bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome-Wide Transcriptional Regulatory Networks Downstream of TAL1/SCL in T-Cell Acute Lymphoblastic Leukemia.
Authors: Palomero Teresa , Odom Duncan T. , O’Neil Jennifer , Ferrando Adolfo A. , Winter Stuart S. , Larson Richard S. , Young Richard A. , Look A. Thomas 
Published: April 2020

Abstract
Aberrant expression of transcription factor oncogenes is a common feature in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. We used chromatin immunoprecipitation combined with promoter microarrays (ChIP on chip) to identify direct transcriptional targets of TAL1/SCL, a bHLH transcription factor aberrantly expressed in 60% of patients with T-ALL. Using the Hu12K arrays that contain over 12,000 human promoter regions, we have identified 71 direct targets of TAL1 in the Jurkat T-ALL cell line by ChIP on chip, and have been able to validate 80% of them by conventional chromatin immunoprecipitation followed by quantitative real time PCR. It has previously been demonstrated that TAL1 needs to bind to E proteins (E2A, HEB or E2-2) to achieve an efficient interaction with DNA. We verified that promoters occupied by TAL1 are bound by both the E-proteins E2A and HEB in at least 50% of TAL1-bound promoter regions, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in the transformation of T-cell precursors. Using RNA interference to knock down TAL1, we have demonstrated functional regulation through TAL1-binding of a significant percentage of the targets identified by ChIP on chip. Results demonstrate that some TAL1 direct targets are repressed by the binding of this transcription factor, while others seem to be activated or are not altered, indicating a high level of complexity in the organization of TAL1-mediated transcriptional regulatory networks. Also, specific association of the expression levels of TAL1 targets in TAL1 positive primary leukemia samples has been shown by comparing gene expression arrays from the different T-ALL subsets. Our results indicate that TAL1 acts as a master transcriptional regulator at the top of a complex regulatory network that contributes to leukemogenesis by regulating T-cell differentiation, proliferation and survival.
Gene Expression Profiles for Normal Human Bone Marrow-Derived CD34+ Stem Cells and CD34−/CD33+ Myeloid-Committed Progenitor Cells in Response to Daily Granulocyte-Colony-Stimulating Factor (G-CSF) Treatment.
Authors: Aprikyan Andrew A.G. , Pritchard David , Liles Conrad W. , Schwartz Steve , Dale David C. 
Published: April 2020

Abstract
G-CSF is widely used to accelerate marrow recovery after cancer chemotherapy, to facilitate collection of hematopoietic progenitor cells, and to treat severe chronic neutropenia. Although G-CSF was originally defined as a stimulus for myeloid cell proliferation, it has potent anti-apoptotic properties, affects synthesis of proteins stored in neutrophil granules, and has many other effects on cells of the myeloid lineage. To improve understanding of the molecular and cellular effects of G-CSF, particularly related to its use for the treatment of severe chronic neutropenia, we performed gene expression profile studies using Affymetrix oligonucleotide arrays and purified bone marrow cell sub-populations from normal volunteers treated with daily subcutaneous G-CSF (300 mcg/sc/qd) for five days. Under local anaesthesia, paired marrow aspirates were obtained from the posterior iliac crest before and after 5 daily doses of G-CSF. CD34+ and CD34−/CD33+ cells were purified using Miltenyi immunomagnetic beads. Two rounds of amplification of total RNA isolated from purified CD34+ or CD33+cells was used to obtain sufficient cRNA for hybridization. Expression data from scanned chips were first analyzed using the RMA algorithm. The limma package of the Bioconductor project was used to identify differentially expressed genes. Limma uses an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The statistical analysis of CD33+ cells revealed that 150 of more than 12,000 genes examined were up- or down-regulated &gt;2-fold in response to G-CSF treatment. The top 10 genes with up- or down-regulated level of expression include clusterin, neutrophil elastase, two transcription factors, gelsolin, Grb2, phospholipase D3, protein kinase C, the major vault protein, and serine-threonine kinase. In the myeloid-committed CD34-/CD33+ progenitor cells, genes with altered expression level represent those with gene products involved in the cell cycle, regulation of apoptosis, the cytoskeleton, the inflammatory response, or serine proteases and transcription factors. Most of the genes up-regulated in CD33+ cells (e.g. neutrophil elastase, phospholipase D, protein kinase C) were down-regulated in CD34-positive cells in response to G-CSF. The results of the comparative analyses revealed the normal signature gene expression profiles for CD34+ and CD34−/CD33+ cells and identified genes that may mediate specific G-CSF effects.
Natural Interferon Producing Cells Develop from Murine CD31+(high)/Ly6C- Marrow Progenitors.
Authors: Kreisel Friederike , Blasius Amanda , Colonna Marco , Cella Marina 
Published: April 2020

