Characterization of HOX Gene Expression During Myelopoiesis: Role of HOX A5 in Lineage Commitment and Maturation

During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.

Download Full-text

Regulation of erythropoiesis in long-term hamster marrow cultures: role of bone marrow-adherent cells

Blood ◽

10.1182/blood.v65.3.736.bloodjournal653736 ◽

1985 ◽

Vol 65 (3) ◽

pp. 736-743

Author(s):

CE Eastment ◽

FW Ruscetti

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Erythroid Differentiation ◽

Adherent Cells ◽

Erythroid Progenitors ◽

Suspension Cells ◽

Liquid Suspension ◽

Adherent Culture ◽

Macrophage Colony

The initial establishment of hamster long-term bone marrow (LTBM) cultures requires formation of an adherent stromal layer, but continued long-term proliferation of these cultures is best accomplished by removal of the suspension cells from the adherent layer and subsequent incubation in liquid suspension culture. Continued maintenance of bone marrow cells in the presence of the adherent layer for more than four to six weeks leads to a decline and eventual disappearance of erythroid and multipotent colony-forming cells. Addition of erythropoietin to LTBM suspension cultures produces mature, hemoglobinized erythrocytes. Incubation of the same cells plus erythropoietin in the presence of autologous parental adherent layers markedly inhibits both terminal erythroid differentiation and the number of detectable erythroid burst- forming units (BFU-Es). This erythroid inhibition occurs primarily within the first 24 hours with little or no effect on CFU-GEMMs or granulocyte-macrophage colony-forming units (GM-CFUs). However, continued incubation for seven days produces a reduction in all parameters. Removal of suspension cells from the adherent layer and restimulation with erythropoietin allows regeneration of erythropoiesis. Pretreatment of suspension cells with erythropoietin for 96 hours before exposure to the adherent culture only slightly inhibits erythropoiesis, suggesting that more mature erythroid progenitors are unaffected. Conditioned medium from the marrow adherent layer (ALCM) produces similar erythroid inhibitory effects in LTBM cultures with as little as two hours of incubation. The inhibition is actively produced by the adherent cells, since cycloheximide abolishes its production, while indomethacin has no apparent effect. Adherent marrow stromal cells may regulate hemopoiesis through negative as well as positive humoral signals, and they are particularly effective in erythroid regulation.

Download Full-text

Regulation of erythropoiesis in long-term hamster marrow cultures: role of bone marrow-adherent cells

Blood ◽

10.1182/blood.v65.3.736.736 ◽

1985 ◽

Vol 65 (3) ◽

pp. 736-743 ◽

Cited By ~ 1

Author(s):

CE Eastment ◽

FW Ruscetti

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Erythroid Differentiation ◽

Adherent Cells ◽

Erythroid Progenitors ◽

Suspension Cells ◽

Liquid Suspension ◽

Adherent Culture ◽

Macrophage Colony

Abstract The initial establishment of hamster long-term bone marrow (LTBM) cultures requires formation of an adherent stromal layer, but continued long-term proliferation of these cultures is best accomplished by removal of the suspension cells from the adherent layer and subsequent incubation in liquid suspension culture. Continued maintenance of bone marrow cells in the presence of the adherent layer for more than four to six weeks leads to a decline and eventual disappearance of erythroid and multipotent colony-forming cells. Addition of erythropoietin to LTBM suspension cultures produces mature, hemoglobinized erythrocytes. Incubation of the same cells plus erythropoietin in the presence of autologous parental adherent layers markedly inhibits both terminal erythroid differentiation and the number of detectable erythroid burst- forming units (BFU-Es). This erythroid inhibition occurs primarily within the first 24 hours with little or no effect on CFU-GEMMs or granulocyte-macrophage colony-forming units (GM-CFUs). However, continued incubation for seven days produces a reduction in all parameters. Removal of suspension cells from the adherent layer and restimulation with erythropoietin allows regeneration of erythropoiesis. Pretreatment of suspension cells with erythropoietin for 96 hours before exposure to the adherent culture only slightly inhibits erythropoiesis, suggesting that more mature erythroid progenitors are unaffected. Conditioned medium from the marrow adherent layer (ALCM) produces similar erythroid inhibitory effects in LTBM cultures with as little as two hours of incubation. The inhibition is actively produced by the adherent cells, since cycloheximide abolishes its production, while indomethacin has no apparent effect. Adherent marrow stromal cells may regulate hemopoiesis through negative as well as positive humoral signals, and they are particularly effective in erythroid regulation.

