losartan-potassium and Leukemia--Erythroblastic--Acute

Topics: Aged; Erythropoietin; Fatal Outcome; Hematopoiesis, Extramedullary; Humans; Iron Overload; Leukemia, Erythroblastic, Acute; Male; Postoperative Complications; Primary Myelofibrosis; Spleen; Splenectomy; Splenomegaly; Transfusion Reaction

The production of erythroid cells is a dynamic and exquisitely regulated process. The mature red cell is only the final phase of a complex but orderly series of genetic events that are initiated at the time a multipotent stem cell becomes committed to expressing the erythroid programme. Aberrations either in the intrinsic generation and/or amplification of functional erythroid cells or in the regulatory influences of microenvironment or cytokines form the basis for a number of blood diseases. In this review we focus upon abnormalities in red blood cell production and discuss how alterations in cytokine regulation of red blood cell production may contribute to these disease processes. We discuss clinical states in which blood red cell numbers are altered, including primary familial and congenital polycythaemia, the myeloproliferative disorder polycythaemia vera, erythroleukaemia, and Diamond-Blackfan anaemia. These disorders are briefly described and evidence supporting a potential role of specific cytokine receptor signalling defects as contributing to these phenotypes is discussed.

Topics: Erythropoietin; Fanconi Anemia; Hematologic Diseases; Hematopoiesis; Humans; Leukemia, Erythroblastic, Acute; Mutation; Polycythemia; Polycythemia Vera; Receptors, Cytokine; Receptors, Erythropoietin; Signal Transduction

The Friend spleen focus-forming virus has been a valuable tool for understanding the molecular events involved in the multiple stages of leukaemia. As summarized in Figure 3, the primary effect of SFFV, which occurs within days, is to cause a polyclonal proliferation of erythroid precursor cells that can proliferate in the absence of their normal regulator erythropoietin. This is the direct result of the unique envelope glycoprotein encoded by SFFV, which is transported to the cell surface and apparently interacts with the EpoR or another component of the multimeric EpoR complex, resulting in the constitutive activation of the Epo signal transduction pathway. Within this proliferating population of erythroid cells is a rare cell that has undergone several genetic changes due to the integration of the viral genome in specific sites in the mouse DNA. This leads to the activation of a gene encoding the PU.1 transcription factor, whose high expression in erythroid cells may be the cause of the block in differentiation that is characteristic of SFFV-transformed erythroid cells. SFFV integration can also lead to the inactivation of the p53 tumour supressor gene, giving these cells a growth advantage in the mouse. The disease induced by SFFV in mice is very similar to polycythaemia vera in humans (Golde et al, 1981). The major clinical feature of polycythaemia vera is the continuous expansion of the number of mature red blood cells in the presence of low serum Epo levels. Also, BFU-E and CFU-E from these patients can form in the absence of Epo like the analogous cells from SFFV-infected mice (Casadevall et al, 1982). It is possible that haematopoietic cells from individuals suffering from this disease express a protein similar to the envelope glycoprotein of SFFV that can interact with the EpoR and lead to its constitutive activation. Alternatively, these patients may contain a mutant EpoR gene that is constitutively activated like the mutant EpoR described earlier. As we understand more fully how the SFFV envelope protein constitutively activates te EpoR complex, we can begin to design therapies to counteract its action that can then be applied to treating patients with polycythaemia vera or other human diseases associated with uncontrolled erythropoiesis.

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Defective Viruses; DNA-Binding Proteins; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Genes, env; Genome, Viral; Helper Viruses; Hyperplasia; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mutagenesis, Insertional; Receptors, Erythropoietin; Retroviridae Infections; Retroviridae Proteins, Oncogenic; Signal Transduction; Spleen Focus-Forming Viruses; Tumor Virus Infections; Viral Envelope Proteins; Virus Replication

Topics: Animals; Cell Line; Erythrocytes; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Receptors, Erythropoietin; Signal Transduction; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; Endoplasmic Reticulum; Erythropoietin; Friend murine leukemia virus; Gene Products, env; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Receptors, Erythropoietin; Viral Envelope Proteins

Molecular analysis of the human erythropoietin receptor (EpoR) promises to yield a greater mechanistic understanding of erythropoiesis and disease states that affect red cell production. The cloned receptor molecule is a 66 kDa membrane protein that is structurally related to a large superfamily of haemopoietin/growth factor receptors. The 66 kDa EpoR alone is capable of binding to erythropoietin (Epo) with nanomolar affinity. The native EpoR may form dimers before or after binding Epo. EpoR dimers and/or associated molecules are probably necessary for high-affinity Epo binding. The 66 kDa EpoR probably exists as a protein complex with as yet unidentified proteins of 100 and 85 kDa. The molecular mechanism of Epo signal transduction remains largely undefined. The possible role of the EpoR in human diseases has been studied in a variety of clinical conditions. A structurally abnormal EpoR gene has been identified in a human erythroleukemia cell line. In polycythemia vera, red cell progenitors exhibit exaggerated sensitivity to Epo and express only low-affinity EpoR. Some cases of hereditary polycythemia may be due to a mutant EpoR conferring enhanced Epo sensitivity. Other pathologic conditions may also be associated with abnormalities of the EpoR or its associated molecules. Soluble, immunoreactive EpoR is detectable in human serum, but its physiological significance is unknown.

Topics: Animals; Cloning, Molecular; Cross-Linking Reagents; Erythropoiesis; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Polycythemia; Protein Conformation; Receptors, Erythropoietin; Red-Cell Aplasia, Pure

Topics: Amino Acid Sequence; Animals; DNA; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Receptors, Erythropoietin; Signal Transduction; Spleen Focus-Forming Viruses; Viral Envelope Proteins

Topics: Anemia; Animals; Cell Line; Endocytosis; Erythroid Precursor Cells; Erythropoietin; Friend murine leukemia virus; Glycosylation; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Protein Binding; Protein Processing, Post-Translational; Receptors, Cell Surface; Receptors, Erythropoietin; Tumor Cells, Cultured

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Erythropoietin; Friend murine leukemia virus; Humans; Leukemia, Erythroblastic, Acute; Lymphokines; Tissue Inhibitor of Metalloproteinases

Topics: Anemia, Pernicious; Cell Differentiation; Cell Separation; Cells, Cultured; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Iron; Leukemia, Erythroblastic, Acute

Topics: Animals; Cell Division; Cortisone; Culture Techniques; Endocrine Glands; Erythropoietin; Glycoproteins; Gonadal Steroid Hormones; Growth Hormone; Growth Inhibitors; Hematopoiesis; Hematopoietic Stem Cells; Hormones; Humans; Leukemia; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Neurotransmitter Agents; Radiation Chimera; Remission, Spontaneous; Thrombopoietin; Thymus Gland

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2

Topics: Amino Acid Sequence; Cell Differentiation; Cell Line, Tumor; Clone Cells; ErbB Receptors; Erythroblasts; Erythropoietin; Gene Expression; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mutation; Polycythemia Vera; Primary Myelofibrosis; Protein Multimerization; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction

Expression levels of MIR144 and MIR451 increase during erythropoiesis, a pattern that is conserved from zebrafish to humans. As these two miRs are expressed from the same polycistronic transcript, we manipulated MIR144 and MIR451 in human erythroid cells individually and together to investigate their effects on human erythropoiesis. Inhibition of endogenous human MIR451 resulted in decreased numbers of erythroid (CD71(hi) CD235a(hi) CD34(-) ) cells, consistent with prior studies in zebrafish and mice. In addition, inhibition of MIR144 impaired human erythroid differentiation, unlike in zebrafish and mouse studies where the functional effect of MIR144 on erythropoiesis was minimal. In this study, we found RAB14 is a direct target of both MIR144 and MIR451. As MIR144 and MIR451 expression increased during human erythropoiesis, RAB14 protein expression decreased. Enforced RAB14 expression phenocopied the effect of MIR144 and/or MIR451 depletion, whereas shRNA-mediated RAB14 knockdown protected cells from MIR144 and/or MIR451 depletion-mediated erythropoietic inhibition. RAB14 knockdown increased the frequency and number of erythroid cells, increased β-haemoglobin expression, and decreased CBFA2T3 expression during human erythropoiesis. In summary, we utilized MIR144 and MIR451 to identify RAB14 as a novel physiological inhibitor of human erythropoiesis.

