bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.
Authors: Hong Jie , Stamatoyannopoulos George , Song Chao-Zhong 
Published: April 2020

Abstract
Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.
The Structure of the Gene for miR-223 in Humans, a MicroRNA Highly Expressed in Hematopoietic Tissues.
Authors: Silva Wilson A. , Kodama Kelson R. , Molfetta Greice A. , Zanette Dalila L. , Valente Valeria , Araujo Amelia G. , Rego Eduardo M. , Zago Marco A. 
Published: April 2020

Abstract
MicroRNAs (miRNA) are highly conserved noncoding regulatory RNA found abundantly in multicellular organisms. They derive from long primary transcripts (pri-miRNA) and act in a variety of regulatory pathways, including cell proliferation, apoptosis, fat metabolism and organogenesis. Over 200 human miRNA were identified by computational analysis and little is known about the gene hosts. Thus far, only 4 genes specific for microRNA have been characterized. Here we describe the structure of the gene for miR-223, a microRNA highly abundant in hematopoietic tissues. One cluster among 21,134 ESTs that we obtained from human bone marrow matched GenBank CD109334. Sequencing of cDNA from normal and leukemic cells did not correspond to known or potential protein structure, and revealed two alternative forms, with two or three “exons”. Phylogenetic analysis showed that our 234-nucleotide (nt) sequence matched DNA sequences of pri-miRNA of 6 non-human primates, with absolute identity in a 38-nt region that encompasses the 21-nt of the mature form. Secondary structure analysis of the transcribed RNA using M-Fold showed the stem-loop structure characteristic of a pri-miRNA. Northern blot with a probe designed to span the three exons of the genome sequence confirmed the expression of multiple bands in normal bone marrow, spleen, and acute and chronic leukemias, whereas other tissues were negative. Real time PCR with specific probes demonstrated that the expression of both alternate forms was restricted to the same cells. Northern blot revealed that mature miR-223 was observed only in hematopoietic cells. Thus, we describe for the first time the structure of a human non protein-coding gene specific for miRNA, spanning 4,450 nt of the genome in chromosome Xq12, with two transcript isoforms with 1,096 and 971 nt respectively, comprising three exons with 27, 125 and 944 nt, and show that the mature mRNA derives from the 3rd exon. Finally, there is an inverse relationship between the abundance of primary and mature species of miR-223 in normal and leukemic hematopoietic tissues, probably owing to a balance between transcription and processing of primary miRNA.
TNF Family Protein Regulation in Megakaryocytes and Platelets.
Authors: Crist Scott A. , Sprague Daniel L. , Lentz Steven R. , Ratliff Timothy L. 
Published: April 2020

Abstract
Activated platelets express and release several TNF family proteins with diverse immunological and inflammatory functions. While the biological effects of these potent immunological modifiers are an area of intense study, there are no studies to date that have investigated the mechanism of TNF protein regulation in megakaryocytes. Understanding the mechanisms of TNF protein regulation in megakaryocytes may allow for the design of new therapeutic strategies for the treatment of chronic inflammatory and autoimmune diseases. Our data show that CD40 Ligand, Fas Ligand and TNF-Related Apoptosis Inducing Ligand (TRAIL) expression are induced during megakaryocyte-differentiation of hematopoietic precursor cells. Promoter analysis and expression studies reveal multiple mechanisms of TNF family protein regulation. Megakaryocyte-specific regulation of TRAIL and Fas Ligand are primarily dependent on Sp1 and Sp3 transcription factor binding to proximal Sp1 binding elements within the respective promoter regions. Our preliminary data demonstrates differentiation-induced expression of CD40 Ligand on the other hand, is mediated via an Ets-1 site within 50 nucleotides of the transcription start site. In addition, endogenous CD40 Ligand but not TRAIL or Fas Ligand is constitutively expressed with ectopic expression of Fli-1, in the absence of differentiation-stimuli. Fli-1 is an Ets-1 family transcription factor that modulates several megakaryocyte-restricted genes. Further regulation studies reveal megakaryocyte expression of CD40 Ligand is diminished by the peroxisome proliferator-activated receptor (PPAR) agonist prostaglandin J2 and enhanced by 9-cis retinoic acid. Conversely, TRAIL and Fas Ligand expression are enhanced by prostaglandin J2 and diminished by retinoic acid. Taken from these data is there are differential mechanisms of differentiation-driven and induced regulation of the TNF family proteins. Continuing studies include the determination of the mechanisms of PPAR agonist, retinoic acid and differentiation-modulated TNF protein expression. Our studies demonstrate novel TRAIL and Fas Ligand regulatory pathways in the context of megakaryocyte differentiation and represent the first investigation into megakaryocyte-associated regulatory mechanisms for the platelet-derived CD40 Ligand, Fas Ligand, and TRAIL.
A Stromal Cell Derived Factor 1 (SDF-1) Agonist Stimulates Neutrophil Mobilization in Normal Humans after Subcutaneous Injection.
Authors: Wong Donald , Korz Walter , Merzouk Ahmed , Salari Hassan , William Bensinger I. 
Published: April 2020

