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PubMed1997

Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis.

Several homeobox genes of the HOXA and HOXB clusters are expressed in primitive blood cells, suggesting a role for HOX genes in normal hematopoiesis. The HOXA9 gene is expressed in CD34+ marrow cells and in developing lymphocytes. We examined blood-forming organs of mice homozygous for an interrupted HOXA9 allele to determine if loss of HOX gene function is deleterious to hematopoiesis. HOXA9-/- mice have approximately 30% to 40% reductions in total leukocytes and lymphocytes (P < .001) and a blunted granulocytic response to granulocyte colony-stimulating factor (G-CSF). Homozygous mice have significantly smaller spleens and thymuses. Myeloid/erythroid and pre-B progenitors in the marrow are significantly reduced, but no significant decreases are noted in mixed colonies, day 12 colony-forming units-spleen (CFU-S), or long-term culture-initiating cells (LTC-IC), suggesting little or no perturbation in earlier progenitors. Heterozygous animals display no hematopoietic defects. The abnormalities in leukocyte production are transplantable, indicating that the defect resides in the hematopoietic cells. These studies demonstrate a physiologic role for a HOX gene in blood cell differentiation, with the greatest apparent influence of HOXA9 at the level of the committed progenitor.

Animals↗
PubMed1997

Mammalian homeobox B6 expression can be correlated with erythropoietin production sites and erythropoiesis during development, but not with hematopoietic or nonhematopoietic stem cell populations.

There has been increasing interest in the involvement of mammalian homeobox (HOX) genes in hematopoietic regulation. The HOX genes are clustered in 4 chromosomes in mice and humans. In general, 5' end HOX gene expression is predominant in hematopoietic stem cell populations, whereas 3' end HOX gene expression are primarily found in committed progenitor cells. Furthermore, HOX genes of the A cluster are generally found in myelomonocytic cells, B cluster genes in erythropoietic cells, and C cluster genes in lymphoid cells. The results presented here concentrate on a single gene, namely HOX B6. Preliminary observations using whole mount in situ hybridization showed that both HOX B6 and erythropoietin (EPO) gene expression occurred in exactly the same areas of the 8.5-day mouse embryo. As a consequence, we studied the expression of HOX B6 and EPO gene expression from 6.5 to 19.5 days of gestation, in the neonate, and in the adult. It was found that the sequential transfer of erythropoiesis in different organs during development was followed by a similar transfer of HOX B6 and EPO gene expression. Between days 16.5 and 17.5, both HOX B6 and EPO gene expression decrease in the fetal liver, even though hepatic erythropoiesis continues to decline and is transferred to the fetal spleen. Precisely at this time point, HOX B6 and EPO gene expression are transferred to both the fetal spleen and fetal kidney. However, surprisingly, expression of both genes increases again in the fetal liver just before birth. HOX B6 is expressed in cells from in vitro erythropoietic colonies (colony-forming unit-erythroid and burst-forming unit-erythroid) and TER-119+ erythroid cells but not in hematopoietic or nonhematopoietic stem cell populations. When the latter two populations are allowed to differentiate into erythropoietic cells, HOX B6 and erythroid-relevant markers are expressed. The results indicate that HOX B6 is intimately involved in the regulation of the erythropoietic system and could be a marker for this lineage.

Animals↗
PubMed1997

Frequency of fetal cells in sorted subpopulations of nucleated erythroid and CD34+ hematopoietic progenitor cells from maternal peripheral blood.

Fetal cells that circulate in maternal peripheral blood (PB) during pregnancy offer a potential source of nucleated fetal material for noninvasive prenatal diagnosis. Fluorescence-activated cell sorting was used to target two populations of fetal cells: nucleated erythroid cells (NECs; CD71/glycophorin-A+ CD45(lo-int) CD34-) and hematopoietic progenitor cells (CD34+ cells; CD34++ CD71/glycophorin-A- CD45(int)). Fetal cells were detected by fluorescence in situ hybridization (FISH) using directly conjugated chromosome X and Y probes in 65% (13 of 20) of the maternal PBs (fetal karyotype 46,XY). The frequency of fetal cells isolated from the NEC and CD34+ fractions was, respectively, 0 to 14 and 0 to 7 cells per 2 x 10(7) previously frozen maternal cells (approximately 20 mL of blood). In nonfrozen samples, the yield and recovery of fetal cells was moderately improved. Culturing the CD34+ sorted fractions in serum-free media with cytokines improved the quality of the FISH preparations and resulted in a slight expansion in detectable fetal cells. The frequency of fetal cells isolated from cultured CD34+ fractions was 0 to 35 and 0 to 93 cells per 2 x 10(7) previously frozen and nonfrozen maternal PB cells, respectively. These results document the isolation, characterization, and enumeration of fetal cells from the maternal periphery that appear to be present in most, but not all, samples analyzed.

Antigens, CD34↗
PubMed1997

Retinoblastoma gene deficiency has mitogenic but not tumorigenic effects on erythropoiesis.