Topics: Cell Line, Tumor; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lentivirus; Leukemia, Erythroblastic, Acute; MicroRNAs; rab GTP-Binding Proteins; Recombinant Proteins; Repressor Proteins; RNA Interference; RNA, Small Interfering; Transduction, Genetic; Tumor Suppressor Proteins

Erythropoiesis is the process of proliferation, differentiation, and maturation of erythroid cells. Understanding these steps will help to elucidate the basis of specific diseases associated with abnormal production of red blood cells. In this study, we continued our efforts to identify genes involved in erythroid proliferation. Lentivirally transduced UT-7/Epo erythroleukemic cells expressing ribosomal protein L11 (RPL11) or retinol dehydrogenase 11 (RDH11) could proliferate in the absence of erythropoietin, and their cell-cycle profiles revealed G0/G1 prolongation and low percentages of apoptosis. RPL11-expressing cells proliferated more rapidly than the RDH11-expressing cells. The antiapoptotic proteins BCL-XL and BCL-2 were expressed in both cell lines. Unlike the parental UT-7/Epo cells, the expression of hemoglobins (Hbs) in the transduced cells had switched from adult to fetal type. Several signal transduction pathways, including STAT5, were highly activated in transduced cells; furthermore, expression of the downstream target genes of STAT5, such as CCND1, was upregulated in the transduced cells. Taken together, the data indicate that RPL11 and RDH11 accelerate erythroid cell proliferation by upregulating the STAT5 signaling pathway with phosphorylation of Lyn and cyclic AMP response element-binding protein (CREB).

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Erythroid Cells; Erythropoietin; Gene Expression; HEK293 Cells; Humans; Immunohistochemistry; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Oxidoreductases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; src-Family Kinases; STAT5 Transcription Factor

A 31-year-old male patient presented with fever and pancytopenia. He was diagnosed as a case of chronic anemia since early childhood. The etiology of the anemia was not known. The patient was transfusion dependent, and he had been maintained on erythropoietin for three years prior to admission. A bone marrow examination revealed prominent proliferation of immature and dysplastic erythroid precursors. Acute erythroid leukemia of the pure erythroid subtype was suspected. However, because of the history of erythropoietin therapy a definite diagnosis was not made. On follow-up one month later, the marrow changes had reversed to normal.

Topics: Adult; Biopsy; Bone Marrow; Erythroblasts; Erythropoietin; Follow-Up Studies; Humans; Leukemia, Erythroblastic, Acute; Male; Staining and Labeling; Vacuoles

Recombinant erythropoietin (rHuEPO) is used extensively to treat anaemia associated with chronic kidney disease. However, the development of neutralizing antibodies (NAbs) to rHuEPO can result in the development of antibody-mediated pure red cell aplasia (PRCA). The detection of NAb in patient sera by in vitro bioassay relies on the inhibition of a cellular response to rHuEPO. Current bioassays for rHuEPO measure proliferation in responsive cell lines such as the erythroleukaemic cell lines, UT-7 and UT-7/EPO, the latter sensitized to EPO. Using these cell lines, we show the dose-responsive induction of both PIM1 and EGR1 gene expression in UT-7 cells and of EGR1 in UT-7/EPO cells. The expression of EGR1 in UT-7/EPO cells in response to rHuEPO was comparable to the proliferative response measured by (3)H-thymidine incorporation and could be inhibited by serum from a patient with NAb-mediated PRCA in a dilution-dependent manner. Bioassays based on the induction of endogenous gene expression are comparable to current bioassays but are considerably quicker given that incubation time is decreased from 2-3 days to 50 min. Measurement of EGR1 gene expression in response to rHuEPO in UT-7/EPO cells offers a rapid, non-radioactive and automatable alternative to current assays for the detection of rHuEPO NAbs.

Topics: Animals; Antibodies, Neutralizing; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Early Growth Response Protein 1; Erythropoietin; Gene Expression; Humans; Immune Sera; Immunologic Techniques; Leukemia, Erythroblastic, Acute; Recombinant Proteins; Red-Cell Aplasia, Pure; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Serum; Sheep; Time Factors

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.

Topics: alpha-Globins; Aminolevulinic Acid; Butyric Acid; Cell Differentiation; Cell Proliferation; Erythroid Cells; Erythropoiesis; Erythropoietin; Ferrochelatase; Glycophorins; Heme; Hemoglobins; Humans; Hydrolysis; Hydroxymethylbilane Synthase; K562 Cells; Leukemia, Erythroblastic, Acute; Levulinic Acids; Prodrugs; RNA, Messenger

Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

Topics: Azepines; Cell Line, Tumor; Cell Proliferation; Erythroid Cells; Erythropoiesis; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Triazoles

We evaluated the molecular mechanism of telomerase activation by erythropoietin (EPO) in human erythroleukemic JAS-REN-A cells. Telomerase activity increased 3-4 fold after 3-24h of culture with EPO and was associated with increases in c-myc mRNA after 1-3h, of c-Myc protein after 3-6h, and of human telomerase reverse transcriptase (hTERT) mRNA and hTERT protein after 6-24h. Simultaneously EPO induced phosphorylation of signal transducer activator of transcription 5 (STAT5), AKT, and extracellular signal-regulated kinase (ERK). Telomerase activity induced by EPO was significantly inhibited by AG490, PD98059, and LY294002. AG490 downregulated c-myc and hTERT mRNA expression with inhibited STAT5 and AKT phosphorylation. PD98059 also reduced c-myc and hTERT expression and inhibited ERK phosphorylation. However, LY294002 did not inhibit c-myc or hTERT mRNA expression despite inhibiting STAT5 and AKT phosphorylation. These results suggest that EPO activates telomerase in JAS-REN-A cells through dual regulation: hTERT gene transcription by Janus tyrosine kinase 2/STAT5/c-Myc and hTERT protein phosphorylation by phosphatidylinositol 3'-kinase/AKT.

Topics: Blotting, Western; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Erythroblastic, Acute; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Processing, Post-Transcriptional; RNA, Messenger; Signal Transduction; Telomerase; Transcription, Genetic; Tumor Cells, Cultured

Erythroid differentiation of human erythroleukemia cell line K562 induced by erythropoietin is a complex process that involves modifications at nuclear level, including nuclear translocation of phosphatidyl-inositol 3-kinase. In this work we show that erythropoietin stimulation of K562 cells can induce nuclear translocation of active Akt, a downstream molecule of the phosphatidyl-inositol 3-kinase signaling pathway. Akt shows a peak of activity in whole cell homogenates at earlier stage when compared to the nucleus, which shows a peak delayed of 10 min. Akt increases its intranuclear amount and activity rapidly and transiently in response to EPO. Almost all Akt kinase that translocates to the nucleus shows a marked phosphorylation on serine 473. Nuclear enzyme translocation is blocked by the phosphatidyl-inositol 3-kinase inhibitor Ly294002 or Wortmannin. The specific Akt pharmacological inhibitor VI, VII and VIII that act as blocking enzyme activation inhibited translocation as well, whereas Akt inhibitor IX, that inhibits Akt activity, did not block Akt nuclear translocation. When cells were treated by means of siRNA sequences or with the Akt inhibitors the differentiation process was arrested, thus showing the requirement of the nuclear translocation of the active enzyme to differentiate. These findings strongly suggest that the intranuclear translocation of active Akt kinase represents an important step in the signaling pathway that mediates erythropoietin-induced erythroid differentiation.

