV-1-infected (C) CD34+ HPCs. Region I represents the CD34+/HTLV p19gag+ cell subpopulation used for further analysis. (D): Representative scatter plot showing CD38 expression on CD34+/HTLV p19gag+ cells. Regions II and III (red squares) represent gating on CD34+/CD38-/HTLV p19gag+ and CD34+/CD38+/HTLV p19gag+ cells, respectively. Representative histograms showing intracellular survivin expression in CD34+/CD38− HPCs infected with HTLV-1 (blue lines), HTLV-2 (pink lines), or mock-infected (green lines) at 48 (E) and 72 (F) hrs post infection. (G, H): Representative histograms showing intracellular survivin expression in CD34+/CD38+ HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (G) and 72 (H) hrs postinfection. Red lines represent cells stained with secondary antibody only. Data are representative of four separate experiments using purified CD34+ HPCs from three different donors. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. Figure 5. Open in new tabDownload slide HTLV-1 downmodulates survivin expression in CD34+/CD38− hematopoietic stem cells and CD34+/CD38+ hematopoietic progenitor cells (HPCs). (A): HTLV-1-infected cells stained with isotype control. (B, C): Representative histograms demonstrating intracellular expression of HTLV-1 p19gag and CD34 on mock-infected (B) and HTLV-1-infected (C) CD34+ HPCs. Region I represents the CD34+/HTLV p19gag+ cell subpopulation used for further analysis. (D): Representative scatter plot showing CD38 expression on CD34+/HTLV p19gag+ cells. Regions II and III (red squares) represent gating on CD34+/CD38-/HTLV p19gag+ and CD34+/CD38+/HTLV p19gag+ cells, respectively. Representative histograms showing intracellular survivin expression in CD34+/CD38− HPCs infected with HTLV-1 (blue lines), HTLV-2 (pink lines), or mock-infected (green lines) at 48 (E) and 72 (F) hrs post infection. (G, H): Representative histograms showing intracellular survivin expression in CD34+/CD38+ HPCs infected with HTLV-1 (blue lines), infected with HTLV-2 (pink lines), or mock-infected (green lines) at 48 (G) and 72 (H) hrs postinfection. Red lines represent cells stained with secondary antibody only. Data are representative of four separate experiments using purified CD34+ HPCs from three different donors. Abbreviations: hrs, hours; HTLV, human T-cell lymphotropic virus. Tax1 Differentially Modulates Survivin Expression in CD4+ T Lymphocytes and CD34+ HPCs Primary CD4+ T cells are one of the targets of HTLV-1 infection in vivo and infection results in immortalization and transformation for growth in vitro [61]. Freshly isolated CD4+ T cells and CD34+ HPCs were transduced with lentiviral vectors encoding for GFP and coexpressing either Tax1, Tax2, or Tax1(−) (an antisense Tax1 construct) [33]. CD34+/GFP+ HPCs transduced with Tax1 showed significantly lower levels of endogenous survivin expression in comparison with mock-transduced or Tax1(−) transduced CD34+ HPCs (Fig. 6A). In contrast, the levels of survivin expression were strikingly similar in Tax1-, Tax2-, and Tax1(−)-transduced CD4+ lymphocytes (Fig. 6B). These data suggest that modulation of endogenous survivin by Tax1 is cell-type-specific for HP/HSCs and that Tax1 is sufficient for the robust repression of intracellular survivin in CD34+ HP/HSCs. Figure 6. Open in new tabDownload slide Human T-cell lymphotropic virus type 1 (HTLV-1) Tax1 transduction in CD4+ T lymphocytes fails to alter survivin expression levels. CD34+ hematopoietic progenitor cells and CD4+ T lymphocytes were transduced with lentivirus vectors expressing HTLV-1 Tax (Tax1) and HTLV-2 Tax (Tax2) or an antisense Tax (Tax1(−)) as a control. (A): Histogram of intracellular survivin expression in CD34+/Tax1-green fluorescent protein (GFP) (blue line), CD34+/Tax2-GFP (pink line), CD34+/Tax1(−)-GFP (black line), or mock-infected CD34+ (green line) cells at 48 hrs postinfection. Red lines represent cells stained with secondary antibody in the absence