Abstract
Dendritic cells (DC) play a critical role in the initiation and regulation of immune responses. Based on immunophenotypic and functional charcateristics, murine CD11c+ DC can be classified into CD11c+, CD11b+, and B220− “conventional” DC and CD11c+, CD11b−/dim, B220+, and Gr-1+ interferon-producing cells (IPC) or plasmacytoid dendritic cells (pDC). The developmental origin of IPC remains controversial. In this study, we investigated the capacity of different murine bone marrow subpopulations to acquire immunophenotypic, morphologic and functional characteristics of IPC after in vitro culture with Flt-3L. Total bone marrow (BM) from 129/SvJ mice was sorted into six distinct BM subsets based on surface expression of the myeloid markers CD31 and Ly6C and subsequently cultured with Flt-3L for 8 days. Purified CD31high/Ly6C− BM cells were the only subset that consistently developed characteristics closely resembling that of IPC. Specifically, these cells expressed high levels of CD11c, low levels of CD11b, the lymphoid marker B220, and the IPC-specific marker 440C. Furthermore, these cells displayed the typical plasmacytoid morphology with eccentrically located nucleus showing mature lymphoid chromatin and lack of nucleoli. Functionally, these cells showed a high proliferation rate as demonstrated by the [3H] thymidine release assay and secreted detectable levels of IFN-alpha when stimulated with CPG ODN 2216. In contrast, all other sorted subsets failed to proliferate in the presence of Flt-3L. Similar results were obtained using BM from B6-RAG−/− mice. These findings indicate that, although both the CD31high/Ly6C− and CD31int/Ly6C− BM subsets contain early progenitor cells corresponding to “cobblestone area forming cells”, only the CD31high/Ly6C− is able to give rise to IPC when cultured with Flt-3L. Interestingly, CD31high/Ly6C− BM cells have been previously shown to generate also conventional DC if cultured with GM-CSF. Thus, IPC and DC may derive from common or closely related progenitors. Moreover, our results demonstrate that IPC development, in contrast to that of lymphocytes, does not require RAG proteins.
More Evidence That the Phenomenon of “Plasticity” of Hematopoietic Stem Cells (HSC) and Potential Involvement of BM-Derived Cells in Organ Regeneration Could Be Explained by the Presence of a Heterogenous Population of Tissue Committed Stem Cells (TCSC) Residing/Hiding out in Bone Marrow.
Authors: Kucia Magda , Wysoczynski Marcin , Reca Ryan , Majka Marcin , Ratajczak Janina , Ratajczak Mariusz Z. 
Published: April 2020

