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PubMed2004

Multiple functional domains of the oncoproteins Spi-1/PU.1 and TLS are involved in their opposite splicing effects in erythroleukemic cells.

The hematopoietic transcription factor Spi-1/PU.1 is an oncoprotein participating to the malignant transformation of proerythroblasts in the Friend erythroleukemia or in the erythroleukemic process developed in spi-1 transgenic mice. Overexpression of Spi-1 in proerythroblasts blocks their differentiation. We have shown that Spi-1 promotes the use of the proximal 5'-splice site during the E1A pre-mRNA splicing and interferes with the effect of TLS (Translocated in LipoSarcoma) in this splicing assay. TLS was identified from chromosomal translocations in human liposarcoma and acute myeloid leukemia. Here, we determine the function of Spi-1 domains in splicing and in the interference with TLS. In transient transfection assays in erythroid cells, we show that the DNA binding domain cooperates with the transactivation domain or the PEST region of Spi-1 to modify the function of TLS in splicing. Interestingly, the 27 C-terminal amino acids, which determine the DNA binding activity of Spi-1, are necessary for the splicing function of Spi-1 as well as for its ability to interfere with TLS. Finally, we demonstrate that in leukemic proerythroblasts overexpressing Spi-1, TLS has lost its splicing effect. Thus, we hypothesize that oncogenic pathways in proerythroblasts may involve the ability of Spi-1 to alter splicing.

Adenovirus E1A Proteins↗
PubMed1995

Early events in erythroid differentiation: accumulation of the acidic peroxidoxin (PRP/TSA/NKEF-B).

The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.

Animals↗
PubMed2002

Effect of growth factors on ex vivo bone marrow cell expansion using three-dimensional matrix support.

To develop a culture system for bone marrow (BM) cell expansion, we examined the effect of growth factors (GFs) on the proliferation and differentiation of BM cells cultured in three-dimensional (3D) scaffolds of porous polyvinyl formal (PVF) resin. Murine BM cells were cultured for 2 weeks in the PVF resin or in culture dishes as a control, in the presence or absence of 4 GFs (erythropoietin, stem cell factor, interleukin [IL]-3, and IL-6). These GFs remarkably stimulated cell proliferation both in PVF and dish cultures. In addition, the PVF cultures showed enhanced cell proliferation in comparison with the corresponding dish cultures. Moreover, PVF cultures with GFs revealed the highest number of colony-forming units and the highest percentage of hematopoietic progenitor cells (HPCs) among all the cultures examined. Therefore, this 3D PVF culture system with GFs is considered as a potential alternative method for the ex vivo expansion of HPCs.

Animals↗
PubMed2002

Effects of interleukin-11 on the hematopoietic action of granulocyte colony-stimulating factor.

Interleukin-11 (IL-11, CAS 145941-26-0) is a cytokine that ameliorates thrombocytopenia induced by chemotherapy. Granulocyte colony-stimulating factor (G-CSF) is frequently used clinically as an adjuvant therapy to ameliorate neutropenia. In this study, it has been investigated whether IL-11 influences the hematopoietic action of G-CSF in vitro. IL-11 alone did not stimulate CFU-Meg colony formation in the absence of interleukin-3 (IL-3), but did in the presence of IL-3. However, IL-11 alone stimulated the formation of CFU-E, BFU-E and CFU-GM colonies, which were further enhanced by IL-3. G-CSF alone stimulated in a concentration dependent manner colony formation of CFU-GM and CFU-E, but not BFU-E or CFU-Meg. The addition of IL-11 enhanced the stimulatory effect of G-CSF on CFU-GM colony formation, and enabled G-CSF to stimulate colony formation of BFU-E, but not CFU-E. The addition of IL-3 further enhanced the stimulatory effect of G-CSF on CFU-E, BFU-E, and CFU-GM colony formation. However, G-CSF was unable to stimulate CFU-Meg colony formation for any of the cytokine combinations tested. These studies demonstrate that IL-11 has significant stimulatory effects on G-CSF-induced CFU-GM colony formation, suggesting that the therapeutic combination of the two growth factors could be beneficial for the treatment of myelosuppression induced by chemotherapy.

Animals↗
PubMed1998

Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia.

