scholarly journals Erythropoietin receptor signals both proliferation and erythroid-specific differentiation

1993 ◽  
Vol 90 (23) ◽  
pp. 11351-11355 ◽  
Author(s):  
E Liboi ◽  
M Carroll ◽  
A D D'Andrea ◽  
B Mathey-Prevot
Keyword(s):  
Cell Line ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Beta Globin ◽  
Globin Mrna ◽  
Interleukin 3 ◽  
Cellular Factors ◽  
Proliferation And Differentiation ◽  
Dependent Cell

Ectopic expression of the erythropoietin receptor (EPO-R) in Ba/F3, an interleukin 3-dependent progenitor cell line, confers EPO-dependent cell growth. To examine whether the introduced EPO-R could affect differentiation, we isolated Ba/F3-EPO-R subclones in interleukin 3 and assayed for the induction of beta-globin mRNA synthesis after exposure to EPO. Detection of beta-globin mRNA was observed within 3 days of EPO treatment, with peak levels accumulating after 10 days. When EPO was withdrawn, expression of beta-globin mRNA persisted in most clones, suggesting that commitment to erythroid differentiation had occurred. Although EPO-R expression also supports EPO-dependent proliferation of CTLL-2, a mature T-cell line, those cells did not produce globin transcripts, presumably because they lack requisite cellular factors involved in erythrocyte differentiation. We conclude that the EPO-R transmits signals important for both proliferation and differentiation along the erythroid lineage.

scholarly journals Erythropoietin and interleukin-3 induce distinct events in erythropoietin receptor-expressing BA/F3 cells

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 50-56 ◽  
Author(s):  
J Krosl ◽  
JE Damen ◽  
G Krystal ◽  
RK Humphries
Keyword(s):  
Kinase Inhibitor ◽  
Flow Cytometric Analysis ◽  
S Phase ◽  
Erythropoietin Receptor ◽  
Growth Delay ◽  
Beta Globin ◽  
Globin Mrna ◽  
Interleukin 3 ◽  
Cell Clones ◽  
Protein Kinase C Inhibitor

To compare the signal transduction pathways used by erythropoietin (Ep) and interleukin-3 (IL-3), the cDNA for the murine erythropoietin receptor (EpR) was introduced into the IL-3-responsive cell lines Ba/F3 and DA-3 using retrovirally mediated gene transfer. After selection in G418 and IL-3, clones expressing comparable levels of cell surface EpR were identified using biotinylated Ep and flow cytometry. A comparison of the effects of Ep and IL-3 on these cells showed that most EpR+ Ba/F3 clones, when first exposed to Ep, dramatically increased their levels of beta-globin mRNA. The kinetics of appearance of this message after exposure to Ep varied considerably from clone to clone, with some clones showing a marked increase in beta-globin mRNA within 1 hour, while others required several days before an increase was observed. Interestingly, not only was this increase not seen with IL-3, but IL-3 prevented the Ep-induced appearance of beta-globin message. On the other hand, none of the EpR+ DA-3 cell clones tested increased their levels of beta-globin mRNA in response to Ep. While the EpR+ DA-3 clones showed identical proliferative responses to IL-3 and Ep, most EpR+ Ba/F3 clones displayed a marked, albeit transient, proliferative lag when first exposed to Ep. This was manifested as both an increased doubling time in liquid culture and a decreased colony size in methylcellulose. Plating efficiencies of EpR+ Ba/F3 cells in methylcellulose, however, were identical in response to IL-3 and Ep, suggesting that the Ep-induced lag in proliferation reflected a growth delay of the entire population of cells to Ep rather than a selection of an Ep-responsive subpopulation. Flow cytometric analysis established that this growth delay was due to a lengthening of the first G1 period after exposure to Ep. Interestingly, this Ep-induced delay in entry into the S phase was not detected in cells stimulated with both Ep and IL-3 nor in EpR+ Ba/F3 cell clones that did not show an increase in beta-globin mRNA in response to Ep. Thymidine-induced growth arrest, however, showed that delaying entry into S phase alone was not sufficient to stimulate beta-globin mRNA in the absence of Ep. Further studies established that the Ep-induced increase in beta-globin mRNA could be inhibited by the tyrosine kinase inhibitor genistein and the protein kinase C inhibitor Compound 3.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Identification of an erythropoietin-sensitive cell line

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1782-1785
Author(s):  
DR Branch ◽  
JM Turc ◽  
LJ Guilbert
Keyword(s):  
Cell Line ◽  
Biochemical Analysis ◽  
Erythroid Differentiation ◽  
Sensitive Cell Line ◽  
Gm Csf ◽  
Dependent Cell ◽  
Macrophage Colony Stimulating Factor ◽  
Survival And Growth ◽  
Macrophage Colony ◽  
Stimulating Factor

The murine lymphoblastic cell line DA-1 has been characterized as dependent upon both interleukin-3 (IL-3, multicolony-stimulating factor [multi-CSF]) and granulocyte-macrophage colony-stimulating factor (GM- CSF) for survival and growth. Here we demonstrate that it is responsive to a third hematopoietic factor, the erythroid-specific hormone, erythropoietin (Epo). DA-1 cells are stimulated to proliferate by partly purified natural murine and human Epo, and pure recombinant human Epo. Antibody to Epo specifically blocks Epo-stimulated growth. Maximal growth stimulated by Epo and GM-CSF is similar, and considerably less than that stimulated by multi-CSF. Proliferation stimulated by Epo and GM-CSF is transient, decreasing within 24 to 48 hours of exposure. However, Epo acts cooperatively with GM-CSF to sustain proliferation. With or without GM-CSF, no obvious erythroid differentiation of DA-1 cells occurs after exposure to Epo for up to 72 hours. This is the first report of a growth factor-dependent cell line also responsive to Epo for survival and growth. The availability of this cell line model should greatly facilitate biochemical analysis of the mechanism of Epo growth-stimulating action.