Topics: Active Transport, Cell Nucleus; Androstadienes; Cell Differentiation; Cell Lineage; Cell Nucleus; Chromones; Erythrocytes; Erythropoietin; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Wortmannin

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Topics: Alleles; Animals; Cell Line, Tumor; Cells, Cultured; Colony-Forming Units Assay; Drug Evaluation, Preclinical; Erythropoietin; Humans; Inhibitory Concentration 50; Janus Kinase 1; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Mutation; Oncogene Proteins, Fusion; Polycythemia Vera; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptors, Thrombopoietin; STAT Transcription Factors; Substrate Specificity

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

Topics: Active Transport, Cell Nucleus; Anemia; Animals; Binding Sites; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fibroblasts; Glycophorins; Humans; Kidney; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Polypyrimidine Tract-Binding Protein; Protein Structure, Tertiary; Recombinant Proteins; RNA Interference; RNA-Binding Proteins; Skin; Thrombospondins; Time Factors; Transduction, Genetic; Zidovudine

In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36P-Gab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2-SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Cell Line, Tumor; Cell Proliferation; Chromones; Enzyme Inhibitors; Erythroid Cells; Erythropoietin; Gene Knockdown Techniques; GRB2 Adaptor Protein; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Phosphatidylinositol 3-Kinases; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Receptors, Erythropoietin; SOS1 Protein

The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been linked to inflammation- and cancer-related anemia, which reduces both quality of life and prognosis of patients. The aim of this study was to reveal molecular mechanisms linked to the inhibition of erythroid differentiation by TNFalpha. In this study, we showed that the inhibition of erythropoietin (Epo)-mediated differentiation by TNFalpha lead to a downregulation of hemoglobin synthesis and was correlated to a modulation of key erythroid transcription factors. Thus, a reverse of the transcription factor GATA-1/GATA-2 balance normally present during erythropoiesis, as well as a downregulation of the cofactor of GATA-1, friend of GATA-1 (FOG-1), and the coregulating transcription factor nuclear factor erythroid 2 (NF-E2) was observed after TNFalpha treatment. Moreover, we showed a reduction of GATA-1/FOG-1 interaction due to a reduced transcription of GATA-1 and a proteasome-dependent FOG-1 degradation after TNFalpha treatment. These changes led to an inhibition of erythroid gene expression including Epo receptor (EpoR), alpha- and gamma-globin, erythroid-associated factor (ERAF), hydroxymethylbilane synthetase (HMBS), and glycophorin A (GPA). An analysis of distinct signaling pathway activations then revealed an activation of p38 by TNF, as well as a corresponding involvement of this mitogen-activated protein kinase (MAPK) in the cytokine-dependent inhibition of erythroid differentiation. Indeed the p38 inhibitor, SB203580, abrogated the inhibitory effect of TNFalpha on the major erythroid transcription factor GATA-1 as well as erythroid marker expression in Epo-induced TF-1 cells. Overall, these data contribute to a better understanding of cytokine-dependent anemia, by giving first hints about key erythroid transcription factor modulations after TNFalpha treatment as well as an involvement of p38 in the inhibition of erythroid differentiation.

Topics: Cell Differentiation; Cell Line, Tumor; Down-Regulation; Erythropoiesis; Erythropoietin; GATA1 Transcription Factor; GATA2 Transcription Factor; Humans; Leukemia, Erythroblastic, Acute; MAP Kinase Signaling System; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Transcription Factors; Tumor Necrosis Factor-alpha; Up-Regulation

HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.

Topics: Cell Division; Cell Line, Tumor; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; G1 Phase; Histocompatibility Antigens Class I; HLA Antigens; HLA-G Antigens; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Microspheres; Mutation, Missense; Phosphorylation; Point Mutation; Polycythemia Vera; Protein Processing, Post-Translational; Receptors, Erythropoietin; Recombinant Fusion Proteins; STAT3 Transcription Factor; STAT5 Transcription Factor

Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes up-regulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1-mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching.

Topics: Apoptosis Regulatory Proteins; Cell Cycle; Cell Differentiation; Cell Line; Cell Line, Tumor; DNA Primers; Erythropoietin; GATA1 Transcription Factor; Globins; Humans; Leukemia, Erythroblastic, Acute; Nuclear Proteins; Transcription Factors

The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.

Topics: Artifacts; Blotting, Western; Cell Line; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Erythropoietin; False Positive Reactions; Flow Cytometry; Humans; Janus Kinases; Leukemia, Erythroblastic, Acute; Phosphorylation; Receptors, Erythropoietin; Reproducibility of Results; STAT5 Transcription Factor

Overexpression of the transcription factor Spi-1/PU.1 in mice leads to acute erythroleukemia characterized by a differentiation block at the proerythroblastic stage. In this study, we made use of a new cellular system allowing us to reach graded expression of Spi-1 in preleukemic cells to dissect mechanisms of Spi-1/ PU-1 in erythroleukemogenesis. This system is based on conditional production of 1 or 2 spi-1-interfering RNAs stably inserted into spi-1 transgenic proerythroblasts. We show that Spi-1 knock-down was sufficient to reinstate the erythroid differentiation program. This differentiation process was associated with an exit from the cell cycle. Evidence is provided that in the presence of erythropoietin (Epo), Spi-1 displays an antiapoptotic role that is independent of its function in blocking erythroid differentiation. Apoptosis inhibited by Spi-1 did not involve activation of the Fas/FasL signaling pathway nor a failure to activate Epo receptor (EpoR). Furthermore, we found that reducing the Spi-1 level yields to ERK dephosphorylation and increased phosphorylation of AKT and STAT5, suggesting that Spi-1 may affect major signaling pathways downstream of the EpoR in erythroid cells. These findings reveal 2 distinct roles for Spi-1 during erythroleukemogenesis: Spi-1 blocks the erythroid differentiation program and acts to impair apoptotic death in cooperation with an Epo signaling.

Topics: Animals; Apoptosis; Base Sequence; Cell Cycle; Cell Differentiation; Erythroblasts; Erythropoiesis; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Receptors, Erythropoietin; RNA, Small Interfering; Signal Transduction; Trans-Activators

MicroRNAs (miRNAs) are a newly discovered class of posttranscriptional regulatory noncoding small RNAs. Recent evidence has shown that miRNA misexpression correlates with progression of various human cancers. Friend erythroleukemia has been used as an excellent system for the identification and characterization of oncogenes and tumor suppressor genes involved in neoplastic transformation. Using this model, we have isolated a novel integration site designated Fli-3, from a Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. The Fli-3 transcription unit is a murine homologue of the human gene C13orf25 that includes a region encoding the mir-17-92 miRNA cluster. C13orf25 is the target gene of 13q31 chromosomal amplification in human B-cell lymphomas and other malignancies. The erythroleukemias that have acquired either insertional activation or amplification of Fli-3 express higher levels of the primary or mature miRNAs derived from mir-17-92. The ectopic expression of Fli-3 in an erythroblastic cell line switches erythropoietin (Epo)-induced differentiation to Epo-induced proliferation through activation of the Ras and PI3K pathways. Such a response is associated with alteration in the expression of several regulatory factors, such as Spi-1 and p27 (Kip1). These findings highlight the potential of the Fli-3 encoding mir-17-92 in the development of erythroleukemia and its important role in hematopoiesis.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Proliferation; Erythropoietin; Friend murine leukemia virus; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; Humans; Leukemia, Erythroblastic, Acute; Mice; MicroRNAs; Multigene Family; Mutagenesis, Insertional; Transcription, Genetic; Viral Proteins

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistep process. At disease onset, preleukemic cells are arrested in differentiation at the proerythroblast stage (HS1 stage) and their survival and growth are under the tight control of erythropoietin (Epo). During disease progression, malignant proerythroblasts characterized by Epo autonomous growth and in vivo tumorigenicity can be isolated (HS2 stage). During analysis of transcriptional profiling representive of discrete stages of leukemic progression, we found that the phosphatidylinositol 4-phosphatase type II gene was turned off in malignant cells. PI-4-phosphatase II is an enzyme that hydrolyses the 4-phosphate position of phosphatidylinositol-3-4-bisphosphate (PtdIns(3,4)P(2)) to form PtdIns(3)P. Using malignant cells engineered to stably express PI-4-phosphatase II, we showed that PI-4-phosphatase II reduced Akt activation level. Moreover, stimulation of malignant cells with Epo-induced PI-4-phosphatase II transcription pointing this gene as an Epo-responsive gene. This study provides first insight for a physiological role of PI-4-phosphatase II in the proerythroblast by controlling Epo responsiveness through a negative regulation of the PI3K/Akt pathway.