Download Full-text

Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity

Pharmaceuticals ◽

10.3390/ph14050459 ◽

2021 ◽

Vol 14 (5) ◽

pp. 459

Author(s):

Mariya Pykhtina ◽

Svetlana Miroshnichenko ◽

Vladimir Romanov ◽

Antonina Grazhdantseva ◽

Galina Kochneva ◽

...

Keyword(s):

Bone Marrow Cells ◽

E Coli ◽

Gm Csf ◽

Human Apolipoprotein ◽

Proliferative Effect ◽

Erythroleukemia Cells ◽

Blast Cells ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

Download Full-text

Identification of an erythropoietin-sensitive cell line

Blood ◽

10.1182/blood.v69.6.1782.bloodjournal6961782 ◽

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.

Download Full-text

Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽

10.1182/blood.v93.8.2578 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2578-2585 ◽

Cited By ~ 80

Author(s):

Carinne Lecoq-Lafon ◽

Frédérique Verdier ◽

Serge Fichelson ◽

Stany Chrétien ◽

Sylvie Gisselbrecht ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Intracellular Signaling ◽

Direct Consequence ◽

Receptor Activation ◽

Phosphatidylinositol 3 Kinase ◽

Erythroid Progenitors ◽

Epo Receptor ◽

Gm Csf ◽

Macrophage Colony ◽

Phosphatidylinositol 3

Abstract Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

Download Full-text

Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1

Blood ◽

10.1182/blood.v73.2.375.375 ◽

1989 ◽

Vol 73 (2) ◽

pp. 375-380 ◽

Cited By ~ 102

Author(s):

T Kitamura ◽

A Tojo ◽

T Kuwaki ◽

S Chiba ◽

K Miyazono ◽

...

Keyword(s):

Cell Line ◽

Binding Sites ◽

Bone Marrow Cells ◽

Scatchard Analysis ◽

Hematopoietic Growth Factors ◽

Single Class ◽

Gm Csf ◽

Molecular Weights ◽

Human Erythropoietin ◽

Macrophage Colony

Abstract We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

Download Full-text

Mcl-1 Expression in Human Neutrophils: Regulation by Cytokines and Correlation With Cell Survival

Blood ◽

10.1182/blood.v92.7.2495 ◽

1998 ◽

Vol 92 (7) ◽

pp. 2495-2502 ◽

Cited By ~ 240

Author(s):

Dale A. Moulding ◽

Julie A. Quayle ◽

C. Anthony Hart ◽

Steven W. Edwards

Keyword(s):

Acute Inflammation ◽

Molecular Mechanisms ◽

Human Neutrophils ◽

Potential Mechanism ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Regulated Gene Expression ◽

Neutrophil Survival ◽

Stimulating Factor

Abstract Human neutrophils possess a very short half-life because they constitutively undergo apoptosis. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), and other agents can rescue neutrophils from apoptosis but the molecular mechanisms involved in this rescue are undefined. Here, we show by Western blotting that human neutrophils do not express Bcl-2 or Bcl-X but constitutively express Bax. However, cellular levels of these proteins are unaffected by agents which either accelerate or delay neutrophil apoptosis. In contrast, neutrophils express the antiapoptotic protein Mcl-1 and levels of this protein correlate with neutrophil survival. Thus, cellular levels of Mcl-1 decline as neutrophils undergo apoptosis and are enhanced by agents (eg, GM-CSF, interleukin-1β, sodium butyrate, and lipopolysaccharide) that promote neutrophil survival. Neutrophils only possess few, small mitochondria, and much of the Mcl-1 protein seems to be located in nuclear fractions. These observations provide the first evidence implicating a Bcl-2 family member in the regulation of neutrophil survival. Moreover, this work also provides a potential mechanism whereby cytokine-regulated gene expression regulates the functional lifespan of neutrophils and hence their ability to function for extended time periods during acute inflammation.