BACKGROUND: In alloimmune anemia of the newborn, the level of hemolysis caused by the presence of antibodies to antigens of the Kell blood-group system is less than that caused by antibodies to the D antigen of the Rh blood-group system, and the numbers of reticulocytes and normoblasts in the baby's circulation are inappropriately low for the degree of anemia. These findings suggest that sensitization to Kell antigens results in suppression of fetal erythropoiesis as well as hemolysis. METHODS: We compared the growth in vitro of Kell-positive and Kell-negative hematopoietic progenitor cells from cord blood in the presence of human monoclonal anti-Kell antibodies and anti-D antibodies and serum from women with anti-Kell antibodies. RESULTS: The growth of Kell-positive erythroid progenitor cells (erythroid burst-forming units and colony-forming units) from cord blood was markedly inhibited by monoclonal IgG and IgM anti-Kell antibodies in a dose-dependent fashion (range of concentrations, 0.2 to 20 percent), but monoclonal anti-D antibodies had no effect. The growth of these types of cells from Kell-negative cord blood was not affected by either type of antibody. Neither monoclonal anti-Kell antibodies nor monoclonal anti-D antibodies inhibited the growth of granulocyte or megakaryocyte progenitor cells from cord blood. Serum from 22 women with anti-Kell antibodies inhibited the growth of Kell-positive erythroid burst-forming units and colony-forming units but not of Kell-negative erythroid burst-forming units and colony-forming units (P<0.001 for the difference between groups). The maternal anti-Kell antibodies had no inhibitory effects on granulocyte-macrophage or mega-karyocyte progenitor cells from cord blood. CONCLUSIONS: Anti-Kell antibodies specifically inhibit the growth of Kell-positive erythroid burst-forming units and colony-forming units, a finding that supports the hypothesis that these antibodies cause fetal anemia by suppressing erythropoiesis at the progenitor-cell level.

Anemia, Hemolytic, Autoimmune↗
PubMed2002

Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin.

BACKGROUND: Within a period of three years, we identified 13 patients in whom pure red-cell aplasia developed during treatment with recombinant human erythropoietin (epoetin). We investigated whether there was an immunologic basis for the anemia in these patients. METHODS: Serum samples from the 13 patients with pure red-cell aplasia were tested for neutralizing antibodies that could inhibit erythroid-colony formation by normal bone marrow cells in vitro. The presence of antierythropoietin antibodies was identified by means of binding assays with the use of radiolabeled intact, deglycosylated, or denatured epoetin. RESULTS: Serum from all 13 patients blocked the formation of erythroid colonies by normal bone marrow cells. The inhibition was reversed by epoetin. Antibodies from 12 of the 13 patients bound only conformational epitopes in the protein moiety of epoetin; serum from the remaining patient bound to both conformational and linear epitopes in erythropoietin. In all the patients, the antibody titer slowly decreased after the discontinuation of treatment with epoetin. CONCLUSIONS: Neutralizing antierythropoietin antibodies and pure red-cell aplasia can develop in patients with the anemia of chronic renal failure during treatment with epoetin.

Adult↗
PubMed2003

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells.

p38alpha, p38beta, p38gamma, and p38delta are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38alpha and p38gamma transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38delta mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38beta is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38alpha, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38alpha, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38alpha does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38alpha and p38delta isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor alpha, an inflammatory cytokine that is modulated by p38alpha, is expressed by differentiating erythroblasts and inhibition of p38alpha or tumor necrosis factor alpha results in reduction in differentiation. Taken together, our data demonstrate that both p38alpha and delta isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Base Sequence↗
PubMed2003

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Amino Acid Sequence↗
PubMed1996

Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.

The interaction of the hormone erythropoietin and its receptor (EpoR) is though to be required for normal hematopoiesis. To define the role of EpoR in this process, the murine EpoR was disrupted by homologous recombination. Mice lacking the EpoR died in utero at embryonic day 11-12.5 with severe anemia. Embryonic erythropoiesis was markedly diminished, while fetal liver hematopoiesis was blocked at the proerythroblast stage. Other cell types known to express EpoR, including megakaryocytes, mast, and neural cells were morphologically normal. Reverse transcription-coupled PCR analysis of RNA from embryonic yolk sac, peripheral blood, and fetal liver demonstrated near normal transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, GATA-1, GATA-2, and alpha- and embryonic beta H1-globin but non for adult beta maj-globin. While colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies were not present in cultures derived from EpoR-/- liver or yolk sac cells, hemoglobin-containing BFU-E colonies were detected in cultures treated with recombinant Tpo and Kit ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E colonies expressed adult beta-globin and c-MPL and appeared morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo receptor c-MPL stimulates proliferation and terminal differentiation of these progenitors in vitro.