Abstract
Recently we identified a mobile, SDF-1 responsive population of CXCR4+ bone marrow mononuclear cells that express mRNA for various markers of early tissue committed stem cells (TCSC) in bone marrow (BM) by employing chemotactic isolation to an SDF-1 gradient combined with real time RT-PCR and immunohistochemical analysis (Leukemia2004:18;29-40). In the current work we report new data showing that murine and human TCSC (i) are highly mobile and respond robust to HGF and LIF gradients, in addition to an SDF-1 gradient, (ii) are released (mobilized) from BM into peripheral blood during organ injury (e.g., stroke, heart infarct, partial body irradiation), (iii) are highly enriched in mRNA for pluripotent stem cell transcription factors such as Oct-4, Rex-1 and Nanog, iv) reside in a population of BM-derived Sca-1+ (mice) and CD34+AC133+ (human) non-hematopoietic CD45 negative cells, v) are small (~7 mm in diameter), vi) contain supercoiled DNA, vii) rapidly adhere to/undergo emperipolesis in fibroblasts and thus are enriched in a fraction of BM-derived adherent cells, viii) their number is highest in BM from young (1–2 month-old) mice and decreases in 1-year-old animals, and similarly ix) these cells are significantly diminished in short living DBA/2J mice as compared to long living B6 mice. Thus, these findings further support our theory of the BM as a “hideout” of TCSC and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as trans-dedifferentiation/plasticity of HSC. Since we noticed that not only SDF-1 but also HGF and LIF are upregulated in damaged tissues (e.g., brain, heart or liver), we postulate that TCSC that express CXCR4, c-Met, and LIF-R after being mobilized from the BM into PB, may be subsequently chemoattracted to damaged organs where they play a role in tissue repair/regeneration. Moreover, our observation that the number of TCSC is the highest in BM of young animals and decreases with age and is significantly lower in short-living murine strains provides a novel insight into aging and may explain why the regeneration process becomes less effective in older individuals. Based on our observation, we conclude that BM-derived HSC are not plastic but BM in addition to HSC also contains heterogenous populations of TCSC.
Cellular Aggregates Induced from Monolayer Cultures of Human Pancreatic Cells Correct Hyperglycemia in Diabetic Mice and Demonstrate Physiological Production of Human C-Peptide.
Authors: Wu Fred D. , Jagir Michael R. , Luu Thomas T. , Powell Jerry S. 
Published: April 2020

Abstract
Diabetes is due to loss of appropriate insulin production by pancreatic islet cells, resulting in hyperglycemia and significant morbidity. We have developed cell cultures of pancreas-derived precursor cells that can be maintained for more than 20 population doublings in culture, growing as monolayers of undifferentiated cells. These monolayer cells can then be induced to differentiate into cellular aggregates that resemble islets when grown on different extracellular matrix components, including matrigel, or poly-d-lysine. The induced cellular aggregates, but not monolayer cells, correct hyperglycemia in diabetic (streptozotocin) SCID (severe combined immunodeficiency) mice after implantation under the kidney capsule. This animal model can be used to follow the process of differentiation from undifferentiated precursor cell to fully functional insulin producing beta cells, and to identify key proteins and genes involved in differentiation of pancreatic islet cells. Here we report that in the mice, after implantation of cellular aggregates, human c-peptide concentrations are controlled physiologically appropriately in response to glucose tolerance testing (challenged with 2 grams of glucose injected intraperitoneally) in the treated diabetic SCID mice (n = 6) as well as in normal mice that received implanted cellular aggregates (n = 6). Human c peptide is not detected in any of the control animals, nor in animals implanted with monolayer cells. Comparative RNA microarray data analyses, using U133 arrays (Affymetrix), were compared between the monolayer cells in culture and the cellular aggregates 48 hours after induction with poly-d-lysine. We consider of particular interest the up-regulation of BMP, CXCR4, and HGF in the cell aggregates, and the down-regulation of VCAM-1. We believe that these genes are necessary for either aggregation or for physiologically functional insulin secretion. When examined for greater than two-fold gene expression differences between monolayer and aggregated cells, 424 gene sequences were upregulated and 690 genes down regulated. In addition, RAGE display analysis for tyrosine kinases also showed that the kinases Erb-B2 and PDGFR-B were upregulated at 48 hours in the aggregating process. In contrast, DDR2, a collagen I receptor tyrosine kinase, was expressed equally in both monolayer and aggregated cells. In summary, the results suggest: 1) that a population of pancreatic stem cells can be isolated and cultured in vitro, 2) that these cells can be induced to form functional islet cells that correct hyperglycemia in diabetic mice, 3) that human c-peptide is physiologically regulated in aggregate implanted mice in response to glucose challenge, and 4) HGF, along with other genes of potential interest, is upregulated in the process of differentiation from monolayer to aggregated cell phenotype.
Ubiquitin-Proteasome-Dependent Degradation of GATA-2 and Its Contribution to the UV-C-Induced Down-Regulation.
Authors: Minegishi Naoko , Suzuki Norio , Kawatani Yukie , Yamamoto Masayuki 
Published: April 2020