Download Full-text

Immunostimulatory Effects of Live Lactobacillus sakei K040706 on the CYP-Induced Immunosuppression Mouse Model

Nutrients ◽

10.3390/nu12113573 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3573

Author(s):

Seo-Yeon Kim ◽

Ji-Sun Shin ◽

Kyung-Sook Chung ◽

Hee-Soo Han ◽

Hwi-Ho Lee ◽

...

Keyword(s):

Cell Proliferation ◽

Molecular Mechanisms ◽

Nk Cell ◽

Cell Activity ◽

Lactobacillus Sakei ◽

Gm Csf ◽

Positive Effects ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Immunosuppressed Mice

Our previous studies have shown that heat-killed Lactobacillus sakei K040706 exerts immunostimulatory and anti-inflammatory activities in macrophages, cyclophosphamide (CYP)-treated mice, and dextran sulfate sodium–induced colitis mice. However, the immunostimulatory effects of live Lactobacillus sakei K040706 (live K040706) against CYP-induced immunosuppression and its underlying molecular mechanisms remain unknown. Therefore, we investigated the immunostimulatory effects of live K040706 (108 or 109 colony forming unit (CFU)/day, p.o.) in CYP-induced immunosuppressed mice. Oral administration of live K040706 prevented the CYP-induced decreases in body weight, thymus index, natural killer (NK) cell activity, T and B cell proliferation, and cytokine (interferon (IFN)-γ, interleukin (IL)-2, and IL-12) production. The administration of live K040706 also exerted positive effects on the gut microbiota of CYP-induced mice, resulting in a microbiota composition similar to that of normal mice. Moreover, live K040706 significantly enhanced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the splenocytes and Peyer’s patch (PP) cells of mice and increased bone marrow (BM) cell proliferation. Taken together, our data indicate that live K040706 may effectively accelerate recovery from CYP-induced immunosuppression, leading to activation of the immune system. Therefore, live K040706 may serve as a potential immunomodulatory agent against immunosuppression.

Download Full-text

Identification of an erythropoietin-sensitive cell line

Blood ◽

10.1182/blood.v69.6.1782.1782 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1782-1785 ◽

Cited By ~ 23

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.

Download Full-text

Characterization of a novel form of transferrin receptor preferentially expressed on normal erythroid progenitors and precursors

Blood ◽

10.1182/blood.v73.1.214.214 ◽

1989 ◽

Vol 73 (1) ◽

pp. 214-221 ◽

Cited By ~ 23

Author(s):

T Cotner ◽

AD Gupta ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Transferrin Receptor ◽

Bone Marrow Cells ◽

Single Gene ◽

Cell Types ◽

Surface Proteins ◽

Antigenic Determinants ◽

Transferrin Receptors ◽

Erythroid Progenitors ◽

Iron Requirement

Abstract A panel of monoclonal antibodies (MoAbs) against cell surface proteins of early BFUe progeny was characterized. Five of these antibodies (Abs) reacted with normal erythroid, but not myeloid, bone marrow cells. Each of the five antibodies, typified by Ab 69.20, immunoprecipitated a dimeric complex of 185,000, which is composed of two identical disulfide-bonded subunits. This antigen had affinity for transferrin, and was essentially identical in biochemical characteristics to transferrin receptors precipitated with the well-characterized MoAbs OKT9 and 5E9. However, this form of transferrin receptor lacked both the OKT9 and 5E9 antigenic determinants and, moreover, the 69.20 epitope was absent from the conventional transferrin receptor, as defined by Abs OKT9 and 5E9. Modulation experiments demonstrated that both 69.20 and OKT9 modulated large, virtually independent populations of transferrin receptors. Both forms of transferrin receptor appeared to be derived from the product of a single gene, but the form defined by MoAb 69.20 apparently predominates in cells of the erythroid lineage and some transformed cell types that manifest a special requirement for iron. These data suggest that cells with a high iron requirement synthesize two forms of transferrin receptor, possibly by means of differential mRNA splicing or by posttranslational modification of the transferrin receptor.

Download Full-text