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Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region
Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of... | {
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Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with a typical three phased course (chronic, accelerated and blastic phase) reflecting the loss of differentiation and malignant progress which inevitably leads to death after the blastic phase (1,2). The hallmark genetic aberration of CML is a reci... | {
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"text": "bcr-abl fusion gene",
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Cell lines
K-562, Jurkat and U-937 were obtained from the ATCC (American Type Culture Collection, Rockville, USA) and EM-2, LAMA-84, CML-T1, BV-173, SD-1 and RPMI-8226 from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). All cell lines, except BV-173, SD-1 and RPMI-8226, w... | {
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... | [] |
Cell culture and stimulation
All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco/BRL Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco/BRL), 1% penicillin/streptomycin (Biochrom, Berlin, Germany). When indicated, cells were treated with 5-aza-2-deoxycytidine (AzadC) or... | {
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Sequencing of the IRF-4 promoter
For analysis of the IRF-4 promoter region for permanent aberrations such as insertions/deletions or mutation, we PCR-amplified two fragments from genomic DNA, which was extracted from depicted cell lines with a commercial kit (Qiagen, Hilde, Germany) as recommended. The primers were 1-f... | {
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Expression analysis
To analyze the IRF-4 transcriptional level, RNA was extracted from cells using the commercial RNAzol-kit (Paesel, Frankfurt, Germany). An aliquot of 1 mug total RNA was used for cDNA synthesis as described previously (27). RNA expression analysis for IRF-4 and the reference gene beta-actin was carri... | {
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"text": "(DNMT1",
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Methylation-specific restriction-PCR-assay
DNA was extracted with a commercial kit (Qiagen) as recommended. Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas,... | {
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Bisulfite treatment
DNA was extracted as described above. Bisulfite treatment of DNA, leading to conversion of unmethylated cytosine to uracil residues and no change of methylated cytosine residues, was performed as described as follows. Briefly, 1 microg of DNA and 2 microg of poly(dA-dT)(poly(dA-dT) copolymers (Amers... | {
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In vitro methylation and reporter gene assays
The IRF-4 promoter-reporter gene construct was generously provided by J. Hiscott (31). Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A).... | {
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Absence of IRF-4 expression in leukemia cells is not due to promoter alterations
We have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3). Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemi... | {
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Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression. A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A). Several chemical substa... | {
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Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells
To further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplifica... | {
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Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethyla... | {
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"text": "IRF-4",
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In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity
To provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation. A complete methylation of these con... | {
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mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation
Since abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation ... | {
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Many genetic lesions are known to influence gene expression of tumor suppressor genes. Whereas mutations and deletions or insertions have permanent effects, reversible mechanisms are gene methylation, or expression and activation of transcription factors, respectively. We studied a putative cause for absent IRF-4 expre... | {
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"text": "PU.1",
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Foxp3 Represses Retroviral Transcription by Targeting Both NF-kappaB and CREB Pathways
Forkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathway... | {
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Immunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1]. Suppression of pathogenic T cell responses is mediated by natu... | {
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Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation
To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead... | {
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The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-kappaB Activation in T Cells
To define the requirements of Foxp3 with respect to inhibition of NF-kappaB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and ... | {
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Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-kappaB
If Foxp3 functions as a repressor of NF-kappaB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-kappaB. To... | {
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The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3
Previous studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-kappaB activation as well as tumor necrosis factor-alpha-stimulated NF-kappaB activation [15]. Our next step was to determine whether Foxp3... | {
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Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load
Since Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients. We there... | {
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CREB Is a Target for Transcriptional Repression by Foxp3
Although Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D). These ... | {
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Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment
Stimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinas... | {
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In the present study, we show that Foxp3 functions as a potent repressor of NF-kappaB- and CREB-dependent transcriptional activation. Furthermore, the carboxyl-terminal FKH domain appears to be dispensable for mediating these effects, at least in T cell populations. This observation may become important in light of rec... | {
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Cell culture.
HEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California, United States). Jurkat T cells and primary human CD4+ T cells were cultured in RPMI-1640 medium (Invitrogen). Media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mug/ml streptomycin (Cam... | {
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Patients and cell preparation.
PBMCs were prepared by centrifugation over Ficoll-Hypaque gradients (BioWhittaker, Walkersville, Maryland, United States) from eight HAM/TSP patients and eight ACs, and the cells were viably cryopreserved in liquid nitrogen until tested. HAM/TSP was diagnosed according to WHO guidelines [... | {
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Plasmids.
Expression vectors encoding human Foxp3 (pCMV-Foxp3-IRES-EGFP) and human Foxp3 lacking the forkhead (FKH) domain (pCMV-deltaFKH-IRES-EGFP) were generous gifts from S. Ziegler (Benaroya Research Institute). pEGFP-C2 was provided by I. Lipinski (NIDDK/NIH). pcDNA3 was provided by K. T. Jeang (NIAID/NIH). pCMV4-... | {
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Isolation of primary human CD4+ T cells.
CD4+ T cells were isolated from cryopreserved healthy donor PBMCs by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's guidelines. Purity of negatively selected CD4+ T cells was consistently higher... | {
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Transient expression and luciferase assays.
HEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent (Roche, Basel, Switzer... | {
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Foxp3 and HTLV-I Tax expression analysis by real-time RT-PCR.
Real-time RT-PCR analysis of Foxp3 and HTLV-I Tax expression was performed as previously described [8,47]. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to manufacturer's guidelines, and cD... | {
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Real-time PCR.
Real-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described [47,50]. DNA was extracted from 1 x 106 cells using Puregene DNA Isolation Kit (Gentra, Minneapolis, Minnesota, United States), and 100 ng of the sample DNA solution was analyzed by this system. The HTLV-I proviral... | {
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Western blot analysis.
HEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 mug total) using FuGene 6 transfection reagent (Roche). Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA... | {
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Transcription factor DNA-binding analysis (TF-ELISA).
CREB-1 and ATF-2 DNA-binding activity was analyzed with the TransFactor Profiling (Inflammation 1) Kit (BD Biosciences) according to the manufacturer's protocol. Nuclear extracts were prepared from HEK 293T cells transfected with a control vector (EGFP) or Foxp3 exp... | {
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Flow cytometric analysis of Foxp3 protein expression.
Cryopreserved PBMCs from HAM/TSP patients or HTLV-I-infected ACs were thawed and washed with FACS buffer (1x PBS, 0.1% NaN3, 5% FBS). Cells (1.5 x 106) were fixed by sequential formaldehyde/methanol fixation as follows. Cells were carefully resuspended in FACS buffe... | {
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Statistical analyses.
The Mann-Whitney U test was used to compare the data between patients with HAM/TSP and AC. | {
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EBV Latent Membrane Protein 1 Activates Akt, NFkappaB, and Stat3 in B Cell Lymphomas
Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a p... | {
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Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1]. EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and ... | {
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High Levels of LMP1 Expression Correlates with the Development of Lymphoma
LMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer. It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detec... | {
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LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (kappa, lambda) were analyzed by flow cytometry. Simil... | {
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LMP1 Promotes B Cell Survival and Proliferation In Vitro
Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32]. To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a ... | {
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Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling
To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA). IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell li... | {
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LMP1 Upregulates IL10 and Constitutively Activates Stat3
To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNg... | {
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LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5]. Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40]. To determine if Akt signaling is activated... | {
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LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFkappaB, and Stat3 Pathways
To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFkappaB, Stat3... | {
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