of a primary antibody. (B): Histogram demonstrating survivin expression in CD4+/Tax1-GFP (blue line), CD4+/Tax2-GFP (pink line), CD4+/Tax1(−)-GFP (black), or mock-infected CD4+ (green line) cells at 48 hrs postinfection. Red lines represent cells stained with secondary antibody only. Abbreviation: hrs, hours. Figure 6. Open in new tabDownload slide Human T-cell lymphotropic virus type 1 (HTLV-1) Tax1 transduction in CD4+ T lymphocytes fails to alter survivin expression levels. CD34+ hematopoietic progenitor cells and CD4+ T lymphocytes were transduced with lentivirus vectors expressing HTLV-1 Tax (Tax1) and HTLV-2 Tax (Tax2) or an antisense Tax (Tax1(−)) as a control. (A): Histogram of intracellular survivin expression in CD34+/Tax1-green fluorescent protein (GFP) (blue line), CD34+/Tax2-GFP (pink line), CD34+/Tax1(−)-GFP (black line), or mock-infected CD34+ (green line) cells at 48 hrs postinfection. Red lines represent cells stained with secondary antibody in the absence of a primary antibody. (B): Histogram demonstrating survivin expression in CD4+/Tax1-GFP (blue line), CD4+/Tax2-GFP (pink line), CD4+/Tax1(−)-GFP (black), or mock-infected CD4+ (green line) cells at 48 hrs postinfection. Red lines represent cells stained with secondary antibody only. Abbreviation: hrs, hours. Tax1 Transcriptionally Represses Survivin Promoter Activity Although the Tax1 gene product functions primarily as a transcriptional transactivator, Tax1 has previously been shown to repress transcription of select cellular genes. To determine whether Tax1 directly repressed transcription from the survivin promoter, transient transfection assays were performed using survivin promoter-luciferase constructs and bicistronic lentivirus vectors (LVs) encoding Tax1, Tax2, and Tax1(−) (Fig. 7A) [33, 34]. Cotransfection of Tax1 with a construct containing 2.8 kb of the survivin promoter (pLuc-2870) resulted in a 36-fold reduction in luciferase expression, in comparison with mock-transfected cultures (Fig. 7B). Tax1 also repressed expression from a truncated survivin promoter construct encoding 178 bp (pLuc-178) of the promoter sequence. Cotransfection of pLuc-2870 with Tax2 reduced luciferase activity by 1.5–2-fold, in comparison with mock-transfected cells, demonstrating that Tax2 modestly repressed expression from the survivin promoter. Increasing amounts of Tax1 vector in cotransfection experiments with pLuc-178 demonstrated a dose-responsive dependent repression (10–15-fold) of luciferase activity (Fig. 7C). The p53 tumor suppressor protein has previously been shown to repress transcription from the survivin promoter [62, 63–64]. Cotransfection of a construct expressing p53 (pc53-SN3) resulted in repression of the survivin promoters at levels that were comparable to the repression observed by Tax1 (Fig. 7D). The repression of endogenous survivin by Tax1 in 293T cells was detected by Western blot analysis following transfection with Tax1 (Fig. 7E). It is noteworthy that Tax1 has previously been shown to repress expression of cellular genes containing promoter E-box motifs (CANNTG) [24, 25, 28, 30]. In addition, Tax1 has also been shown to transcriptionally express genes that encode a CBP/p300 binding site. Sequence analysis of the human survivin promoter revealed the presence of 9 E-box motifs as well as three CBP/p300 binding sites (Fig. 7A). These data demonstrate that Tax1 directly represses transcriptional activity from the survivin promoter. Figure 7. Open in new tabDownload slide Tax1-mediated repression of transcription from the human survivin promoter. (A): The survivin proximal promoter sequence was analyzed for transcription factor binding sites using the MAPPER database [45]. The database located nine E-box motifs (CANNTG) within 3,000 base pairs upstream of the survivin start codon. Also identified were p300 binding sites (−1,545, −431, −46), as well as two CREB binding sites (−2,234, −865). (B): Cotransfection of