Topics: Animals; Blotting, Northern; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Erythroblasts; Erythropoietin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

Normal and malignant hematopoietic cells are shown to express and secrete various cytokines and chemokines, some of which are believed to play an important role in normal and abnormal hematopoiesis in an autocrine/paracrine manner. To explore the possibility of a cytokine/chemokine network participating in the pathophysiology of anemic disorders, we evaluated the ability of inflammatory cytokines to induce chemokine expression using erythroid progenitor cells.. Erythropoietin-dependent human leukemia cell line AS-E2 was used as a model of erythroid colony-forming unit (CFU-E) cells. The expression of mRNA of 8 chemokines was examined using RT-PCR, before and after TNF-alpha, IFN-gamma, and IL-1beta stimulation. For MIP-3alpha, the promoter activity was analyzed by luciferase assay and secretion was confirmed by ELISA. The expression of CCR6, the specific receptor for MIP-3alpha, was analyzed by RT-PCR and flow cytometry.. Unstimulated AS-E2 cells constitutively expressed transcripts for MCP-4, IP-10, PF-4, IL-8, and MIP-3alpha. Stimulation with TNF-alpha, IFN-gamma, and IL-1beta upregulated MIP-3alpha mRNA expression and induced its protein secretion. Luciferase assay revealed that these cytokines could upregulate promoter activity of the MIP-3alpha gene, possibly through the NF-kappaB pathway. CCR6 mRNA was detected and its intracellular expression was confirmed.. These data suggest that inflammatory cytokine-stimulated erythroid progenitors secrete MIP-3alpha, which may function in an autocrine/paracrine manner. Furthermore, the existence of intracellular CCR6 suggests the involvement in cytokine signaling of a MIP-3alpha-dependent internal autocrine mechanism. These mechanisms may play a role in pathophysiology of anemic disorders, such as secondary anemia and bone marrow failure syndromes.

Topics: Cell Line, Tumor; Chemokine CCL20; Chemokines; Chemokines, CC; Cytokines; Enzyme-Linked Immunosorbent Assay; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Humans; Inflammation; Interleukin-1; Leukemia, Erythroblastic, Acute; Macrophage Inflammatory Proteins; Promoter Regions, Genetic; Receptors, CCR6; Receptors, Chemokine; RNA, Messenger; Tumor Necrosis Factor-alpha

Erythropoietin (EPO) therapy is widely used for the prevention and treatment of anemia resulting from cancer chemotherapy. Native EPO regulates erythropoiesis, at least in part, by protecting erythroid progenitor cells from apoptotic cell death. The recent discovery of the EPO receptor (EPOR) on cancer cells raises the concern that EPO therapy might stimulate tumor growth and/or protect cancer cells from drug-induced apoptosis. Therefore, the capacity of EPO to interfere with the effects of conventional chemotherapeutic drugs on proliferation, apoptosis, and the induction of senescence was investigated in MCF-7 and MDA-MB231 breast tumor cells, which express the EPOR as well as in F-MEL erythroleukemia cells.. Breast cancer cells and F-MEL leukemic cells were cultured in the presence or absence of EPO and then exposed to antitumor drugs. Cell proliferation was assessed by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay 72 hours after drug exposure. Cytotoxicity was monitored by clonogenic survival. Apoptosis was evaluated either by the terminal deoxyribonucleotide transferase-mediated nick-end labeling assay or fluorescence-activated cell sorting analysis, and senescence was monitored by beta-galactosidase staining. EPO signaling was assessed by monitoring the phosphorylation/activation of specific signaling proteins.. EPO failed to stimulate the proliferation of MCF-7 or MDA-MB231 breast tumor cells or F-MEL leukemic cells. EPO treatment also failed to interfere with the antiproliferative and/or cytotoxic effects of Adriamycin, Taxol, and tamoxifen in breast tumor cells (or of cytarabine and daunorubicin in F-MEL cells). EPO failed to prevent apoptosis induced by Taxol or senescence induced by Adriamycin in MCF-7 cells. EPO stimulated the activation of extracellular signal-regulated kinase, p38, and c-Jun-NH(2)-kinase in MCF-7 cells but did not activate Akt or signal transducers and activators of transcription 5 (STAT5). EPO failed to activate any of these signaling pathways in MDA-MB231 cells. Cytarabine and daunorubicin interfered with EPO signaling in F-MEL cells.. These findings suggest that EPO is unlikely to directly counteract the effectiveness of cancer chemotherapeutic drugs. This may be a consequence of either ineffective signaling through the EPOR or drug-mediated suppression of EPO signaling.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Doxorubicin; Drug Antagonism; Drug Screening Assays, Antitumor; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Paclitaxel; Signal Transduction; Structure-Activity Relationship; Tamoxifen; Tumor Cells, Cultured

In concert with its ligand, the stem cell factor (SCF), the receptor tyrosine kinase c-Kit acts as a key signaling molecule for a number of cell types, including hematopoietic stem cells, mast cells, melanocytes and germ cells. Gain-of-function mutations in c-Kit have been described in a number of human cancers, including testicular germinomas, acute myeloid leukemia and gastrointestinal stromal tumors. Yet their contribution to neoplastic growth is incompletely understood. Now Kosmider et al report the acquisition of Kit mutations in 86% of late-stage eryhtroleukemias in Spi-1/PU.1 transgenic mice. Without Kit mutations, these mice suffer from a benign disease whose hallmark is erythropoietin-dependent expansion of undifferentiated red blood cell precursors. Newly acquired Kit mutations affect codon 814 or 818, and ectopic expression of these mutants in nonmalignant pro-erythroblasts confers erythropoietin independence and tumorigenicity. Using tyrosine kinase inhibitors PP1, PP2, and imatinib mesylate (a.k.a. Gleevac), the authors demonstrate that Kit mutations are important for the autonomous expansion of malignant cells via the MEK/Erk1/2 and PI3K/Akt pathways. These findings validate the notion that one differentiation-blocking (e.g., PU.1 activation) and one proliferative (e.g., c-Kit mutations) event are required for the development of frank leukemia.

Topics: Animals; Codon; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute; Mice; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Trans-Activators

The characteristic progression and specificity of Friend virus for the erythroid lineage have allowed for the identification of a number of host-encoded loci that are required for disease progression. Several of these loci, including the Friend virus susceptibility gene 2 (Fv2), dominant white spotting gene (W), and Steel gene (Sl), regulate the initial polyclonal expansion of infected erythroid progenitor cells. W and Sl encode the Kit receptor tyrosine kinase and its ligand, stem cell factor, respectively. W mutant mice are severely anemic, and earlier work suggested that this defect in erythroid differentiation is the cause for the resistance to Friend virus-induced erythroleukemia. Here we show that in bone marrow, W/W(v) mice have near normal numbers of target cells and the initial infection of bone marrow occurs normally in vivo. In contrast, spleen cells from W/W(v) mice infected both in vitro and in vivo with Friend virus failed to give rise to erythropoietin-independent colonies at any time following Friend virus infection, suggesting that mutation of the Kit receptor specifically affects target cells in the spleen, rendering the mutant mice resistant to the development of Friend virus-induced erythroleukemia. In addition, we show that the Kit+ pathogenic targets of Friend virus in the spleen are distinct from the pathogenic targets in bone marrow and this population of spleen target cells is markedly decreased in W/W(v) mice and these cells fail to express Sf-Stk. These results also underscore the unique nature of the spleen microenvironment in its role in supporting the progression of acute leukemia in Friend virus-infected mice.

