scholarly journals A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277 ◽  
Author(s):  
G D Longmore ◽  
P N Pharr ◽  
H F Lodish
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Active Form ◽  
Erythropoietin Receptor ◽  
Mutant Form ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

scholarly journals A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277
Author(s):  
G D Longmore ◽  
P N Pharr ◽  
H F Lodish
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Active Form ◽  
Erythropoietin Receptor ◽  
Mutant Form ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

scholarly journals Ex Vivo-Generated CD36+ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2470-2476 ◽  
Author(s):  
Susan Wong ◽  
Ning Zhi ◽  
Claudia Filippone ◽  
Keyvan Keyvanfar ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Real Time ◽  
Cell Lines ◽  
Ex Vivo ◽  
Parvovirus B19 ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Human Virus ◽  
Erythroid Progenitor Cells ◽  
Large Numbers

ABSTRACT The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

A Minimal Cytoplasmic Subdomain of the Erythropoietin Receptor Mediates Erythroid and Megakaryocytic Cell Development

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3381-3387 ◽  
Author(s):  
Chris P. Miller ◽  
Zi Y. Liu ◽  
Constance T. Noguchi ◽  
Don M. Wojchowski
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Erythroid Progenitor Cells ◽  
Fetal Liver Cells

Signals provided by the erythropoietin (Epo) receptor are essential for the development of red blood cells, and at least 15 distinct signaling factors are now known to assemble within activated Epo receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal liver cells suggest that most of these factors and signals may be functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells expanded ex vivo, and this EE372 chimera was observed to support mitogenesis and red blood cell development at wild-type efficiencies both independently and in synergy with c-Kit. In addition, the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and 2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine 343 site (each retained within EE372), appear to provide all of the signals necessary for the development of committed progenitor cells within both the erythroid and megakaryocytic lineages.

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 Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 651-657 ◽  
Author(s):  
Xingwei Sui ◽  
Sanford B. Krantz ◽  
Zhizhuang Zhao
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Normal Cells ◽  
Protein Tyrosine ◽  
Erythroid Progenitor Cells

Abstract Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

PU.1 determines the self-renewal capacity of erythroid progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3615-3623 ◽  
Author(s):  
Jonathan Back ◽  
Andrée Dierich ◽  
Corinne Bronn ◽  
Philippe Kastner ◽  
Susan Chan
Keyword(s):  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Self Renewal ◽  
Adult Mice ◽  
Erythroid Progenitor Cells ◽  
Low Levels ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

Abstract PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation—it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1-deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary Cultured Human Erythroid Precursor Cells From Apoptosis

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1568-1577 ◽  
Author(s):  
Yoshihito Haseyama ◽  
Ken-ichi Sawada ◽  
Atsushi Oda ◽  
Kazuki Koizumi ◽  
Hina Takano ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Maximal Effect ◽  
Erythroid Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Erythroid Progenitor Cells ◽  
Dependent Cell ◽  
Phosphatidylinositol 3

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34+ cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 μmol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V–binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

scholarly journals Establishment of an erythroid cell line from primary CD36+ erythroid progenitor cells

2010 ◽  
Vol 38 (11) ◽  
pp. 994-1005.e2 ◽  
Author(s):  
Susan Wong ◽  
Keyvan Keyvanfar ◽  
Zhihong Wan ◽  
Sachiko Kajigaya ◽  
Neal S. Young ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

scholarly journals Clonal analysis of the late stages of erythroleukemia induced by two distinct strains of Friend leukemia virus.

1981 ◽  
Vol 1 (8) ◽  
pp. 721-730 ◽  
Author(s):  
D Mager ◽  
M E MacDonald ◽  
I B Robson ◽  
T W Mak ◽  
A Bernstein
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Single Cells ◽  
Leukemia Virus ◽  
Significant Proportion ◽  
Erythroid Progenitor ◽  
Self Renewal ◽  
Friend Leukemia ◽  
P Colonies ◽  
Friend Leukemia Virus

We observed striking differences between the tumorigenic colony-forming cells present in the spleens of mice late after infection with the anemia-inducing strain of Friend leukemia virus (strain FV-A) and those present after infection with the polycythemia-inducing strain (strain FV-P). Cells within primary colonies derived from FV-A- and FV-P-transformed cells (CFU-FV-A and CFU-FV-P, respectively) contained hemoglobin and spectrin, indicating that the CFU-FV-A and CFU-FV-P were transformed erythroid progenitor cells. The proportion of cells containing hemoglobin was relatively high (> 25%) in newly isolated cell lines derived from CFU-FV-P colonies, whereas cell lines derived from CFU-FV-A colonies had only low levels (0 to 2%) of hemoglobin-containing cells. A high proportion of the cell lines derived from CFU-FV-A colonies responded to pure erythropoietin and accumulated spectrin and hemoglobin, whereas the cell lines derived from CFU-FV-P colonies did not. A cytogenetic analysis indicated that primary CFU-FV-P colony cells were diploid, whereas chromosomal aberrations were observed in the immediate progeny of CFU-FV-A. The presence of unique chromosomal markers in the majority of the cells within individual colonies derived from CFU-FV-A suggested that these colonies originated from single cells. Finally, leukemic progenitor cells transformed by strain FV-A appeared to have an extensive capacity to self-renew (i.e., form secondary colonies in methylcellulose), whereas a significant proportion of the corresponding cells transformed by strain FV-P did not. In addition, the self-renewal capacity of both CFU-FV-A and CFU-FV-P increased as the disease progressed. From these observations, we propose a model for the multistage nature of Friend disease; this model involves clonal evolution and expansion from a differentiating population with limited proliferative capacity to a population with a high capacity for self-renewal and proliferation.

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