Interleukin 5 and interleukin 3 induce serine and tyrosine phosphorylations of several cellular proteins in an interleukin 5-dependent cell line

1990 ◽  
Vol 173 (3) ◽  
pp. 1102-1108 ◽  
Author(s):  
Yoshiyuki Murata ◽  
Naoto Yamaguchi ◽  
Yasumichi Hitoshi ◽  
Akira Tominaga ◽  
Kiyoshi Takatsu
Keyword(s):  
Cell Line ◽  
Cellular Proteins ◽  
Interleukin 5 ◽  
Interleukin 3 ◽  
Dependent Cell

scholarly journals Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
M Silva ◽  
D Grillot ◽  
A Benito ◽  
C Richard ◽  
G Nunez ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Survival Factor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

scholarly journals The extracytoplasmic domain of the erythropoietin receptor forms a monomeric complex with erythropoietin

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1713-1719 ◽  
Author(s):  
MG Yet ◽  
SS Jones
Keyword(s):  
Growth Factor ◽  
Cell Line ◽  
Gel Filtration ◽  
Chinese Hamster ◽  
Erythropoietin Receptor ◽  
Apparent Dissociation Constant ◽  
Amino Terminal ◽  
Truncated Receptor ◽  
Monomeric Complex ◽  
Dependent Cell

Abstract We have generated a truncated form of the erythropoietin receptor (EPO- R), the extracytoplasmic ligand-binding domain, that is secreted from a transfected Chinese hamster ovary (CHO) cell line. The truncated receptor is readily purified from CHO conditioned media as a 33-Kd glycosylated protein, which is converted to a 25-Kd species upon treatment with protein N-glycan glycosidase F. Cross-linking of radioiodinated EPO to the secreted receptor yielded a complex of 72 Kd. Also, the growth of the EPO-dependent cell line, FDCPE, was inhibited in a dose-responsive manner by the truncated receptor. The complex of the secreted receptor and EPO was isolated by gel filtration and shown to be a one-to-one complex of the receptor and growth factor by quantitative amino terminal sequencing. Finally, analysis of the interaction of the receptor and growth factor by gel filtration indicated an apparent dissociation constant of 1.1 nmol/L for the truncated receptor.

scholarly journals SHP-1 Phosphatase C-Terminus Interacts With Novel Substrates p32/p30 During Erythropoietin and Interleukin-3 Mitogenic Responses

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3746-3755 ◽  
Author(s):  
Wentian Yang ◽  
Mina Tabrizi ◽  
Karim Berrada ◽  
Taolin Yi
Keyword(s):  
Cell Line ◽  
Hematopoietic Cells ◽  
Signaling Molecules ◽  
Erythropoietin Receptor ◽  
Negative Regulator ◽  
Growth Responses ◽  
Mitogenic Signaling ◽  
Hematopoietic Growth Factors ◽  
Interleukin 3 ◽  
C Terminus

Abstract SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.

scholarly journals Spontaneous delta- to beta-globin switching in K562 human leukemia cells

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 820-825 ◽  
Author(s):  
B Mookerjee ◽  
MO Arcasoy ◽  
GF Atweh
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Culture Conditions ◽  
Human Leukemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Hemoglobin Switching ◽  
Erythroleukemia Cell ◽  
Globin Promoter

Abstract Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta- globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.

scholarly journals Identification of an erythropoietin-sensitive cell line

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1782-1785 ◽  
Author(s):  
DR Branch ◽  
JM Turc ◽  
LJ Guilbert
Keyword(s):  
Cell Line ◽  
Biochemical Analysis ◽  
Erythroid Differentiation ◽  
Sensitive Cell Line ◽  
Gm Csf ◽  
Dependent Cell ◽  
Macrophage Colony Stimulating Factor ◽  
Survival And Growth ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The murine lymphoblastic cell line DA-1 has been characterized as dependent upon both interleukin-3 (IL-3, multicolony-stimulating factor [multi-CSF]) and granulocyte-macrophage colony-stimulating factor (GM- CSF) for survival and growth. Here we demonstrate that it is responsive to a third hematopoietic factor, the erythroid-specific hormone, erythropoietin (Epo). DA-1 cells are stimulated to proliferate by partly purified natural murine and human Epo, and pure recombinant human Epo. Antibody to Epo specifically blocks Epo-stimulated growth. Maximal growth stimulated by Epo and GM-CSF is similar, and considerably less than that stimulated by multi-CSF. Proliferation stimulated by Epo and GM-CSF is transient, decreasing within 24 to 48 hours of exposure. However, Epo acts cooperatively with GM-CSF to sustain proliferation. With or without GM-CSF, no obvious erythroid differentiation of DA-1 cells occurs after exposure to Epo for up to 72 hours. This is the first report of a growth factor-dependent cell line also responsive to Epo for survival and growth. The availability of this cell line model should greatly facilitate biochemical analysis of the mechanism of Epo growth-stimulating action.

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