Topics: Animals; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Receptor Protein-Tyrosine Kinases; Spleen; Tyrosine

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.

Topics: Animals; Butadienes; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Inhibitors; Erythropoietin; Flavonoids; Gene Expression Regulation, Neoplastic; Globins; Imidazoles; Immediate-Early Proteins; Leukemia, Erythroblastic, Acute; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-fos; Pyridines; Transcription Factors; Tumor Cells, Cultured

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope protein, gp55, which interacts with the erythropoietin (Epo) receptor complex, causing proliferation and differentiation of erythroid cells in the absence of Epo. Susceptibility to SFFV-induced erythroleukemia is conferred by the Fv-2 gene, which encodes a short form of the receptor tyrosine kinase Stk/Ron (sf-Stk) only in susceptible strains of mice. We recently demonstrated that sf-Stk becomes activated by forming a strong interaction with SFFV gp55. To examine the biological consequences of activated sf-Stk on erythroid cell growth, we prepared retroviral vectors which express sf-Stk, either in conjunction with gp55 or alone in a constitutively activated mutant form, and tested them for their ability to induce Epo-independent erythroid colonies ex vivo and disease in mice. Our data indicate that both gp55-activated sf-Stk and the constitutively activated mutant of sf-Stk induce erythroid cells from Fv-2-susceptible and Fv-2-resistant (sf-Stk null) mice to form Epo-independent colonies. Mutational analysis of sf-Stk indicated that a functional kinase domain and 8 of its 12 tyrosine residues are required for the induction of Epo-independent colonies. Further studies demonstrated that coexpression of SFFV gp55 with sf-Stk significantly extends the half-life of the kinase. When injected into Fv-2-resistant mice, neither the gp55-activated sf-Stk nor the constitutively activated mutant caused erythroleukemia. Surprisingly, both Fv-2-susceptible and -resistant mice injected with the gp55-sf-Stk vector developed clinical signs not previously associated with SFFV-induced disease. We conclude that sf-Stk, activated by either point mutation or interaction with SFFV gp55, is sufficient to induce Epo-independent erythroid colonies from both Fv-2-susceptible and -resistant mice but is unable to cause erythroleukemia in Fv-2-resistant mice.

Topics: Animals; Cell Line; Erythroid Cells; Erythropoietin; Genetic Vectors; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred C57BL; Point Mutation; Receptor Protein-Tyrosine Kinases; Retroviridae Infections; Spleen Focus-Forming Viruses; Tumor Virus Infections; Viral Envelope Proteins

Binding of erythropoietin (EPO) to its receptor (EPOR) on erythroid cells induces the activation of numerous signal transduction pathways, including the mitogen-activated protein kinase Jun-N-terminal kinase (JNK). In an effort to understand the regulation of EPO-induced proliferation and JNK activation, we have examined the role of potential autocrine factors in the proliferation of the murine erythroleukemia cell line HCD57. We report here that treatment of these cells with EPO induced the expression and secretion of tumor necrosis factor alpha (TNF-alpha). EPO-dependent proliferation was reduced by the addition of neutralizing antibodies to TNF-alpha, and exogenously added TNF-alpha induced proliferation of HCD57 cells. EPO also could induce TNF-alpha expression in BAF3 and DA3 myeloid cells ectopically expressing EPOR. Addition of TNF-alpha activated JNK in HCD57 cells, and the activity of JNK was partially inhibited by addition of a TNF-alpha neutralizing antibody. Primary human and murine erythroid progenitors expressed TNF-alpha in either an EPO-dependent or constitutive manner. However, TNF-alpha had an inhibitory effect on both immature primary human and murine cells, suggestive that the proliferative effects of TNF-alpha may be limited to erythroleukemic cells. This study suggests a novel role for autocrine TNF-alpha expression in the proliferation of erythroleukemia cells that is distinct from the effect of TNF-alpha in normal erythropoiesis.

Topics: Animals; Autocrine Communication; Cell Division; Cell Line; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Erythroblastic, Acute; Mice; Mitogen-Activated Protein Kinases; Signal Transduction; Tumor Necrosis Factor-alpha

Congenital blood disorders are common and yet clinically challenging globin disorders. Gene therapy continues to serve as a potential therapeutic method to treat these disorders. While tremendous advances have been made in vivo, gene delivery protocols and vector prototypes still require optimization. Alternative cis-acting promoter elements derived from VL30 retroelements have been effective in expressing tissue-specific transgene expression in vivo in nonerythroid cells. VL30 promoter elements were isolated from ELM-I-1 erythroid progenitor cells upon erythropoietin (epo) treatment. These promoters were inserted into a VL30-derived expression vector and reintroduced into the ELM-I-1 cells. beta-Galactosidase reporter gene activity from the ELM 5 clone, a BVL-1-like VL30 promoter, was capable of expressing sustained levels of the transgene expression over a 16-week assay period. These findings delineate the potential utility of these retroelement promoters as transcriptionally active, erythroid-specific, long terminal repeat (LTR) components for current globin vector constructs.

Topics: Animals; beta-Galactosidase; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Genes, Reporter; Genetic Vectors; Lac Operon; Leukemia, Erythroblastic, Acute; Mice; Organ Specificity; Promoter Regions, Genetic; Recombinant Fusion Proteins; Retroelements; Transfection; Tumor Cells, Cultured

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)-based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Cell Differentiation; Cysteine Endopeptidases; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Glycophorins; Humans; Leukemia, Erythroblastic, Acute; Membrane Proteins; Multienzyme Complexes; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reticulocytes; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Alternative therapeutic approaches with low dose chemotherapy and differentiative-maturative treatment by growth factors are under consideration for elderly patients with acute leukemia. Two patients with AML-M6 with maturation, one refractory to standard chemotherapy and the other ineligible for cytoxic treatment, obtained complete remission from leukemia using high dose recombinant erythropoietin and granulocyte colony-stimulating factor.

Topics: Acute Disease; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Erythropoietin; Fatal Outcome; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Erythroblastic, Acute; Male; Remission Induction

Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. Previously, we demonstrated that Stat1 and Stat3 are activated by erythropoietin (EPO) in a human EPO-dependent erythroleukemia cell line UT-7/EPO. We report here that Stat1 and Stat3 are constitutively activated in freshly isolated erythroleukemia cells. In addition, EPO promoted cell growth of these cells, accompanied by enhanced activities of Stat1 and Stat3. Furthermore, mutation in the Statl/Stat3-binding sites of the c-myc gene promoter clearly blocked its promoter activity in EPO-stimulated primary erythroleukemia cells. Thus, Stat1 and Stat3 may support cell growth in part via c-myc gene activation in primary erythroleukemia cells.

Topics: Adult; Binding Sites; Cell Division; DNA-Binding Proteins; Erythropoietin; Fatal Outcome; Female; Gene Expression Regulation, Leukemic; Genes, myc; Humans; Leukemia, Erythroblastic, Acute; Milk Proteins; Neoplasm Proteins; Promoter Regions, Genetic; Sequence Deletion; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured

Cancer is a multi-step, multi-genetic event. Whether oncogenic mutations cooperate with one another to transform cells and how is not well understood. The Friend murine retroviral erythroleukemia model involves mitogenic activation of the erythropoietin receptor (EpoR) by the virus env gene (F-gp55), aberrant over-expression of the transcription factor PU.1, and inactivating mutations in p53. In this report we demonstrate that concurrent expression of F-gp55 and PU.1 in erythroid target cells, in vivo, cooperate to accelerate erythroleukemia induction. Early in the disease, prior to the detection of clonal leukemic cells, activation of the EpoR by F-gp55, but not erythropoietin, resulted in transcriptional upregulation of PU.1 through a trans regulatory mechanism. This could occur in the absence of an integrated provirus within the PU.1 gene locus. The regulation of PU.1 transcription in established erythroleukemia cell lines differed depending upon the level of PU.1 protein present. Our results suggest that the action of F-gp55 contributes to both early and late stages of Friend erythroleukemia and that persistence of F-gp55 expression may be required not only to initiate erythroleukemia but to also maintain erythroleukemia following Friend virus infection.