The retinoblastoma protein (Rb), an important ubiquitous cell cycle regulator, was initially identified as the retinoblastoma tumor suppressor. To further address the activities of Rb in proliferation and tumorigenesis in the hematopoietic lineage, we transplanted Rb-/- fetal liver cells into sibling mice and assessed the outcome of Rb-/- hematopoietic cells in both short-term and long-term studies. Rb-/- hematopoietic cells rescued lethally irradiated mice with an efficiency comparable to that of wild-type cells. In spleen colony-forming unit assays, proliferation rates of the Rb-/- cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Rb-/- embryonic livers. Recipients of Rb-/- cells lived for more than 15-18 months, and most blood cell lineages matured normally with the expected switch from fetal to adult hemoglobin. However, the continued presence of nucleated erythrocytes in the peripheral blood and extensive extramedullary erythropoiesis indicated that the Rb-/- erythrocytes were not completely normal. No erythroleukemia developed during the 15-18 month period following transplantation. These results demonstrate the mitogenic effect but not tumorigenic transformation in erythrocyte lineage in the absence of Rb, which is distinct from the effect of Rb deficiency in neuroectodermal cells. The study supports the prevalent model that loss of the ubiquitously expressed tumor suppressor gene predisposes to only a limited spectrum of tumors.

Animals↗
PubMed1997

Disruption of bone marrow stromal cell function by etoposide.

Long-term deficits in hematopoietic reconstitution following aggressive chemotherapeutic regimens used before transplantation may be a result, in part, of damage to the bone marrow microenvironment. Disruption of the hematopoietic microenvironment is indicated by delays in functional recovery of the immune system in spite of delivery of healthy peripheral blood or bone marrow progenitor cells. Cultures of primary human bone marrow stromal cells and a cloned murine stromal cell line, S10, were evaluated for functional changes following in vitro exposure to the chemotherapeutic agent, etoposide (VP-16). Stromal cells had reduced capacity to support lymphoid or myeloid cell proliferation following chemotherapy treatment. A consistent reduction of vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stromal cells also followed VP-16 exposure. These observations indicate that functional disruption of the bone marrow microenvironment by chemotherapeutic regimens must be considered when attempting to optimize patient recovery from hematopoietic transplantation.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Interleukin-10 inhibits erythropoietin-independent growth of erythroid bursts in patients with polycythemia vera.

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10-induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.

Adult↗
PubMed1998

Differential development of fetal and adult haemoglobin profiles in colony culture: isolation of fetal nucleated red cells by two-colour fluorescence labelling.

Fetal cells in maternal peripheral blood are a source of fetal DNA for prenatal genetic diagnosis, but their numbers are so small and variable that a reliable isolation procedure has yet to be demonstrated. The problem of scarcity may be overcome by amplification of fetal progenitor cells in cultures from maternal blood samples. One challenge is to identify post-culture fetal cells and colonies. We have found that the progeny of fetal and adult erythroid progenitors developed differential haemoglobin profiles in co-culture. Fetus-derived cells initially made only fetal haemoglobin (HbF) and began to express adult haemoglobin (HbA) only after intracellular HbF had reached maximum levels, which occurred after c 7 d in culture. By this time the large majority of adult-derived erythroid cells contained already high levels of HbA alone or combined with HbE Using the HbF+ HbA- criterion, we were able to flow-sort fetal cells with up to nearly 50% purity from some post-termination blood cultures, and with >90% purity in cultures from maternal blood spiked with 1% blood from the fetus. Fetal cell purity depended on culture time and serum supplement. After 7-10 d, purity was higher in low concentrations of human cord serum (1-3%) than in the standard 30% fetal calf serum. This was reversed at later times. Thus, if fetal clonogenic erythroid cells were present in maternal blood. their progeny could be isolated from most adult erythoid cells based on haemoglobin profiles. Cultures using CD34+ cells could be performed complementary to other methods targeting more mature fetal cells in the same maternal blood samples, thus increasing the overall chances of finding fetal cells and potentially providing clonal isolation of such cells.

Adult↗
PubMed1999

Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis.

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.

Adult↗
PubMed1993

The two-step liquid culture: a novel procedure for studying maturation of human normal and pathological erythroid precursors.

We have recently described a novel two-phase liquid culture procedure for growing human erythroid cells in vitro. The two phases are 1) an erythropoietin (EPO)-independent phase, in which the cells are first cultured in the presence of a combination of growth factors excluding EPO; during this phase, early erythroid committed progenitors, burst forming units (BFU-e), proliferate and differentiate into colony forming unit (CFU-e)-like progenitors; 2) a second phase, in which the latter cells are cultured in an EPO-supplemented medium, in which the CFU-e-like progenitors continue to proliferate and mature into orthochromatic normoblasts and then enucleated erythrocytes. This procedure yields large (up to 5 x 10(8)) and pure (95-98%) populations of erythroid cells, which allow detailed study of normal and pathologic erythroid maturation, including 1) the effects of growth factors on proliferation and differentiation at various erythroid developmental stages, 2) intracellular iron metabolism in normal and thalassemic erythroid cells and the role of ferritin as an iron donor for heme synthesis, 3) the expression of surface antigens: transferrin receptor, glycophorin, A, B, H, D and I/i antigens, 4) synthesis of erythroid-specific membrane proteins, 5) the kinetics of globin mRNA accumulation during erythroid maturation, 6) the expression of exogenous human beta globin gene in beta-thalassemic cells as a model for gene therapy, and 7) the enhancement of gamma globin chain synthesis by chemical agents.