Abstract
Stromal cell-derived factor-1 (SDF-1) is a well known key signaling molecule in the proliferation, homing, engraftment and expansion of hematopoietic stem cells and leukocytes. CTCE-0214, a peptide produced by rational drug design, is an analog of SDF-1 and agonist of the SDF-1 receptor, CXCR4. In vitro and in vivo models have shown that CTCE-0214 mobilized blood and progenitor cells and enhanced the survival and expansion of cord blood cells (Letters in Drug Design and Discovery.1:126, 2004; Exp Hematol.32:470, 2004; Exp Hematol.32:300, 2004; Stem Cells. In press). In this first phase I clinical trial of CTCE-0214, the safety and tolerability of single doses of CTCE-0214 given subcutaneously to healthy subjects as well as the pharmacokinetic profile and the pharmacodynamic effects were studied. The randomized, double-blinded, placebo-controlled dose-escalation trial enrolled 24 subjects in six dose-escalation groups. Four healthy human subjects in each cohort received either CTCE-0214 or Placebo, randomized in a 3:1 ratio. The starting dose for CTCE-0214 was 0.2 mg/kg. Successive dose cohorts received 0.5 mg/kg, 0.8 mg/kg, 1.5 mg/kg, 2.0 mg/kg, and 3.0 mg/kg. CTCE-0214 was shown to be safe with no serious adverse events reported in any of the dose levels studied. The most common drug related adverse events were injection site pain and erythema which were transient and resolved without intervention. The severity of injection site pain appeared to have an association with the overall quantity of study drug administered. Moderate or severe pain was reported only in subjects who received at least 80 mg of CTCE-0214 per syringe. Dilution across more than one syringe appeared to be effective in reducing injection site pain. Pharmacokinetic analysis of CTCE-0214 plasma concentrations showed that Tmax was reached at 0.25 hours post-administration for all CTCE-0214 treated cohorts. The apparent terminal elimination half-life (t1/2) values were estimated to be 0.41 to 0.32 hours. CTCE-0214 administration was associated with significant dose-dependent increases in total white blood cell and neutrophil counts in treated subjects, peaking at around 6 hours post-injection. In the 3.0 mg/kg arm, the mean difference in neutrophil count from baseline was more than three times the corresponding figure in the Placebo arm. The same trend was apparent in the 2.0 and 1.5 mg/kg arms with a greater than two times and two times increases, respectively. These results suggest that SDF-1 agonists may potentially be used in patients with low neutrophil count receiving chemotherapy with or without the use of G-CSF. The potential for CTCE-0214 to mobilize neutrophils and other blood cells merits serious consideration.
CD34+ Cells Mobilized with the New CXCR4 Antagonist AMD3100 + G-CSF Show Increased Anti-Apoptotic, Cell Cycle, DNA Repair and Cell Motility-Associated Gene Expression When Compared to G-CSF Mobilization Alone.
Authors: Seeger Timon , Maier Patrick , Li Li , Laufs Stephanie , Eckstein Volker , Bridger Gary , Calandra Gary , Wenz Frederik , Goldschmidt Hartmut , Ho Anthony D. , Fruehauf Stefan 
Published: April 2020

