Rbm15 Affects Notch Signaling and Myelopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2545-2545
Author(s):  
Matthew J. Renda ◽  
Ee-Chun Cheng ◽  
Lin Wang ◽  
Xian-Young Ma ◽  
Diane S. Krause
Keyword(s):  
Fusion Protein ◽  
Notch Signaling ◽  
Cell Lines ◽  
Promoter Activity ◽  
Hematopoietic Cell ◽  
Chromosome 1 ◽  
Dominant Negative Effect ◽  
Myeloid Differentiation ◽  
Hematopoietic Cell Lines

Abstract Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation. Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.

scholarly journals Rbm15 Modulates Notch-Induced Transcriptional Activation and Affects Myeloid Differentiation

2007 ◽  
Vol 27 (8) ◽  
pp. 3056-3064 ◽  
Author(s):  
Xianyong Ma ◽  
Matthew J. Renda ◽  
Lin Wang ◽  
Ee-chun Cheng ◽  
Chao Niu ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Myeloid Differentiation ◽  
Fusion Partner ◽  
Hematopoietic Stem ◽  
N Terminus

ABSTRACT RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJκ, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJκ.

scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

Abstract The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

AML1/RUNX1, One of the Most Common Targets of Aberration in Acute Myeloid Leukemia as a Transcriptional Regulator of Vascular Endothelial Growth Factor (VEGFA).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1618-1618
Author(s):  
Arja ter Elst ◽  
Bin Ma ◽  
Frank Scherpen ◽  
Bart-Jan A.T. Wierenga ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Fusion Protein ◽  
Mrna Expression ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Computer Assisted ◽  
Negative Effect

Abstract Cellular VEGFA is an independent adverse prognostic factor related to worse outcome in AML and is significantly upregulated in leukemic blasts (de bont ES. et al., 2002). Transcriptional activation of the VEGFA promoter represents the core mechanism through which expression of VEGFA can be regulated, but is still not identified in AML (Carmeliet P. and Jaine RK., 2000; Kolch W. et al., 1995; Yancoupolus GD. et al., 2000). One of the most common targets of aberrations in acute leukemia is Runt domain transcription factor (AML1/RUNX1), due to point mutations (Roumier C. et al., 2003) or disruption of chromosome 21, which occurs in about 10%-15% of all de novo AML patients. In the current study, functional 5′ deletion analysis of the VEGFA promoter was performed to identify VEGFA promoter regions involved in VEGFA transcriptional regulation in AML. Loss of the region spanning −2274/−507 resulted in a significant increase of promoter activity in two AML cell lines, whereas the promoter activity dropped after deletion of the promoter region −286 to −52. Computer-assisted sequence analysis of the −2247/−507 fragment of the VEGFA promoter, revealed three perfect AML1/RUNX1-binding sites in the first three deletion regions. The loss of each AML1/RUNX1-binding site corresponded to an (further) increase in VEGFA promoter activity. siRNA mediated AML1/RUNX1 depletion caused a 24% to 36% increase in VEGFA mRNA expression in two different AML cell lines (HL-60 and TF-1), whereas VEGFA promoter activity was two-fold increased in HL-60 cells treated with AML1/RUNX1 siRNA compared to the non-silencing control siRNA. In addition, mutation of all three AML1/RUNX1 sites resulted in a 22 fold increase of VEGFA promoter activity in HL-60 cells. VEGFA mRNA expression was found to be higher in AML patients with a t(8;21) translocation compared to patients without this translocation. To investigate whether blocking of the fusion protein AML1-ETO, generated by the t(8;21) translocation, would have an effect on VEGFA mRNA we have used a siRNA against AML-ETO in Kasumi-1. siRNA mediated depletion of AML1-ETO caused a 40% decrease in VEGFA mRNA expression. AML-ETO has the potential to interact with AML1/RUNX1 co-factors such as histone deacetylase (HDAC) which results in a dominant negative effect on AML1/RUNX1 transcriptional regulation. To test whether AML1/RUNX1 transcription repression of VEGFA is histone deacetylase (HDAC) dependent HL-60 cells were treated with 200 nM Trichostatin A (TSA), a potent inhibitor of HDAC, this resulted in an 75% increase of luciferase activity after 24h. In contrast, TSA treatment of Kasumi-1 cells containing the AML-ETO fusion protein had no effect on VEGFA expression. These results indicate that VEGFA is transcriptionally regulated by AML1/RUNX1, one of the most common targets of aberrations in AML, through three AML1/RUNX1 repressor elements located in the promoter of VEGFA. Furthermore, prognostically unfavorable high VEGFA expression in AML is caused by a dominant negative effect of the fusion protein AML-ETO on AML1/RUNX1 mediated repression of VEGFA transcription. These results underscore the importance of AML1/RUNX1 aberrations in AML development and progression and better understanding the molecular basis for aberrant AML/RUNX1 signaling pathway may help design more effective treatment strategies.

scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

Regulation and Functional Role of Beta2-Adrenergic Receptor in Acute Myelogenous Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2563-2563
Author(s):  
Ningfei An ◽  
Yeong-Bin Im ◽  
Amr Abdallah Moh'D Qudeimat ◽  
Luciano J Costa ◽  
Robert K Stuart ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Leukemia Cell ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
Pim Kinases ◽  
Leukemia Cell Lines ◽  
Acute Myelogenous ◽  
Hematopoietic Cell Lines