Tax1, Tax1(−), Tax2, and survivin promoter-luciferase construct. Survivin promoter-luciferase constructs were cotransfected into 293T cells with lentivirus vector plasmids encoding Tax1 (black bar), Tax1(−) (white bar), and Tax2 (gray bar). Mock (striped bar) represents luciferase activity resulting from cotransfection of luciferase construct with pUC19 DNA. (C): Increasing amounts of Tax1 vector (in μg) and 4 μg of pLuc-178 were cotransfected into 293T cells. Experiments were assayed in triplicate, and data shown are from a representative experiment of three independent determinations. Error bars represent 1 SD. (D): Cotransfection of pLuc-2870 with pC53-SN3 (a p53 expression vector; a gift from Dr. Bert Vogelstein) Tax1, or Tax1(−). 293T cells (1 × 105) were cotransfected with 2 μg of pLuc-2870 and increasing amounts of either Tax1 (black bars), Tax1(−) (white bars), or pC53-SN3 (striped bars), as indicated. Experiments were assayed in triplicate, and data shown are from a representative experiment of three independent determinations. Error bars represent 1 SD. (E): Western blot analysis were performed on whole protein lysates from 293T cells (2 × 105) that were transfected with either Tax1 or Tax1(−) (4 μg). Proteins were extracted 48 hours post-transfection, and membranes were stained with a monoclonal antibody against survivin followed by a monoclonal antibody against β-actin. This experiment was performed twice. Abbreviation: CREB, cyclic AMP response element-binding protein. Figure 7. Open in new tabDownload slide Tax1-mediated repression of transcription from the human survivin promoter. (A): The survivin proximal promoter sequence was analyzed for transcription factor binding sites using the MAPPER database [45]. The database located nine E-box motifs (CANNTG) within 3,000 base pairs upstream of the survivin start codon. Also identified were p300 binding sites (−1,545, −431, −46), as well as two CREB binding sites (−2,234, −865). (B): Cotransfection of Tax1, Tax1(−), Tax2, and survivin promoter-luciferase construct. Survivin promoter-luciferase constructs were cotransfected into 293T cells with lentivirus vector plasmids encoding Tax1 (black bar), Tax1(−) (white bar), and Tax2 (gray bar). Mock (striped bar) represents luciferase activity resulting from cotransfection of luciferase construct with pUC19 DNA. (C): Increasing amounts of Tax1 vector (in μg) and 4 μg of pLuc-178 were cotransfected into 293T cells. Experiments were assayed in triplicate, and data shown are from a representative experiment of three independent determinations. Error bars represent 1 SD. (D): Cotransfection of pLuc-2870 with pC53-SN3 (a p53 expression vector; a gift from Dr. Bert Vogelstein) Tax1, or Tax1(−). 293T cells (1 × 105) were cotransfected with 2 μg of pLuc-2870 and increasing amounts of either Tax1 (black bars), Tax1(−) (white bars), or pC53-SN3 (striped bars), as indicated. Experiments were assayed in triplicate, and data shown are from a representative experiment of three independent determinations. Error bars represent 1 SD. (E): Western blot analysis were performed on whole protein lysates from 293T cells (2 × 105) that were transfected with either Tax1 or Tax1(−) (4 μg). Proteins were extracted 48 hours post-transfection, and membranes were stained with a monoclonal antibody against survivin followed by a monoclonal antibody against β-actin. This experiment was performed twice. Abbreviation: CREB, cyclic AMP response element-binding protein. Discussion CD34+ HP/HSCs represent a heterogeneous cell population encompassing early and committed multipotent progenitor cells, uncommitted differentiating cells and stem cells [53, 65, 66]. Our data demonstrate that CD34+/CD38− HSCs are inherently more susceptible to Tax1-mediated modulation of p21 and survivin expression following HTLV-1 infection, in contrast to more mature multipotent progenitor CD34+/CD38+ cells. Both p21 and survivin are key regulatory