Abstract
AMD3100 is a new CXCR4 antagonist which induces a rapid release of hematopoietic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and Non-Hodgkin’s lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPC) when given an mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Since experimental data suggest that A+G-mobilized PBPC are functionally different from G-mobilized PBPC we were interested in an intraindividual comparison of the gene expression profile of CD34+ cells in the two different settings.
To this end peripheral blood CD34+ cells of 3 patients (3 G, 3 A+G samples) were isolated by immunomagnetic followed by flow cytometric sorting to a purity of &gt;99%.
Total RNA was purified. Differentially expressed genes were analysed by using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). The expression analysis was performed with the SAS-MAS 1.0.3 software (SAS Institute Inc.). We found a pattern of unanimously higher (81 genes, fold-change &gt; 2e0.5, p&lt;10e−4) or lower (29 genes, fold-change &gt; 2e−0.4, p&lt;10e−4) expressed genes in the G+A-mobilized vs. G-mobilized CD34+ PBPC. The significant changes noted in the microarray analysis were validated by representative quantitative real-time PCR of four genes in these three patients and in further four independent patients. Genes were grouped according to gene function. Only increased expression was found in the categories anti-apoptosis (e.g. MPO, HSPA1B), cell cycle (e.g. MS4A3, RRM2), replication/DNA repair (e.g. DNTT RRM2), cell motility (e.g. TNFSF4, HMMR) and oxygen transport. Only decreased expression occurred in the pro-apoptosis gene group (e.g. MDA5 BCL10). A mixed pattern of decreased and increased gene expression was found for the categories transcription, cytoskeleton, immune response, metabolism and signalling.
CXCR4 receptor gene expression itself was significantly increased 1.5-fold in the A+G vs. G group. Interestingly also regulating molecules upstream of CXCR4 such as Id1, MYC, CREBP1, CEBP were increased in their expression while molecules downstream of CXCR4, e.g. in the MAP kinase pathway were significantly decreased (EGR1, EGR3) or increased (DUSP1, DUSP6), suggesting activity of feedback loops. We conclude that A+G mobilizes CD34+ PBPC with increased gene expression in categories which potentially promote superior engraftment after myeloablative therapy than G-mobilized CD34+ PBPC.
Quantitative Analysis of Growth Factor Production in the Mechanism of Fibrosis in Agnogenic Myeloid Metaplasia.
Authors: Wang Jen-Chin , Chang Tsong S. , Goldberg Amit , Novetsky Allan D. , Lipton Jeffrey 
Published: April 2020

Abstract
There are previous reports that growth factors including TGF-β1, PDGF and FGF produced by megakaryocytes are responsible for the etiology of fibrosis in agnogenic myeloid metaplasia (AMM). Due to the technical difficulty in isolating enough megakaryocytes from the inaspirable bone marrow from patients with AMM, a quantitative analysis of these growth factors produced by the megakaryocytes and correlated to the degree of myelofibrosis has not been able to be performed. The present study employed the cell culture technique to grow megakaryocytes from blood CD 34+ cells and then perform the quantitative analysis of these growth factors, compared with other sources such as monocyte-macrophage lineages and correlated to the degree of bone marrow fibrosis. We found TGF-β1, PDGF and FGF produced by the megakaryocytes were significantly elevated in AMM compared with normal controls (p&lt;0.05) and TGF-β1 was more abundantly produced than PDGF or FGF. While these growth factors are several fold elevated in AMM compared with other MPD including essential thrombocythemia (ET), polycythemia vera (PV), it was not statistically significant. A quantitative analysis of these growth factors produced by the CD 14+ cells in the blood and bone marrow showed that these growth factors were not significantly elevated in AMM compared with other MPD or controls and were significantly elevated only in some patients (defined as elevation of more than 2 SD of the controls). The correlation of these growth factors produced by the megakaryocytes or monocyte-macrophage lineages with degree of myelofibrosis in 12 patients with AMM were r=0.73 and 0.23 respectively (Non parametric (Spearman) correlation with two-tailed analysis was used to calculate the correlation). We concluded:
In AMM, these fibrosing growth factors are mainly produced by the megakaryocytes and in some patients, monocyte-macrophage lineages may contribute to the production of these growth factor production. TGF-β1 is more abundantly produced from the megakaryocytes than PDGF or FGF confirming TGF-β1 is the most important fibrosing growth factor in the pathogenesis of myelofibrosis in AMM. A statistically significant correlation of the growth factor and degree of myelofibrosis in AMM suggests that these fibroing growth factors produced by the megakaryocytes are main etiology of bone marrow fibrosis in AMM.
Danazol Stimulates Proplatelet Formation of Megakaryocytes Via Rac Activation Pathway.
Authors: Murai Kazunori , Kowata Shugo , Ishiguro Mamiko , Ishida Yoji 
Published: April 2020