Topics: Animals; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Promoter Regions, Genetic; Proto-Oncogene Proteins; Receptors, Erythropoietin; RNA, Messenger; Signal Transduction; Spleen Focus-Forming Viruses; Time Factors; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation; Viral Envelope Proteins; Virus Integration

In a 70 year old man with primary glomerulonephritis, severe anemia occurred after 4 years on hemodialysis and rHu-EPO. The usual mechanisms of EPO-resistance were excluded. A bone marrow sample showed red all aplasia. No circulating EPO could be detected; the serum inhibited the growth of erythroid precursors in bone marrow cultures. Immunoprecipitation identified an IgG anti-EPO, still active against deglycosylated EPO, i.e. directed against the peptidic matrix. Its high neutralising capacity and the absence of any immune abnormality rule out an auto-antibody. Anti-rHu EPO immunisation is a very rare occurrence, made severe by transfusion-dependence and the risk of hemosiderosis. An immuno-modulating treatment can therefore be justified.

Topics: Aged; Anemia; Angiodysplasia; Blood Transfusion; Bone Marrow; Cell Division; Combined Modality Therapy; Digestive System; Drug Resistance; Erythroid Precursor Cells; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Hematemesis; Humans; Immunoglobulin G; Isoantibodies; Kidney Failure, Chronic; Leukemia, Erythroblastic, Acute; Male; Melena; Recombinant Proteins; Renal Dialysis; Tumor Cells, Cultured

During the initial stage of Friend virus-induced erythroleukemia in mice, interaction of the viral protein gp55 with the erythropoietin receptor, and other host factors, drives the expansion of erythroid precursor cells. Recently, we demonstrated that the Friend virus susceptibility locus, Fv2, which is required for the expansion of infected cells, encodes a naturally occurring, N-terminally truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Here we show that in vitro expression of Sf-Stk confers Friend virus sensitivity to erythroid progenitor cells from Fv2(rr) mice. Furthermore, our data reveal that Sf-Stk kinase activity and Y436, but not Y429, are required for Epo-independent colony formation following Friend virus infection. Introduction of a mutation that results in failure to bind Grb2 abrogates the ability of Sf-Stk to induce colonies in response to Friend virus, while the Grb2 binding site from EGFR restores this response. Consistent with the ability of Grb2 to recruit SOS and Gab1, the Ras/MAPK and PI3K pathways are activated by Sf-Stk, and both of these pathways are required for gp55-mediated erythroblast proliferation. These data clearly demonstrate a requirement for signaling through Sf-Stk in the Epo-independent expansion of Friend virus-infected cells, and suggest a pivotal role for Grb2 in this response.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Binding Sites; Cell Division; Cell Line; Cells, Cultured; Erythroblasts; Erythropoietin; Friend murine leukemia virus; GRB2 Adaptor Protein; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Sequence Homology, Amino Acid; Signal Transduction; Tyrosine; Viral Envelope Proteins

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development. (Blood. 2001;97:901-910)

Topics: 3T3 Cells; Animals; Cell Lineage; Colony-Forming Units Assay; DNA, Complementary; Drug Synergism; Dyrk Kinases; Enzyme Induction; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fetal Proteins; Humans; Isoenzymes; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred DBA; Neoplasm Proteins; Oligodeoxyribonucleotides, Antisense; Organ Specificity; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Sequence Alignment; Sequence Homology, Amino Acid; Stem Cell Factor; Substrate Specificity; Tumor Cells, Cultured

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Enzyme Activation; Erythroid Precursor Cells; Erythropoietin; Hemoglobins; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Liver; Mice; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Signal Transduction; src-Family Kinases

Gene expression profiles during erythropoietin (Epo)-induced differentiation of erythroid progenitor cells derived from the Friend virus anaemia (FVA) and phenylhydrazine (PHZ) murine models have been examined using differential display polymerase chain reaction (PCR). Ten cDNA fragments upregulated by Epo were isolated. The ribonuclease protection assay confirmed differential expression between Epo-stimulated and Epo-deprived cells for one of these, provisionally named ERIC-1. Sequencing of the full-length cDNA predicted a protein of 558 amino acids, 17 amino acids longer than mTACC3, the third member of a novel family of proteins that contain a coiled-coil domain. The human homologue, cloned using rapid amplification of cDNA ends (RACE)-PCR, encodes a larger protein of 838 amino acids that is identical to hTACC3. In addition to erythroid precursor cells, ERIC-1/TACC3 is expressed at high levels in the testes, at moderate levels in the thymus and peripheral leucocytes, and at lower levels in the spleen and intestinal tissue. Immunohistochemical analysis using an antibody to a GST fusion product of the C-terminus of hERIC-1/TACC3 revealed that it is localized to Sertoli cells in the human testes. Confocal microscopy demonstrated hERIC-1/TACC3 protein concentrated in the perinuclear vesicles of dermal microvascular endothelial cells. Although ERIC-1/TACC3 is expressed in a wide range of tissues, its upregulation by Epo in erythroid progenitors implies that it has a role in terminal erythropoiesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Gene Expression; Humans; Immunohistochemistry; Leukemia, Erythroblastic, Acute; Male; Mice; Microtubule-Associated Proteins; Molecular Sequence Data; Phenylhydrazines; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sertoli Cells; Tumor Cells, Cultured

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.

Topics: Animals; Bioreactors; Cell Differentiation; Cell Division; Culture Media, Conditioned; Erythrocytes; Erythropoiesis; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Rotation; Tumor Cells, Cultured; United States; United States National Aeronautics and Space Administration; Weightlessness Simulation

Previous reports have shown a direct effect of erythropoietin (Epo) on vascular smooth muscle cells (VSMCs). Our aim was to assess expression of the Epo receptor (EpoR) on VSMCs and to study the activation of two major signaling cascades activated by Epo, namely JAK2/STAT5 and MAPK pathways. All experiments were performed in parallel using the Epo-responsive UT7 cell line. From semiquantitative RT-PCR experiments, VSMCs were estimated to express approximately 30-fold less EpoR mRNA than UT7 cells. Epo-induced phosphorylation of proteins involved in the EpoR/JAK2/STAT5 cascade could not be detected in VSMCs, even using pharmacological doses of Epo (250 IU/ml). In contrast, a strong activation of MAP kinase pathway was detected with as low as 10 IU/ml Epo. We suggest that MAPK activation reflects a physiologically relevant effect of Epo on VSMCs that may be correlated to cell proliferation.

Topics: Animals; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Erythropoietin; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Male; MAP Kinase Signaling System; Milk Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Rats, Inbred SHR; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured

Topics: Animals; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Genes, myc; Humans; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Signal Transduction; STAT3 Transcription Factor; Trans-Activators

The serine/threonine kinase Raf-1 is crucial for transducing intracellular signals emanating from numerous growth factors. Here we used the J2E erythroid cell line transformed by the nu-raf/nu-myc oncogenes to examine the effects of erythropoietin on endogenous Raf-1 activity. Despite the presence of constitutively active v-raf in these cells, Raf-1 exokinase activity increased after erythropoietin stimulation. This increase in enzymatic activity coincided with tyrosine phosphorylation of Raf-1 on residue Y341. Significantly, the tyrosine kinase Lyn coimmunoprecipitated with Raf-1, and Raf-1 was not tyrosine-phosphorylated in a J2E subclone lacking Lyn. Therefore, it was concluded that Lyn may be the kinase responsible for tyrosine phosphorylating Raf-1 and increasing its exokinase activity in response to erythropoietin.