Cells, Cultured↗
PubMedDate not supplied

[Formation of mixed colonies by progenitor cells of chronic myeloid leukemia cultured in vivo].

The clonal growth of multipotential progenitor cells of chronic myeloid leukemia (CML) in diffusion chamber implanted into peritoneal cavity of neutropenic and anemic rats was assessed. CML precursors formed in methylcellulose culture enriched with medium conditioned by phytohemagglutinin activated lymphocytes mixed neutrophilic-erythroid colonies, in number significantly higher then normal cells. Mixed colonies produced by CML cells, in contrast to the normal precursors, contained also macrophages, eosinophils and megakaryocytes. We conclude that modified diffusion chamber culture technique may be a suitable tool for multipotential progenitor study in CML.

Adult↗
PubMed1999

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.

Antigens, CD↗
PubMed2002

A patient with acquired pure red cell aplasia showing a positive antiglobulin test and the presence of inhibitor against erythroid precursors.

A 66-year-old Japanese man developed severe anemia and erythroid hypoplasia in bone marrow without any significant underlying disease. The results of an antiglobulin test were strongly positive, and serum erythropoietin (Epo) was high. The patient was diagnosed as having acquired pure red cell aplasia (PRCA) and was treated with steroids. Anemia was subsided by reticulocyte production in parallel with a decrease in the titer of antiglobulin test and the level of Epo. We studied the immunological mechanism directed against erythroid cells in vitro by using the patient's serum. In vitro analysis indicated the presence of an inhibitor of erythroid precursors at onset, and its disappearance at remission, suggesting the presence of inhibitor against erythroid precursors.

Aged↗
PubMed1994

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level.

Animals↗
PubMed2001

Regulation of the transcription factor GATA-1 at the gene and protein level.

GATA-1, an important hematopoietic transcription factor, plays a critical role in differentiation and maturation of erythroid and megakaryocytic cell lines. GATA-1 appears to serve as a factor for virtually all characterized erythroid and megakaryocytic-expressed genes. Thus, defining the mechanisms by which the GATA-1 gene and protein are regulated should provide important clues regarding the establishment of erythroid and megakaryocytic programs of gene expression in committed cells and their maintenance thereafter in maturing precursors. This review focuses on the regulation of GATA-1 expression and elucidates the regulation of GATA-1 at the gene and protein levels. Such research is expected to provide insights into the mechanisms involved in hematopoietic commitment.

Animals↗
PubMed1990

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia.

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.

Animals↗
PubMed1998

Bcl-3 expression and nuclear translocation are induced by granulocyte-macrophage colony-stimulating factor and erythropoietin in proliferating human erythroid precursors.

Bcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IkappaB family of proteins. In this report, involvement of Bcl-3 in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)-derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation of Bcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-kappaB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus-type 1 (HIV-1) kappaB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of kappaB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a kappaB enhancer in the c-myb promoter together with NF-kappaB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of a c-myb kappaB-TATA-luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain kappaB-containing genes involved in hematopoiesis, including c-myb.

B-Cell Lymphoma 3 Protein↗
PubMed1998

NF-kappaB transcription factors are involved in normal erythropoiesis.

NF-kappaB/Rel designates a widely distributed family of transcription factors involved in immune and acute phase responses. Here, the expression and function of NF-kappaB factors in erythroid proliferation and differentiation were explored. In an erythroleukemia cell line, TF-1, high levels of p105/p50, p100/p52, p65, and IkappaBalpha were detected 24 hours after growth factor deprivation. In response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, significant induction of p52 expression was observed. GM-CSF also induced nuclear translocation of both p52 and p65. No induction of NF-kappaB factors was observed with erythropoietin stimulation of TF-1 cells. Overexpression of p52 and p65 in TF-1 cells by transient transfection resulted in significant induction of a kappaB-TATA-luciferase reporter plasmid, showing that these factors are functional in vivo in erythroid cells. To determine whether NF-kappaB factors may play a role in normal erythropoiesis, levels of these factors were determined in burst-forming unit-erythroid (BFU-E)-derived cells at different stages of differentiation. The NF-kappaB factors p105/p50, p100/p52, and p65 were highly expressed in early BFU-E-derived precursors, which are rapidly proliferating, and declined during maturation. Furthermore, nuclear levels of NF-kappaB factors p50, p52, and p65 were higher in less mature precursors (day 10 BFU-E-derived cells) compared with more differentiated (day 14) erythroblasts. In nuclear extracts from day 10 BFU-E-derived cells, p50, p52, and p65 were able to form complexes, which bound to kappaB sites in the promoters of both the c-myb and c-myc genes, suggesting that c-myb and c-myc may be among the kappaB-containing genes regulated by NF-kappaB factors in normal erythroid cells. Taken together, these data show that NF-kappaB factors are modulated by GM-CSF and suggest they function to regulate specific kappaB containing genes involved in erythropoiesis.

Cell Division↗