scholarly journals Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

1988 ◽  
Vol 8 (12) ◽  
pp. 5545-5548 ◽  
Author(s):  
T Kaneko-Ishino ◽  
T U Kume ◽  
H Sasaki ◽  
M Obinata ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Dimethyl Sulfoxide ◽  
Cell Fusion ◽  
Erythroid Differentiation ◽  
Metallothionein Promoter ◽  
Myc Gene ◽  
Suppressive Action ◽  
Mouse Erythroleukemia

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro

1988 ◽  
Vol 8 (12) ◽  
pp. 5545-5548
Author(s):  
T Kaneko-Ishino ◽  
T U Kume ◽  
H Sasaki ◽  
M Obinata ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Dimethyl Sulfoxide ◽  
Cell Fusion ◽  
Erythroid Differentiation ◽  
Metallothionein Promoter ◽  
Myc Gene ◽  
Suppressive Action ◽  
Mouse Erythroleukemia

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

Transcriptional Profile of Carbohydrate Blood Group Genes in Erythroid Versus Neutrophil Differentiation of Human CD34+ Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4242-4242
Author(s):  
Josefina H. Dykes ◽  
Britt Thuresson ◽  
Louise Edvardsson ◽  
Tor Olofsson ◽  
Martin L. Olsson
Keyword(s):  
Gene Expression ◽  
Blood Group ◽  
Erythroid Differentiation ◽  
Surface Expression ◽  
Blood Groups ◽  
Blood Group A ◽  
Cd34 Cells ◽  
Group A ◽  
Neutrophil Differentiation

Abstract Carbohydrate blood groups and their corresponding antibodies are clinically important, known to be involved in conditions such as hemolytic transfusion reactions, hemolytic disease of the newborn and spontaneous abortion. However, little is understood about the developmental changes in expression of carbohydrate blood groups during hematopoiesis, with preceding studies mainly focusing on protein-based blood group molecules. We have previously identified the carbohydrate blood group A antigen as the earliest, specific marker for definitive erythroid commitment, in preference to other suggested candidates such as Kell or Glycophorin C [Br J Haematol2004;127:451–63]. With regard to this lineage-restriction point we mapped the gene expression of some clinically important carbohydrate blood group systems during erythroid versus neutrophil differentiation in vitro. Human bone marrow CD34+ cells from healthy donors, carrying the blood group A1 allele and functional secretor (FUT2) and Lewis (FUT3) genes, were cultured in vitro towards erythroid or neutrophil development and sorted on a flow cytometer into subpopulations according to their surface expression of blood group A antigen/CD117 and CD15/CD33. Sorted cells were cultured in clonogenic assays in methylcellulose or analyzed by TaqMan real-time reverse transcriptase PCR for gene expression of a number of carbohydrate blood group glycosyltransferases. Surface expression of the blood group A antigen coincided with commitment to erythroid differentiation and the expression of CD15 with neutrophil/monocytic differentiation. In gene expression studies the ABO, H (FUT1), I (IGnT) and Pk (A4GALT) genes were all expressed in freshly isolated and sorted CD34+ cells. The ABO and the H genes were up-regulated in erythroid differentiation and silenced in neutrophil differentiation. The ABO gene expression was markedly decreased in late stages of erythroid maturation. The I gene was expressed both during erythroid and neutrophil development with an increased expression in late erythroid differentiation. The Pk gene retained a low expression throughout neutrophil differentiation and was up-regulated several-fold during erythroid differentiation. There was no detectable expression of FUT3 and the gene suggested being responsible for biosynthesis of the Sda antigen, GALGT2, in either erythroid or neutrophil differentiation. Our data support the identification of the blood group A antigen as an early and specific marker for definitive erythroid differentiation. In mature cells of the myeloid lineage, the results of the gene expression studies are compatible with previous findings of gene and/or surface expression of the I and Pk blood groups but not of ABO and H. The marked increase in expression of the Pk gene during erythroid differentiation may well agree with the fact that the Pk antigen is the precursor structure of globoside, the most abundant neutral glycolipid in the erythrocyte membrane. The absence of hematopoietic FUT3 expression in Lewis gene positive individuals was expected whilst the relevance of undetectable GALGT2 expression in hematopoietic differentiation is uncertain. The role of the GALGT2 gene in surface expression of Sda has not been definitively proven and the molecular basis of different Sda phenotype variants is unknown. In conclusion, our data extend previous findings of carbohydrate blood group distribution, primarily obtained from mature blood cells and leukemic cell lines, to normal human hematopoiesis.

