Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3750-3757 ◽  
Author(s):  
Sandrine Baghdoyan ◽  
Patrice Dubreuil ◽  
Frédéric Eberlé ◽  
Sophie Gomez
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Integration Site ◽  
Gene Trap ◽  
Transcriptional Analysis ◽  
Monocytic Differentiation ◽  
Interleukin 5 ◽  
Gm Csf ◽  
Proviral Integration Site ◽  
G418 Resistance

We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosaβgeo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5′ rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF–treated and –infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACAwas recently defined as a coactivator of c-jun–mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34+ cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis.

Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3750-3757 ◽  
Author(s):  
Sandrine Baghdoyan ◽  
Patrice Dubreuil ◽  
Frédéric Eberlé ◽  
Sophie Gomez
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Integration Site ◽  
Gene Trap ◽  
Transcriptional Analysis ◽  
Monocytic Differentiation ◽  
Interleukin 5 ◽  
Gm Csf ◽  
Proviral Integration Site ◽  
G418 Resistance

Abstract We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosaβgeo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5′ rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF–treated and –infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACAwas recently defined as a coactivator of c-jun–mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34+ cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis.

scholarly journals The importance of the granulocyte-colony stimulating factor in oncology

2015 ◽  
Vol 88 (4) ◽  
pp. 468-472 ◽  
Author(s):  
Sînziana Cetean ◽  
Călin Căinap ◽  
Anne-Marie Constantin ◽  
Simona Căinap ◽  
Alexandra Gherman ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Prophylaxis ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Monocytic Differentiation ◽  
Hematopoietic Stem ◽  
Interleukin 5 ◽  
Gm Csf ◽  
And Migration ◽  
Stimulating Factor

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein, the second CSF, sharing some common effects with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5). G-CSF is mainly produced by fibroblasts and endothelial cells from bone marrow stroma and by immunocompetent cells (monocytes, macrophages). The receptor for G-CSF (G-CSFR) is part of the cytokine and hematopoietin receptor superfamily and G-CSFR mutations cause severe congenital neutropenia.The main action of G-CSF - G-CSFR linkage is stimulation of the production, mobilization, survival and chemotaxis of neutrophils, but there are many other G-CSF effects: growth and migration of endothelial cells, decrease of norepinephrine reuptake, increase in osteoclastic activity and decrease in osteoblast activity.In oncology, G-CSF is utilized especially for the primary prophylaxis of chemotherapy-induced neutropenia, but it can be used for hematopoietic stem cell transplantation, it can produce monocytic differentiation of some myeloid leukemias and it can increase some drug resistance.The therapeutic indications of G-CSF are becoming more and more numerous: non neutropenic patients infections, reproductive medicine, neurological disturbances, regeneration therapy after acute myocardial infarction and of skeletal muscle, and hepatitis C therapy.

Ectopic interleukin-5 receptor expression promotes proliferation without development in a multipotent hematopoietic cell line

1998 ◽  
Vol 111 (6) ◽  
pp. 815-823
Author(s):  
A. Pierce ◽  
A.D. Whetton ◽  
P.J. Owen-Lynch ◽  
J. Tavernier ◽  
E. Spooncer ◽  
...  
Keyword(s):  
Cell Line ◽  
Ectopic Expression ◽  
Hematopoietic Cell ◽  
Receptor Expression ◽  
Alpha Subunit ◽  
Specific Factor ◽  
Interleukin 5 ◽  
Receptor Alpha ◽  
Gm Csf ◽  
Lineage Specificity