Abstract
Danazol (C 22 H 27NO2; 17a-2,4-pregnadien-20-yno[2,3-d]isoxazol-17-ol) have been widely used for patients with endometriosis. Recently, many reports showed that danazol was effective especially in thrombopoietic action in patients with refractory chronic idiopathic thrombocytopenic purpura, apalstic anemia or myelodysplastic syndrome-refractory anemia (MDS/RA). The thrombopoietic mechanism of danazol has been unknown. In this study, we investigated the thrombopoietic effects of danazol on megakaryocytes, especially proplatelet formation (PPF).
Murine megakaryocytes were isolated from bone marrow cells by Percoll®‚ density centrifugation and BSA gradient method. Isolated megakaryocytes (300/well) were cultured for 24 hrs in serum free condition with danazol and several kinase specific inhibitors including PPI (Src family kinase inhibitor), SU6695 (Src family kinase inhibitor), or LY294002 (PI3K inhibitor). PPF stimulation index (%) by danazol was calculated as (PPF with danazol)/(PPF without danazol). It was stimulated by danazol in a dose dependent manner(0μM: 100%, 0.2μM: 118±14.29%, 2μM: 136±10.3% (p&lt;0.05), 12μM:176.7±17.4%(p&lt;0.001), mean±SD, n=5).
The inhibitiory index(%) by kinase specific inhibitor was calculated as (PPF with danazole and each kinase specific inhibitor)/(PPF without them). Src family kinase inhibitor including PPI or SU6656 did not affect the PPF of murine megakaryocytes at 0, 0.2, 2.4, and 12 μM concentrations of danazol. While, LY294002 inhibited PPF of murine megakaryocytes at 0μM of danazol (LY294002; 0μM: 100%, 10μM: 75.33±4.5% (p&lt;0.01), 100μM: 61.3±9.4% (p&lt;0.01) (n=4)), as shown in Figure 1. PPF with danazole 0.2μM/ LY294002 100μM was 86% of that with danazol 0.2μM/ LY294002 0μM, that with danazol 2.4μM/ LY294002 100μM was 78%(p&lt;0.05) of PPF with danazol 2.4μM/ LY294002 0μM, and that with danazol 12μM/ LY294002 100μM was 60%(p&lt;0.01) of PPF with danazol 12μM/ LY294002 0μM (Figure 1). As a result, the similar inhibitory effects of LY294002 on PPF of murine megakaryocytes at 0, 0.2, 2.4 and 12.0μM of danazol were also observed.
These results strongly suggest that danazol is a powerful stimulating agent for PPF and that PI3K activation, followed by Rac activation might be the signal transduction pathway in PPF of megakaryocytes, based on that PI3K and Src family kinase stimulate Rac activation.
Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor) Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor)
The G Protein-Coupled Receptor cysLT1 Signals Via ERK/MAP-Kinase and Pyk2, but Not NF-kappa B, in CD34+ Progenitor Cells, Explaining Its Dual Effect on Both, Cell Migration and Proliferation.
Authors: Boehmler Andreas M. , Seitz Gabriele , Kanz Lothar , Möhle Robert 
Published: April 2020