Topics: Cell Line, Transformed; Erythropoietin; Leukemia, Erythroblastic, Acute; Mitogen-Activated Protein Kinase 1; Oncogene Proteins v-raf; Phosphorylation; Precipitin Tests; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; src-Family Kinases; Tumor Cells, Cultured

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

Topics: Anemia; Animals; Biological Transport; Cation Transport Proteins; CHO Cells; Cricetinae; Endosomes; Erythrocyte Membrane; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Fluorescent Antibody Technique; Heme; Hemoglobins; Homozygote; Iron; Iron-Binding Proteins; Leukemia, Erythroblastic, Acute; Mice; Mice, Mutant Strains; Microscopy, Confocal; Mutation; Phenylhydrazines; Protein Isoforms; Reticulocyte Count; Reticulocytes; Spleen; Transferrin; Tumor Cells, Cultured

The ligand-dependent activation of the JAK/STAT (Januskinase/Signal Transducer and Activator of Transcription) pathway has been implicated in the explanation of cytokine-specific regulation of gene expression. Previous studies have reported conflicting results on the role of the transcription factor STAT5 in erythropoietin (EPO)-induced cellular responses. In this study we focused on the functional importance of STAT5 docking sites in the intracellular EPO receptor (EPOR) domain for the mediation of antiapoptotic activities. We demonstrate that EPO-dependent survival of erythroleukemic cell lines is accompanied by sustained STAT5 DNA-binding activity. The role of single tyrosine residues was dissected by the analysis of myeloid FDCP-1 cells stably expressing mutant EPOR proteins. The data show that receptors having a high potential to mediate antiapoptotic signals also effectively activate STAT5, whereas receptors lacking STAT5 docking sites are diminished in both activities. We conclude that the transcription factor STAT5 is functionally implicated in the EPO-dependent survival of cells.

Topics: Animals; Apoptosis; DNA-Binding Proteins; Erythroblasts; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Milk Proteins; Receptors, Erythropoietin; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

Topics: Animals; Cell Differentiation; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Erythropoietin; Genes, Dominant; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Mutation; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Tumor Cells, Cultured

Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line-the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate.

Topics: Adaptor Proteins, Signal Transducing; Binding Sites; Blood Proteins; Cell Differentiation; Endosomes; Erythroid Precursor Cells; Erythropoietin; Leukemia, Erythroblastic, Acute; Peptide Fragments; Protein Binding; Saccharomyces cerevisiae; src Homology Domains; src-Family Kinases; Tumor Cells, Cultured; Two-Hybrid System Techniques

The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced differentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced differentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undifferentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but significant inactivation of ERK was also observed during EPO-induced differentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid differentiation, while pharmacological inhibition of MAPKK induced differentiation. These findings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid differentiation in this system.

Topics: Androstadienes; Animals; Cell Differentiation; Dimethyl Sulfoxide; Down-Regulation; Enzyme Inhibitors; Erythropoietin; Flavonoids; Friend murine leukemia virus; Gene Transfer Techniques; Genes, Dominant; Globins; JNK Mitogen-Activated Protein Kinases; Leukemia, Erythroblastic, Acute; MAP Kinase Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation; Protein Serine-Threonine Kinases; ras Proteins; Repressor Proteins; Tumor Cells, Cultured; Wortmannin

We have recently isolated the erythroleukemic cell line, HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes terminal differentiation in the presence of Epo alone. Ectopic expression of the ets related transcription factor Fli-1 in these cells resulted in the establishment of the Epo-dependent cell line HB60-ED that proliferates in the presence of Epo. In this study, we utilized these two cell lines to examine the signal transduction pathways that are activated in response to Epo and SCF stimulation. We demonstrate that Epo, but not SCF, phosphorylates STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5 cells while Epo does not. However, both Epo and SCF are capable of activating the Shc/ras pathway in HB60ED cells. Furthermore, enforced expression of gp55 in HB60-5 cells by means of infection with the Spleen Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation of Fli-1. Activation of the Shc/ras pathway is readily detected in gp55 expressing cells in response to both Epo and SCF, and is associated with a block in STAT-5B tyrosine phosphorylation. These results suggest that STAT-5 activation, in the absence of Shc/ras activation, plays a role in erythroid differentiation. Moreover, Fli-1 is capable of switching Epo-induced differentiation to Epo-induced proliferation, suggesting that this ets factor regulated genes whose products modulate the Epo-Epo-R signal transduction pathway.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Cell Differentiation; Cell Division; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Humans; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Milk Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteins; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; ras Proteins; Receptors, Erythropoietin; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; STAT5 Transcription Factor; Stem Cell Factor; Trans-Activators; Tumor Cells, Cultured; Viral Envelope Proteins

Hypoxia is a strong stimulus for the transcription of a set of genes, including erythropoietin and vascular endothelial growth factor. Here we report on the cloning, functional significance, and expression of a complementary DNA (cDNA) that is involved in hypoxia-mediated expression of these 2 genes. The full-length cDNA encodes a predicted protein of 806 amino acids that contains a leucine zipper motif. This protein, termed HAF for hypoxia-associated factor, binds to a 17-base pair (bp) region of the erythropoietin promoter, which was shown earlier to participate in hypoxia-induced expression of the erythropoietin gene. In Hep3B cells, clones modified to express HAF antisense RNA showed an attenuated response to hypoxia-mediated induction of both erythropoietin and vascular endothelial growth factor transcription. HAF showed sequence-specific interaction with a DNA element in the 5' untranslated region of VEGF gene. The HAF 2.6-kilobase (kb) messenger RNA (mRNA) is expressed in most adult tissues. The highest expression occurs in fetal liver and the least in adult liver. HAF is the murine homolog of Sart-1, a 125-kd human protein expressed in the nuclei of normal and malignant cells. (Blood. 2000;96:491-497)

Topics: Animals; Base Pairing; Cell Hypoxia; Cloning, Molecular; DNA, Complementary; Endothelial Growth Factors; Erythropoietin; Gene Expression; Leucine Zippers; Leukemia, Erythroblastic, Acute; Liver; Lymphokines; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Ribonucleoproteins, Small Nuclear; RNA, Antisense; RNA, Messenger; Tissue Distribution; Trans-Activators; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

UT-7/Epo cells are human factor-dependent erythroleukemic cells, requiring erythropoietin (Epo) for long-term growth. Stem cell factor (SCF) stimulates proliferation of UT-7/Epo only transiently, for three to five days. An investigation of the signal transduction pathways activated by these cytokines in UT-7/Epo cells may identify those signals specifically required for sustained growth. Proliferation assays demonstrate that SCF generates a substantial growth response in UT-7/Epo cells; however, the cells do not multiply or survive past five to seven days. While Epo induces the activation of JAK2 and STAT5, SCF stimulation shows no activation of JAK2 or STATs 1, 3, or 5. The activation of MAPK (p42/44) by SCF was transient, lasting only 30 min, in contrast to Epo, which stimulated phosphorylation of p42/44 for up to 2 h. The expression of the early response genes c-fos, egr1, and cytokine-inducible SH2 protein (CIS) in response to SCF or Epo stimulation demonstrated that the transient expression of p42/44 correlated with the transient expression of c-fos and egr1. In addition, CIS was activated by Epo but not SCF. These data indicate that EpoR, JAK2, and STAT5 activation are not required for the initiation of proliferation of these erythroid cells, that the transient activation of p42/44 correlates with the transient gene expression of c-fos and egr1, and sustained expression of c-fos and egr1 as seen in UT-7/Epo cells continuously grown in Epo may be necessary for long-term proliferation.

Topics: Cell Division; Cell Survival; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Activation; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, Immediate-Early; Humans; Immediate-Early Proteins; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Milk Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Recombinant Proteins; Signal Transduction; STAT5 Transcription Factor; Stem Cell Factor; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

During studies of erythroid cell growth and differentiation induced by erythropoietin (Epo), we developed a method that allows the identification and isolation of genes based upon their transcriptional activity. Transcriptionally active genomic DNA fragments from Epo-treated cells and control cells are purified from inactive chromatin using mercury affinity chromatography, based on the mechanism that the thiol groups of histone H3 on transcriptionally active chromatin are exposed to the solvent and therefore are easily accessible. Using the purified genomic DNA fragments from the two populations of cells, a subtractive hybridization strategy is used to isolate and clone genes that are differentially expressed in the absence or in the presence of Epo.