Abstract
Transcription factor GATA-2 is crucial for the development of hematopoietic stem cells. GATA-2 regulates proliferation and survival of hematopoietic stem/progenitor cells. We analyzed the degradation process of GATA-2, assuming that GATA-2 may be under the strict controls of protein degradation like many factors regulating cellular proliferations and apoptosis. The half-life of endogenous GATA-2 in hematopoietic and neuroblastoma cells was short (within 30 min in P815 cells) after cycloheximide treatment. The UV-C irradiation also induced the reduction of endogenous GATA-2 expression. Treatment with a proteasome inhibitor MG132 abrogated the effects of cycloheximide and UV-C irradiation, and increased the expression levels of both endogenous and transfected GATA-2 protein. Experiments with GATA-2 deletion mutants and their GBD-fusion proteins revealed that degradation elements of GATA-2 are within the transactivation domains and within a region containing putative protein modification motifs. We detected poly-ubiquitinated forms of GATA-2 after MG132 treatment, implying that the rapid degradation of GATA-2 is ubiquitin-dependent. Together, these results suggest that the ubiquitination/proteasome-dependent degradation pathway regulates the GATA-2 protein expression and this process may be important for the GATA-2 function.
An Establishment of a System for Conditional Overexpression of Genes in Megakaryocytes and Platelets In Vivo.
Authors: Nguyen Hao G. , Yu Guangao , Makitalo Maria , Jones Matthew , Ravid Katya 
Published: April 2020

Abstract
Expression of foreign or endogenous genes in vivo is an essential tool for following regulatory processes, including the control of gene expression. Constitutive expression of such genes throughout development often results with lethality due to adverse effects during embryogenesis. Several such examples exist with regard to the megakaryocyte (MK)/platelet lineage. Here, we employed the tetracycline (Tet)-off system to conditionally overexpress genes in MKs and platelets in vivo. We generated three transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the megakaryocyte-specific, platelet factor 4 promoter (PF4-Tet). Three responder lines were simultaneously generated, each with a bidirectional minimal CMV-tTA responsive promoter upstream of prokaryotic ß-Galactosidase gene (ß-Gal) (as a marker), with an insertion site in the opposite side for a gene of interest. A transactivator founder line that strongly expressed tTA was cross-bred to the three other responder lines. The resulting double transgenic mouse lines exhibited tetracycline-dependent transgene overexpression in the megakaryocytic lineage. Lineage specificity was confirmed by immunohistochemistry and western blot analyses. By changing tetracycline concentration in the drinking water, we could turn on/off the expression of the gene at any desirable time during the mouse lifespan, and showed that in adult mice under inducing conditions, ß-Gal expression was restricted to MKs and platelets. Hence, we generated a mouse line, PF4-Tet that is available to conditionally overexpress any gene of interest in the MK/platelet lineage in vivo. This system should be valuable for the study of effects of selected proteins on an array of genes expressed in vivo and on consequent megakarypoiesis and/or thrombosis and hemostasis.
Identification of Genes Affected during Myeloid Differentiation Induced by Valproic Acid and Retinoic Acid: The Choice of Array Pre-Processing and Normalization Algorithm Has a Critical Effect on Results.
Authors: Berkofsky-Fessler Windy D. , McConnell Melanie-Jane , Neuberg Donna S. , Licht Jonathan D. 
Published: April 2020

