scholarly journals Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1662-1667 ◽  
Author(s):  
Y Fukuda ◽  
H Fujita ◽  
L Garbaczewski ◽  
S Sassa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Erythroleukemia Cells ◽  
Dmso Treatment ◽  
Free Heme ◽  
Murine Erythroleukemia

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

scholarly journals Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1662-1667 ◽  
Author(s):  
Y Fukuda ◽  
H Fujita ◽  
L Garbaczewski ◽  
S Sassa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Erythroleukemia Cells ◽  
Dmso Treatment ◽  
Free Heme ◽  
Murine Erythroleukemia

Abstract The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

scholarly journals Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 933-939
Author(s):  
R Gambari ◽  
RA Rifkind ◽  
PA Marks
Keyword(s):  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Induced Differentiation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.

scholarly journals Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 933-939 ◽  
Author(s):  
R Gambari ◽  
RA Rifkind ◽  
PA Marks
Keyword(s):  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Induced Differentiation ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Mrna Content ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Abstract Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.

Inhibition of DMSO-induced differentiation by hyperthermia in a murine erythroleukemia cell system

1984 ◽  
Vol 62 (11) ◽  
pp. 1091-1096 ◽  
Author(s):  
G. P. Raaphorst ◽  
E. I. Azzam ◽  
J. Borsa ◽  
M. Einspenner ◽  
J. A. Vadasz
Keyword(s):  
Heat Treatment ◽  
Dimethyl Sulfoxide ◽  
Erythroid Differentiation ◽  
Staining Technique ◽  
Cell System ◽  
Differentiation Process ◽  
Erythroleukemia Cells ◽  
Dmso Treatment ◽  
Erythroleukemia Cell ◽  
Murine Erythroleukemia

Friend erythroleukemia cells were induced by dimethyl sulfoxide (DMSO) into erythroid differentiation, as characterized by the production of hemoglobin. Induction increased with DMSO concentrations up to 1.5% v/v, at which point about 90% of the cell population produced hemoglobin as measured by a benzidine-staining technique. Heat treatment at 39.0–40.5 °C during a 7-day-incubation period, for differentiation in the presence of DMSO, resulted in the inhibition of hemoglobin induction. Also, acute heat treatments at 41.5–46.0 °C before or after the addition of DMSO resulted in the inhibition of DMSO induction. This effect was greatest when DMSO was present during heating. The results support the conclusion that hyperthermia inhibits the differentiation process which is induced by DMSO treatment.

scholarly journals Relationship between globin mRNA accumulation and commitment to terminal cell differentiation in inducer sensitive and resistant erythroleukemia variants

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 302-306
Author(s):  
N Weich ◽  
PA Marks ◽  
RA Rifkind
Keyword(s):  
Cell Differentiation ◽  
Erythroid Differentiation ◽  
Terminal Cell ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Chemical Inducers ◽  
Transformed Cell Line ◽  
Increased Sensitivity ◽  
Differentiation Inducers ◽  
Murine Erythroleukemia

Abstract The relationship between the kinetics of commitment to terminal cell differentiation and the rates of accumulation of globin mRNA has been examined during the induction of erythroid differentiation by polar/apolar chemical inducers in murine erythroleukemia cells (MELC), under conditions of more and less rapid commitment. Two differentiation inducers and three MELC variants have been studied. Hexamethylene bisacetamide (HMBA) initiates more rapid commitment than does dimethylsulfoxide (Me2SO). MELC variant DR10 is resistant to induction by Me2SO and responds sluggishly to HMBA, in comparison with the DS19- Sc9 variant. V3.17, an MELC variant resistant to low concentrations of vincristine, shows increased sensitivity to the inducers and an accelerated rate of commitment to terminal differentiation compared with DS19-Sc9. It is demonstrated that commitment and the actual expression of differentiation, as measured by the accumulation of alpha- , beta maj-, and beta min-globin mRNA, are temporally coordinated functions during induced differentiation of a transformed cell line by exposure to polar/apolar agents.

Nonsense mutations in the human β-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2895-2901 ◽  
Author(s):  
Luı́sa Romão ◽  
Ângela Inácio ◽  
Susana Santos ◽  
Madalena Ávila ◽  
Paula Faustino ◽  
...  
Keyword(s):  
Globin Gene ◽  
Mrna Level ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Globin Genes ◽  
Mrna Accumulation ◽  
Nonsense Mutations ◽  
Differentiation Induction ◽  
Nonsense Codons ◽  
Murine Erythroleukemia

Generally, nonsense codons 50 bp or more upstream of the 3′-most intron of the human β-globin gene reduce mRNA abundance. In contrast, dominantly inherited β-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human β-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG→TGA), 39 (C→T), or 127 (C→T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C→T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C→T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG→TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3′-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

Regulated Expression and Functional Role of the Transcription Factor CHOP (GADD153) in Erythroid Growth and Differentiation

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3369-3378 ◽  
Author(s):  
Margaret Coutts ◽  
Kunyuan Cui ◽  
Kerry L. Davis ◽  
Joan Cleves Keutzer ◽  
Arthur J. Sytkowski
Keyword(s):  
Transcription Factor ◽  
Steady State ◽  
Dimethyl Sulfoxide ◽  
Family Members ◽  
Erythroid Differentiation ◽  
Colony Growth ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Murine Erythroleukemia

The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA ofchop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp β was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression ofchop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)–derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)–derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

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