scholarly journals Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3311-3315 ◽  
Author(s):  
T Kaneko ◽  
T Watanabe ◽  
M Oishi
Keyword(s):  
Protein Synthesis ◽  
Early Stage ◽  
Mitochondrial Protein ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Mitochondrial Protein Synthesis ◽  
Cell Extracts ◽  
Factor Ii ◽  
Mouse Erythroleukemia

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

scholarly journals OncogenicGata1causes stage-specific megakaryocyte differentiation delay

2019 ◽  
Author(s):  
Gaëtan Juban ◽  
Nathalie Sakakini ◽  
Hedia Chagraoui ◽  
Qian Cheng ◽  
Kelly Soady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Detailed Examination ◽  
S Phase ◽  
Myeloproliferative Disorder ◽  
Specific Gene ◽  
Specific Stage

AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3311-3315
Author(s):  
T Kaneko ◽  
T Watanabe ◽  
M Oishi
Keyword(s):  
Protein Synthesis ◽  
Early Stage ◽  
Mitochondrial Protein ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Mitochondrial Protein Synthesis ◽  
Cell Extracts ◽  
Factor Ii ◽  
Mouse Erythroleukemia

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

scholarly journals Synthesis of Combretastatin A-4 and 3′-Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants and Study of their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene Expression

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 660 ◽  
Author(s):  
Raül Agut ◽  
Eva Falomir ◽  
Juan Murga ◽  
Celia Martín-Beltrán ◽  
Raquel Gil-Edo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Parent Compound ◽  
Mitotic Cell ◽  
Tubulin Polymerization ◽  
Microtubule Network ◽  
Cell Network ◽  
Myc Gene ◽  
Cell Arrest

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3′-aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound.

scholarly journals A simple trypsin resistance assay for muscle and other cell fusion.

1986 ◽  
Vol 34 (7) ◽  
pp. 959-962 ◽  
Author(s):  
G Denning ◽  
A B Fulton
Keyword(s):  
Gene Expression ◽  
Cell Fusion ◽  
Muscle Cells ◽  
Cell Loss ◽  
Lugol’S Iodine ◽  
Edta Solution ◽  
Specific Stage ◽  
Independent Assay ◽  
Study Development

Muscle cells fusing in vitro have long provided biologists with a tool to study development and gene expression. However, many such studies used morphological assays of cell fusion. We present here a method for assaying fusion at a specific, operationally defined step. Muscle cells grown in monolayer are exposed to trypsin-EDTA solution at 37 degrees C; the trypsin is inactivated, the cells fixed in Lugol's iodine, and 200 to 300 nuclei are counted as being single or multiple. The presence of EDTA is important under standard conditions for muscle culture; however, little difference is seen in divalent cation-depleted cultures. Therefore, for consistency EDTA can be included in all assays. Samples are stable for over 24 hr, with no cell loss from trypsinization or fixation. This assay exploits a specific stage of muscle fusion, trypsin-resistant contact, to provide a rapid, simple, and observer-independent assay for an early state of muscle fusion. The assay can be used to measure fusion between any nucleated cells.

scholarly journals L-myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1980
Author(s):  
So Hee Kim ◽  
Bokyung Kim ◽  
Jung Hak Kim ◽  
Dong-Hoon Kim ◽  
Seung Hoon Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Soft Agar ◽  
Tumor Formation ◽  
Fibroblast Cells ◽  
Self Renewal ◽  
Fetal Fibroblasts ◽  
Myc Gene ◽  
Pluripotency Genes

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.

scholarly journals ARHGEF18/p114RhoGEF coordinates PKA/CREB signaling and actomyosin remodeling to drive trophoblast cell-cell fusion during placenta morphogenesis

2020 ◽  
Author(s):  
Robert Beal ◽  
Ana Alonso-Carriazo Fernandez ◽  
Dimitris K. Grammatopoulos ◽  
Karl Matter ◽  
Maria S. Balda
Keyword(s):  
Gene Expression ◽  
Cell Fusion ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Trophoblast Cell ◽  
Cell Junctions ◽  
Nucleotide Exchange ◽  
Cell Cell

SUMMARYCoordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating PKA signalling, and is required for PKA-induced actomyosin remodelling, CREB-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signalling, gene expression and cell-cell fusion.

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