Abstract Abstract 2563 Acute myelogenous leukemia (AML) occurs with an incidence of 2.7 per 100,000 population in the year of 2009, and is associated with significant mortality and morbidity. Despite recent advances in molecular and cytogenetic analytic technologies, initial treatment for AML patients has remained essentially the same over the last 30 years; and the treatment outcome is dreadful with a 5-year survival rate of ∼25%. In an effort to gain a better understanding of AML cell biology and to develop more effective treatments for AML, we have been exploring the roles of the b2-adrenergic receptor (B2-AR) in AML. B2-AR is a G-protein- coupled catecholamine receptor and was recently found to play a direct role in the engraftment, proliferation and migration of hematopoietic stem cells [Spiegel, A., et al. Nat Immunol8, 1123–1131 (2007)]. However, very little is known about the roles of B2-AR in AML. We first screened a number of leukemia/hematopoietic cell lines, including K562, NBAL3, Jurkat, RPMI8226, U937, HEL, HL60, NB4, THP-1, and MV4;11, for B2-AR expression using flow cytometry. We found that B2-AR expression was not uniform in the leukemia/hematopoietic cell lines we tested. B2-AR was absent in acute non-myelogenous leukemia/hematopoietic cell lines such as K562, NBAL3, Jurkat or RPMI8226. In contrast, B2-AR was expressed on all acute myelogenous leukemia cell lines tested, with the highest expression on 2 myelomonocytic leukemia cell lines (THP-1 and MV4;11). We next examined the roles of B2-AR in leukemia cell survival, in leukemia cell responses to chemotherapy, and in leukemia cell migration in response to stromal derived factor-1 (SDF-1). We found that treatment with a B2-AR antagonist (ICI115881) modestly inhibited leukemia cell growth. Interestingly, treatment with a B2-AR agonist (i.e., isoproteronol or clenbuterol) inhibited leukemia cell migration to SDF-1. Additionally, combined treatment of MV4-11 cells with Isoproterenol and SDF-1 increased downstream ERK phosphorylation synergistically, suggesting a potential interaction or reciprocal regulation between B2-AR and CXCR4 receptor. To further understand the regulation and functional role of B2-AR in AML cells, we have been focusing on two biphenotypic leukemia cell lines, that is, MV4;11 and RS4;11 cells. Both MV4;11 and RS4;11 cells carry the t(4;11)(q21;q23) chromosomal translocation. While RS4;11 cells bear only the wild-type version of the Flt3 gene, MV4;11 cells express exclusively the mutated Flt3-ITD gene. Interestingly, we found that in contrast to MV4;11 leukemia cells, RS4;11 cells did not express surface B2-AR (panel A), although the total amount of B2-AR in the whole cell lysate was comparable between these two cell lines (panel B). This significantly different B2-AR expression pattern between these 2 cell lines may be related to the difference in the expression level of proviral insertion in murine lymphoma (Pim) kinases (panel B); RS4;11 has reduced/absent expression of Pim kinases compared to MV4;11 cells. Consistent with the potential regulation of B2-AR expression by Pim kinases, we found that Pim 2 and 3 double knockout mice had reduced B2-AR surface expression in peripheral blood mononucleated cells. Additional experiments are currently ongoing to further dissect the interaction between B2-AR and Pim kinases. Taken together, our current studies demonstrated a novel role of B2-AR in AML and a potential functional interaction between B2-AR, CXCR4, Pim kinase, and Flt3 gene. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

Malignant hematopoietic cell lines: In vitro models for the study of primary mediastinal B-cell lymphomas

2015 ◽  
Vol 39 (1) ◽  
pp. 18-29 ◽  
Author(s):  
Hans G. Drexler ◽  
Stefan Ehrentraut ◽  
Stefan Nagel ◽  
Sonja Eberth ◽  
Roderick A.F. MacLeod
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
In Vitro Models ◽  
Hematopoietic Cell ◽  
B Cell Lymphomas ◽  
Hematopoietic Cell Lines

scholarly journals Collagen type VI in the human bone marrow microenvironment: a strong cytoadhesive component

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1740-1748 ◽  
Author(s):  
G Klein ◽  
CA Muller ◽  
E Tillet ◽  
ML Chu ◽  
R Timpl
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Triple Helix ◽  
Collagen Type ◽  
Hematopoietic Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Collagen Vi ◽  
Collagen Type Vi ◽  
Hematopoietic Cell Lines

Collagen type VI, which forms characteristic microfibrillar structures, is assembled from three individual alpha(VI) chains that form a short triple helix and two adjacent globular domains. Expression of all three alpha (VI) collagen chains in the human bone marrow (BM) microenvironment could be detected by chain-specific antibodies in tissue sections and in the adherent stromal layer of long-term BM cultures. In functional studies, collagen type VI was shown to be a strong adhesive substrate for various hematopoietic cell lines and light-density BM mononuclear cells. The adhesive site within the molecule seems to be restricted to the triple helical domain of all three alpha (VI) chains, because individual alpha (VI) chains were not active in the attachment assays. Adhesion of the hematopoietic cell lines to collagen VI was dose-dependent and could be inhibited by heparin. Although the triple helix contains several RGD sequences, adhesion of the hematopoietic cell types to collagen VI could be blocked neither by RGD-containing peptides nor by a neutralizing antibody to the beta 1 integrin subunit. In combination with an antiadhesive substrate, the binding properties of collagen VI could be downregulated. These data suggest that this collagen type may play an important role in the adhesion of hematopoietic cells within the BM microenvironment.

scholarly journals Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43696 ◽  
Author(s):  
Colin Correnti ◽  
Vera Richardson ◽  
Allyson K. Sia ◽  
Ashok D. Bandaranayake ◽  
Mario Ruiz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hematopoietic Cell ◽  
Gentisic Acid ◽  
Hematopoietic Cell Lines
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