The interleukin-5 (IL-5) receptor is a heterodimer that consists of an IL-5 specific alpha subunit and a common ssc chain that is shared with the receptors for granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). In contrast to IL-5, which acts mainly as an eosinophil lineage specific factor in vivo, IL-3 and GM-CSF stimulate the survival, proliferation and development of various hematopoietic cell lineages and also multipotent progenitor cells. IL-5 has little effect on the survival or proliferation of the multipotent stem cell line FDCP-Mix A4 but does promote some eosinophil development. To investigate whether the lineage specificity of IL-5 is due to the restricted expression of the IL-5 receptor alpha subunit we transfected the FDCP-Mix A4 cells with a retroviral vector containing this alpha subunit. The ectopic expression of the IL-5 receptor alpha subunit in the FDCP-Mix cells did not increase the observed eosinophilic development but did stimulate survival and proliferation of the transfected cells when IL-5 was added. IL-5 thus acts like IL-3 in these cells, promoting proliferation and survival. The results suggest that IL-5, whilst having a capacity to promote proliferation, does not influence eosinophilic lineage commitment in these multipotent cells. The results further argue that the observed lineage specificity of IL-5 is probably due to factors in addition to the restricted expression of the IL-5 receptor alpha subunit.

scholarly journals The role of the homeobox gene, HOX B7, in human myelomonocytic differentiation

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 692-697 ◽  
Author(s):  
MC Lill ◽  
JF Fuller ◽  
R Herzig ◽  
GM Crooks ◽  
JC Gasson
Keyword(s):  
Cell Line ◽  
Vitamin D3 ◽  
Critical Role ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Hl60 Cell ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Gm Csf

Homeobox genes encode transcription factors known to be important morphogenic regulators during embryogenesis. An increasing body of work implies a role for homeobox genes in both hematopoiesis and oncogenesis. We have analyzed the role of the homeobox gene, HOX B7, in the program of differentiation of the biphenotypic myeloid cell line, HL60. Induction of monocytic differentiation in HL-60 cells by vitamin D3 resulted in rapid expression of HOX B7 mRNA, but stimulation with phorbol ester or dimethyl sulfoxide (DMSO) did not. Constitutive overexpression of HOX B7 in the HL60 cell line inhibited the granulocytic differentiation associated with stimulation with DMSO or retinoic acid, but had no effect on the monocytic differentiation induced by vitamin D3. Normal human monocytes do not constitutively express HOX B7, nor are they able to be induced to do so by stimulation with colony-stimulating factor 1 (CSF-1) and gamma interferon (IFN gamma), or with vitamin D3 and lipopolysaccharide. Human bone marrow (BM) cells were found to express HOX B7 in response to granulocyte- macrophage CSF (GM-CSF) and antisense oligonucleotides directed against HOX B7 inhibited the formation of colonies derived from GM-CSF- stimulated BM. These data suggest a critical role for HOX B7 in myelomonocytic differentiation.

scholarly journals A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413 ◽  
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

scholarly journals New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 867
Author(s):  
Bruno Oyallon ◽  
Marie Brachet-Botineau ◽  
Cédric Logé ◽  
Thomas Robert ◽  
Stéphane Bach ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Cancer Progression ◽  
Kinase Inhibitors ◽  
Integration Site ◽  
Leukemia Virus ◽  
Biological Evaluation ◽  
Design Synthesis ◽  
Proviral Integration Site ◽  
Quinoxaline Derivatives

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

scholarly journals From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation

Blood ◽  
2015 ◽  
Vol 126 (24) ◽  
pp. 2561-2569 ◽  
Author(s):  
Tarik Möröy ◽  
Lothar Vassen ◽  
Brian Wilkes ◽  
Cyrus Khandanpour
Keyword(s):  
Zinc Finger ◽  
Target Genes ◽  
Integration Site ◽  
Wide Spectrum ◽  
Human Leukemia ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Proviral Integration Site ◽  
Cell Type Specific Expression

AbstractThe DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type–specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

scholarly journals Transcriptional Analysis of the Adeno-Associated Virus Integration Site

2009 ◽  
Vol 83 (23) ◽  
pp. 12512-12525 ◽  
Author(s):  
Nathalie Dutheil ◽  
Els Henckaerts ◽  
Erik Kohlbrenner ◽  
R. Michael Linden
Keyword(s):  
Transcriptional Activity ◽  
Integration Site ◽  
Regulatory Elements ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Complex Interactions ◽  
Site Specific Integration ◽  
Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

1992 ◽  
Vol 3 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B C Gliniak ◽  
L S Park ◽  
L R Rohrschneider
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Mrna Degradation ◽  
Receptor Expression ◽  
Metabolic Inhibitors ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

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