Abstract
The G protein-coupled receptor (GPCR) cysLT1 is expressed in CD34+ cell lines and immature hematopoietic progenitor cells (HPC), while its ligand leukotriene D4 (LTD4) is produced by bone marrow endothelium and stromal cells when deprived of hematopoietic cells. In the present study, we analyzed the signal transduction pathways of cysLT1 in progenitor cell lines and primary CD34+ HPC isolated from the mobilized peripheral blood. Of note, the effects of LTD4 on intracellular calcium fluxes, a typical event related to signaling of Gi-coupled GPCR, were substantially stronger than those observed after stimulation of the HPC-associated chemokine receptor CXCR4 with optimal doses of the corresponding ligand stromal cell-derived factor-1 (SDF-1, CXCL12). In cytokine-supplemented liquid cultures in vitro (containing interleukin-3, stem cell factor, and FLT3-ligand), addition of LTD4 resulted in a significantly greater expansion of both, committed and immature CD34+ progenitors. The effect of LTD4 in these cultures was comparable to that induced by the early acting hematopoietic growth factor FLT3-ligand, whereas the CXCR4 ligand SDF-1 (CXCL12) was unable to expand the number of CD34+ HPC when added to similar cytokine-supplemented liquid cultures. In both, CD34+ cell lines and primary CD34+ progenitor cells, LTD4 induced a rapid phosphorylation of ERK/MAP-kinase, a signaling step related to cell proliferation, and Pyk2, which is connected to downstream signaling pathways resulting in cell migration. Signaling through NF-kappa B, representing an alternative pathway of GPCR signaling associated with cell proliferation, was unaffected. In conclusion, the GPCR cysLT1 activates multiple signal transduction pathways in CD34+ HPC leading to cell migration and proliferation. In contrast to the CXCR4 ligand SDF-1 (CXCL12), the effect of the cysLT1 ligand LTD4 on cell proliferation was more pronounced and even comparable to that induced by early acting hematopoietic growth factors.
Comparison of Human IL-11 and PTH: Effect on Blood and Bone Parameters in Adult Ovariectomized Mice.
Authors: Liu Ling , Zeng Qing Qiang , Zeng Wei , Cain Ricky , Ma Yan Fei 
Published: April 2020

Abstract
Interleukin-11 (IL-11) is a multi-functional cytokine with effects on hematopoiesis and bone remodeling. IL-11 is known to promote osteoclast formation by RANKL-dependent and independent processes. Recently, it was reported that IL-11 receptor signaling is required for normal bone turnover. To understand recombinant human IL-11 (rhuIL-11) effects on bone remodeling in vivo, we compared rhuIL-11 and PTH treatment in adult ovariectomized mice. IL -11 or PTH was administered s.c. daily for 6 weeks to 7 month-old mice that had been permitted to lose bone for 1 month post-ovariectomy. IL-11 at either 50 or 200 microg/kg/day increased platelets and neutrophils in the peripheral blood indicating that a therapeutic dose was achieved, but there was not a significant effect of rhuIL-11 on the plasma concentrations of osteocalcin as compared to vechicle control. In contrast, treatment with 30 micro/kg/day PTH significantly increased plasma osteocalcin. Interestingly, both IL-11 and PTH increased bone mineral density (BMD) of whole femur at the end of the 6-week study. Further studies are required to understand the effects of IL-11 on bone formation and resorption.
Enhanced Apoptosis in Response to Chemotherapeutic Agents in Leukemic Cells Is Associated with Post-Transcriptional Modification of Cyclin A1.
Authors: Ekberg Jenny , Brunhoff Cecilia , Landberg Göran , Perssson Jenny L. 
Published: April 2020

Abstract
A critical cell cycle regulatory protein, cyclin A1 has previously been demonstrated to initiate acute myeloid leukemia (AML) in transgenic mice. We have recently shown that the cellular level and distribution of cyclin A1 correlates with the status of the leukemic phenotype. The aim of our present study is to investigate the role of cyclin A1 in mediating the response of leukemic cells to drug-induced apoptosis, and to define the molecular pathways that cooperate with cyclin A1 to enhance drug-induced apoptosis. We have shown that apoptosis was enhanced in U-937 cells when treated with chemotherapeutic agents including staurosporine and TNF. Interestingly, the increased rate of apoptosis was associated with concomitant increase in cyclin A1 expression. Treatment with a general caspase inhibitor blocked the cleavage of caspase-3 but did not prevent the up-regulation of cyclin A1. We have found that cyclin A1 protein is normally undergoing ubiquitin-mediated degradation in myeloid cells. However, in cells undergoing apoptosis induced by TNF, cyclin A1 protein is prevented from degradation and ubiquitination, suggesting that post-transcriptional modification of cyclin A1 protein is involved. Two dimensional gel analyses further verified that cyclin A1 protein is modified following the increased rate of apoptosis induced by TNF. We have further established a link between cyclin A1 and CDC25C, which might contribute to the protein modification of cyclin A1 and thus contribute to the enhanced apoptosis in response to TNF in leukemic cells. To further define the functional role of cyclin A1 in drug-induced apoptosis, we are currently using myeloid cells from cyclin A1 deficient mice and wild-type controls.