Animals↗
PubMed1997

Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)-/- fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation in vitro by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred in vivo before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in (Epo-R)-/- fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF/KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.

Animals↗
PubMed1997

Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors.

Homodimerization of the erythropoietin (EPO) receptor (EPO-R) in response to EPO binding transiently activates the receptor-associated protein tyrosine kinase JAK2. Tyrosine phosphorylation of the EPO-R creates "docking sites" for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 and SH-PTP2, phosphoinositide 3-kinase (PI3 kinase), and STAT5. However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors. Here we show that an EPO-R containing only one cytosolic (phospho)tyrosine residue, Y479, induces a signal transduction pathway sufficient for proliferation and differentiation of fetal liver progenitors of erythroid colony-forming units from EPO-R(-/-) mice as well as for proliferation of cultured hematopoietic cells. This cascade involves sequential EPO-induced recruitment of PI3 kinase to the EPO-R and activation of mitogen-activated protein kinase activity, independent of the Shc/Grb2-adapter pathway and of STAT5. Protein kinase C epsilon may be one of the mediators connecting PI3 kinase with the mitogen-activated protein kinase signaling cascade. Our results identify a signaling cascade important in vivo for erythroid cell proliferation and differentiation.

Animals↗
PubMed2001

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.

Animals↗
PubMed1995

E-cadherin is functionally involved in the maturation of the erythroid lineage.

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.

Base Sequence↗
PubMed2004

Btk is required for an efficient response to erythropoietin and for SCF-controlled protection against TRAIL in erythroid progenitors.

Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCgamma1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

A new methodology of fetal stem cell isolation, purification, and expansion: preliminary results for noninvasive prenatal diagnosis.

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p < 0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Antigens, CD34↗
PubMed1995

Interferon-alpha inhibits erythropoietin-induced proliferation, but not differentiation, and restricts erythroleukemia development.

The immature erythroid cell line J2E responds to erythropoietin (Epo) by proliferating and terminally differentiating into hemoglobin-synthesizing red blood cells. These cells produce a rapid, fatal erythroleukemia in mice characterized by hepatosplenomegaly and severe anemia. The aim of this study was to investigate the effects of murine interferons-alpha (MuIFN-alpha) on J2E cells in vitro and in vivo. Here we show that in culture MuIFN-alpha inhibited the Epo-induced proliferation of J2E cells but did not interfere with differentiation. When mice with J2E erythroleukemias were treated with MuIFNs in vivo, an extension of their life span was observed. Moreover, numerous necrotic lesions of infiltrating leukemic cells were detected in the spleens of these mice. Finally, ex vivo treatment of leukemic bone marrow cells with Epo and MuIFNs delayed mortality even further. It was concluded that MuIFNs (1) suppressed the proliferation of J2E cells in vitro but did not affect Epo-induced differentiation, and (2) inhibited the progress of erythroleukemias, especially in combination with Epo.

Animals↗
PubMed1993

Antigenic analysis of hematopoiesis: a review.

The ability to immunophenotype the heterogeneous populations of cells within bone marrow has given investigators the tools to identify, enumerate, and isolate cells of any lymphohematopoietic lineage. These cells can, in turn, be studied in morphologic, growth, biochemical, and molecular biological assays, and used for clinical transplantation, with or without in vitro expansion. Although the map of normal marrow lymphohematopoiesis has been largely determined, there is still some controversy regarding the identity and maturation pathways of lymphohematopoietic progenitors and stem cells. The currently available information on this area is reviewed and interpreted in this article.

ADP-ribosyl Cyclase↗
PubMed2002

Differentiation of monkey embryonic stem cells into neural lineages.

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

Animals↗