molecules involved in the modulation of cell cycle progression of CD34+ HP/HSCs [13, 14, 38, 67, 68–69]. We speculate that HTLV-1 infection of HSCs induces cell cycle arrest and quiescence, allowing HTLV-1 latency to be established in a subset of the uncommitted progenitor stem cells. Tax1 has previously been demonstrated to induce p21 expression, although the cellular physiological response to elevated p21 appears to be cell type-specific [70, 71]. It has previously been shown that Tax1 upregulates the expression of p21 in HTLV-1-infected CD4+ T-cell lines, leading to the abrogation of key cell cycle checkpoints and resulting in cellular transformation [70, 71–72]. Tax1 can transcriptionally activate p21 expression, as well as post-transcriptionally stabilizing p21 levels through unscheduled activation of anaphase-promoting complex [73, 74]. The response of CD34+ HP/HSCs to Tax1 transduction is distinct and different in comparison with Tax1 transduction of mature CD4+ lymphocytes. We have previously demonstrated that Tax1-induced p21 RNA expression in CD34+ HP/HSCs results in G0/G1 cell cycle arrest and suppression of clonogenic colony formation [31, 33]. In the current study, knockdown of p21 expression in HTLV-1-infected CD34+ HPCs relieved cell cycle arrest, directly demonstrating that p21 induction contributes to cell cycle arrest by HTLV-1. Survivin plays an important role in cell cycle entry, maturation, and survival of CD34+ HP/HSCs and is necessary for normal lymphopoiesis. Inactivation of the endogenous survivin gene has been shown to adversely affect T lymphopoiesis [60, 75]. Notably, deletion of survivin has been shown to block transition and maturation of CD4−/CD8− (DN) thymocytes into CD4+/CD8+ (DP) cells in transgenic mice, and DN cells exhibited cell cycle arrest [75]. Downmodulation of survivin expression in HTLV-1-infected CD34+/CD38− HSCs may be a critical mechanism by which this human retrovirus deregulates hematopoiesis and initiates leukemogenesis. Modulation of survivin in CD34+ HP/HSCs may also help facilitate the proper environment for the establishment of HTLV-1 latency by allowing infected cells to return to a quiescent state. Survivin expression has been reported to be upregulated in ATL cells from patients and in HTLV-1-infected T-cell lines via activation of NF-κB [76, 77–78], which contrasts with Tax1-mediated suppression of survivin in HP/HSCs. It should be noted that ATL cells universally fail to express detectable levels of Tax1, suggesting that survivin induction in these leukemic cells occurs independently of HTLV-1 gene expression. Tax1-mediated transformation of mature CD4+ T lymphocytes also occurs by activation of NF-κB [79]. Notably, we did not detect cellular transformation following HTLV-1 infection or Tax1 transduction in CD34+ HPCs. Naïve CD34+/CD38− HSCs display very little NF-κB activity [80], in contrast to CD4+ T lymphocytes, and this may account for the distinct biological activities of Tax1 [81, 82]. The reported absence of NF-κB activity in naïve CD34+/CD38− HSCs [80] leads us to speculate that HP/HSCs may provide a unique microenvironment for HTLV-1 infection that stands in stark contrast to the cellular environment provided by CD4+ T lymphocytes. Tax1 has previously been demonstrated to repress the expression of a number of cellular genes, including β-polymerase, cyclin A, p53, and bax. Notably, Tax1-mediated repression of β-polymerase, p53, bax, and MyoD is attributed to the interactions with factors that bind E-box motifs [24, 25, 28, 30]. Another reported mechanism of transcriptional repression by Tax1 is by binding and sequestration of the cellular coactivator protein CBP/p300 [29, 83, 84–85]. Context-specific binding of Tax1 with CREB/ATF has been implicated in Tax1-mediated suppression of cyclin A and cyclin D3 expression [26]. The presence of multiple E-box motifs in the survivin promoter, as well as p300 binding sites (−1,545, −431, −46) and CREB binding sites (−2,234, −865), implies that Tax1 repression of survivin expression may occur through collective mechanisms involving these transcription regulatory motifs. p53 has previously been shown to repress survivin gene expression, and we detected similar effects on survivin transcriptional repression by Tax1 and p53 in 293T cells [30, 62]. Although we previously reported that survivin mRNA levels were unaltered following HTLV infection or Tax1 transduction of CD34+ HPCs [31], we speculate that our assay (reverse transcription-PCR) may not have been properly designed to detect a downregulation of survivin RNA or, alternatively, that the turnover rate of survivin mRNA in CD34+ cells is relatively slow. Analysis of survivin protein levels in CD34+ HPCs by flow cytometry more precisely assays the combined transcriptional and post-transcriptional effects of Tax1 on survivin protein levels. It will be important to further define the role of Tax1 in regulating expression from the survivin promoter, particularly since survivin participates in modulation of fundamental cellular processes such as hematopoiesis, apoptosis, and leukemogenesis. Conclusion ATL is etiologically linked with neonatal or perinatal transmission of HTLV-1 infection, and the disease develops decades after the initial infection. HTLV-1-mediated suppression of multilineage hematopoiesis and cell-specific cell cycle arrest in CD34+ HP/HSCs identifies a unique mechanism by which this human retrovirus may establish viral latency and avoid immune surveillance in humans, accounting for the relatively long persistence of HTLV-1 infection demonstrated in ATL patients. Although both HTLV-1 and HTLV-2 can establish latent infections in mature lymphocytes, only HTLV-1 demonstrates the ability to arrest hematopoietic stem cells in G0/G1. This is also in accordance with previous reports that the bone marrow, a site enriched with CD34+ HPCs, may be a target for HTLV-1 latency and an in vivo reservoir of infected cells [86, 87]. Moreover, we have recently detected proviral sequences in purified CD34+ HPCs from HTLV-1-infected patient PBLs, suggesting that HTLV-1 infection of CD34+ HPCs is a biologically relevant phenomenon (P. Banerjee and G. Feuer, unpublished data). In addition, since submission of this article HTLV-1 infection and Tax expression have been reported to arrest cells in G1 [88]. Interestingly, other viruses, such as CMV, have been shown to achieve a latent infection in CD34+/CD38− stem cells [3, 89]. This indicates that HTLV-1 may also target stem cells and achieve long-term latency in CD34+ HSCs. HTLV-1-mediated suppression of hematopoiesis by G0/G1 cell cycle arrest in CD34+ HPCs, and in particular in HSCs, can be attributed to the concurrent robust activation of p21 and repression of survivin by Tax1. This observation is consistent with our previously published data showing that Tax1 transduction of CD34+ HPCs using LVs was sufficient for cell cycle arrest and induction of suppression of multilineage hematopoiesis in vitro [31, 33]. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: P.B. and M.S.: participation in design and performance of the research, control and analysis of data, manuscript writing, final approval of manuscript; E.S.: performance of experiments, final approval of manuscript; G.F.: oversight of experimental design, manuscript writing, final approval of manuscript. P.B. and M.S. contributed equally to this study. Copyright © 2008 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Human T-Cell Lymphotropic Virus Type 1 Infection of CD34+ Hematopoietic Progenitor Cells Induces Cell Cycle Arrest by Modulation of p21cip1/waf1 and Survivin
JF - Stem Cells
DO - 10.1634/stemcells.2008-0353
DA - 2008-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/human-t-cell-lymphotropic-virus-type-1-infection-of-cd34-hematopoietic-1h05BpChcd
SP - 3047
EP - 3058
VL - 26
IS - 12
DP - DeepDyve
ER -