Abstract
Valproic acid is an anti-epileptic drug with histone deacetylase inhibitor activity currently in clinical trial for the treatment of leukemia. In order to understand the effects of this agent in myeloid differentiation and leukemia therapy we determined the changes in gene expression that occurred when the myeloid cell line ML-1 was treated with valproic acid (VPA), all-trans retinoic acid (ATRA) or the combination for 24 or 96 hours. Each condition was compared to no drug treatment at each time point, as full biological triplicates. RNA samples were labeled and hybridized to Affymetrix HGU133A genechips. The data was processed using the Affymetrix MAS5.0 calculated intensity signals or was pre-processed using three different algorithms to normalize data: GC-RMA (GeneChip-Robust Microarray Analysis), RMA (Robust Microarray Analysis) or MBEI (Model-Based Expression Index), using the original Affymetrix CEL files. From the resulting normalized data sets, we then identified genes that were up or down regulated at least two-fold by the combination VPA + ATRA treatment when compared to the no drug condition or either drug alone. If any of these genes were affected by either ATRA or VPA, we selected only those with an additional change of 50% or more with the combination therapy. There were striking differences in the gene sets obtained from each normalization algorithm with respect to both list size and content. GC-RMA identified 100 genes significantly differentially expressed according to the above criteria at the 24 hour time point, MBEI found 19 and MAS5.0 found 39. Using the RMA algorithm, 33 genes were determined to be significantly affected by the combination treatment and all of these were also found with GC-RMA suggesting that GC-RMA has a greater sensitivity than RMA. This may be because GC-RMA, a refinement of RMA, takes into account mismatched probe set signals and probe set sequence content. Only 12 genes were common to all lists, including endothelin receptor B, myosin binding protein H, IL8, and G0S2. We are currently using real-time PCR to validate the extent of differential regulation of the commonly identified genes in reponse to VPA+ATRA. This will be contrasted with the levels of regulation of gene sets found by only one or two algorithms in order to determine the sensitivity and specificity of these methods. These data indicate the potential power of new bioinformatic tools for microarray analysis and the need for systematic validation to ensure biological relevance.
Interferon-gamma Has Dual Potential in Inhibiting or Promoting Survival and Growth of Haematopoietic Progenitors: Interactions with Stromal Cell-Derived Factor-1.
Authors: Jo Deog-Yeon , Hwang Jin-Hee , Kim Seong-Woo , Yun Hwan-Jung , Kim Samyong 
Published: April 2020

Abstract
Despite many apparently contradictory reports, IFN-γ is well known as a hematopoiesis-inhibitory cytokine and is implicated in the pathogenesis of aplastic anemia. It has been shown that IFN-γ intrinsically and simultaneously induces specific and conflicting signalling pathways and transcriptional programs that contribute to the potential dual effects of IFN-γ in promoting or inhibiting proliferation of murine pro-B cells (Asao et al, J Biol Chem275: 867, 2000). We explored the dual roles of IFN-γ in the inhibition or promotion of the survival and growth of hematopoietic progenitors, especially with regard to interactions with stromal cell-derived factor-1 (SDF-1). IFN-γ partially rescued normal bone marrow CD34+ cells and colony-forming cells from apoptosis induced by serum- and growth factor-deprivation. SDF-1 further enhanced cell survival, in synergy with IFN-γ. Short-term IFN-γ treatment of CD34+ cells enhanced the clonal growth of the cells in synergy with SDF-1. In contrast, IFN-γ inhibited the clonal growth of hematopoietic progenitor cells in a standard methylcellulose clonogenic assay and inhibited the growth factor-mediated survival of normal CD34+ cells, CD34+ cells from patients with chronic myeloid leukaemia, and MO7e cells. The addition of SDF-1 did not alter these outcomes. IFN-γ did not activate PI3K/Akt, enhance SDF-1-induced activation of PI3K/Akt, or up-regulate the expression of CXCR4 or its chemotactic function in bone marrow CD34+ cells. IFN-γ up-regulated the expression of Socs1 mRNA in CD34+ cells and MO7e cells. Referring to a previous report describing that Socs1 binds to multiple signalling proteins and suppresses stem cell factor-dependent proliferation of bone marrow-derived mast cells (De Sepulveda et al, EMBO J 18: 904,1999), it is suggested that IFN-γ-induced inhibition of the growth factor-dependent survival of CD34+ cells might be mediated, at least in part, through the induction of Socs1. Paradoxically, IFN-γ down-regulated SDF-1 production in primary human bone marrow stromal cells. These results indicate that IFN-γ, partly in concert with SDF-1, exerts dual effects on the survival and growth of hematopoietic progenitor cells; the effects of IFN-γ on hematopoietic progenitor cells can differ depending on the particular in vitro experimental conditions, especially the presence of hematopoietic growth factors.