Topics: Animals; Blotting, Northern; Cell Nucleus; Cloning, Molecular; DNA; Erythropoietin; Gene Expression Regulation; Genetic Techniques; Leukemia, Erythroblastic, Acute; Mercury; Mice; Nucleosomes; Polymerase Chain Reaction; Sequence Analysis, DNA; Transcription, Genetic; Tumor Cells, Cultured

Hematopoietic progenitor kinase-1 (HPK1), which is expressed predominantly in hematopoietic cells, was identified as a mammalian Ste20 homologue that, upon transfection, leads to activation of JNK/SAPK in nonhematopoietic cells. The JNK/SAPK pathway is activated by various environmental stresses and proinflammatory and hematopoietic cytokines. Upstream activators of HPK1 currently remain elusive, and its precise role in hematopoiesis has yet to be defined. We therefore examined the possible involvement of HPK1 in erythropoietin (Epo) and environmental stress-induced JNK/SAPK activation in the Epo-dependent FD-EPO cells and Epo-responsive SKT6 cells. We found that Epo, but not environmental stresses, induced rapid and transient activation of HPK1, whereas both induced activation of JNK/SAPK. A screen for HPK1 binding proteins identified the hematopoietic cell-specific protein 1 (HS1) as a potential HPK1 interaction partner. We found HPK1 constitutively associated with HS1 and that HS1 was tyrosine-phosphorylated in response to cellular stresses as well as Epo stimulation. Furthermore, antisense oligonucleotides to HPK1 suppressed Epo-dependent cell growth and Epo-induced erythroid differentiation. We therefore conclude that Epo induces activation of both HPK1 and HS1, whereas cellular stresses activate only HS1, and that the HPK1-JNK/SAPK pathway is involved in Epo-induced growth and differentiation signals.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Division; Cell Line; Enzyme Activation; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides, Antisense; Osmolar Concentration; Peptide Fragments; Phosphorylation; Protein Serine-Threonine Kinases; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; src Homology Domains; Transcription, Genetic; Tumor Cells, Cultured

The dynamics of gene expression during terminal erythroid differentiation have been examined in three murine models; the erythroleukaemia cell line HCD-57 and splenic erythroblasts isolated from mice treated with either the anaemia-inducing strain of Friend virus (FVA cells) or the haemolytic agent phenylhydrazine (PHZ cells). In response to erythropoietin (EPO) and haemin, HCD-57 cells proliferated and synthesized haemoglobin, but failed to complete terminal differentiation as indicated by lack of change in both gene expression and morphological appearance. In contrast, EPO-induced terminal differentiation in FVA and PHZ cells in vitro was accompanied by increases in haemoglobin positivity, morphological maturation and a shared pattern of gene expression. EPO receptor (EPO-R) mRNA levels peaked before globin gene expression which was maximal at 24 h. Peak GATA-1 and EKLF mRNA levels also preceded the globin gene peak, but the highest NF-E2 levels coincided with maximal globin levels, suggesting a role for NF-E2 in the maintenance, rather than the initiation of globin gene expression. Peak expression of delta-aminolaevulinic acid synthase (ALAS) coincided with peak globin expression. FVA and PHZ cells represent more effective models than the HCD-57 cell line for the investigation of erythroid gene expression during EPO-regulated terminal erythropoiesis.

Topics: Animals; Cell Size; Erythroblasts; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Gene Expression; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Phenylhydrazines; Tumor Cells, Cultured

Macromolecular centromere-kinetochore complex plays a critical role in sister chromatid separation, but its complete protein composition as well as its precise dynamic function during mitosis has not yet been clearly determined. Here we report the isolation of a novel mouse kinetochore protein, CENP-H. The CENP-H, with an apparent molecular mass of 33 kDa, was found to contain a coiled-coil structure and a nuclear localization signal. The CENP-H transcripts were relatively scarce but were detectable in most tissues and embryos at various stages of development. Immunofluorescence stainings of mouse fibroblast cells with anti-CENP-H-specific antibody demonstrated that the CENP-H is specifically and constitutively localized in kinetochores throughout the cell cycle; this was also confirmed by stainings with anti-centromere-specific antibody. Thus the newly isolated CENP-H may play a role in kinetochore organization and function throughout the cell cycle.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Chromosomal Proteins, Non-Histone; Cloning, Molecular; Embryonic and Fetal Development; Erythropoietin; Female; Gene Expression Regulation, Developmental; Kinetochores; Leukemia, Erythroblastic, Acute; Male; Mice; Molecular Sequence Data; Molecular Weight; Recombinant Proteins; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Erythroleukemias induced by Friend Murine Leukemia Virus (F-MuLV) involve the insertional activation of the proto-oncogene Fli-1, and the inactivation of the p53 tumor suppressor gene. While the activation of Fli-1 is an early, primary transforming event, p53 mutations are correlated with the immortalization of erythroleukemic cells in culture. In this study we have further analysed the role of p53 loss in F-MuLV induced erythroleukemias by examining the progression of this disease in p53 deficient mice. We found that p53-/- mice succumb to the disease more rapidly than p53+/+ littermates. Additionally, of the 112 tumors generated, 19 gave rise to immortal cell lines, eight of which were derived from p53-/- mice, and ten of which were from p53+/- mice. The ability of these primary tumor cells to grow in culture was associated with the complete loss of wild-type p53 in these cell lines. However, cells from many of the tumors induced in p53-/- hosts did not survive in vitro. These results suggest that the loss of p53 does not directly immortalize tumor cells. Instead, we have evidence to suggest that the loss of p53 promotes the accumulation of mutations that are required for survival in culture and that are capable of accelerating tumor progression in vivo. Indeed, mutations causing expression of the growth factor gene erythropoietin (Epo), were detected in two of seven Epo-independent cell lines from p53 deficient primary erythroleukemias. Moreover, the mechanism of activation of the Epo gene in one of these two Epo-independent cell lines involved genomic rearrangement, that is a hallmark of genetic instability. We propose that, in F-MuLV induced-erythroleukemias, p53 loss may encourage the accumulation of further mutations, subsequently conferring a growth advantage and immortality to the transformed erythroblasts.

Topics: Animals; Animals, Newborn; Blood Proteins; Cell Division; Cellular Senescence; Disease Progression; DNA-Binding Proteins; Erythroid Precursor Cells; Erythropoietin; Female; Friend murine leukemia virus; Gene Deletion; Gene Expression Regulation, Leukemic; Genes, p53; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Mutagenesis; Neoplasm Proteins; Neoplasm Transplantation; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; Retroviridae Infections; Trans-Activators; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Virus Infections

The glucocorticoid receptor (GR) coordinates a multitude of physiological responses in vivo. In vitro, glucocorticoids are required for sustained proliferation of erythroid progenitors (ebls). Here, we analyze the impact of the GR on erythropoiesis in vivo, using GR-deficient mice or mice expressing a GR defective for transactivation. In vitro, sustained proliferation of primary ebls requires an intact GR. In vivo, the GR is required for rapid expansion of ebls under stress situations like erythrolysis or hypoxia. A particular, GR-sensitive progenitor could be identified as being responsible for the stress response. Thus, GR-mediated regulation of ebl proliferation is essential for stress erythropoiesis in vivo.

Topics: Anemia; Animals; Cells, Cultured; Chickens; Culture Media, Serum-Free; Dimerization; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Hematopoiesis, Extramedullary; Hematopoietic Stem Cell Transplantation; Hemolysis; Hypoxia; Leukemia, Erythroblastic, Acute; Liver; Mice; Mice, Knockout; Radiation Chimera; Receptors, Glucocorticoid; Stem Cell Factor; Stress, Physiological; Transcriptional Activation

The SH2 domain-containing tyrosine phosphatase SHP1 is known to play a crucial role in the regulation of hematopoiesis. It has been shown previously that SHP1 associates with the activated erythropoietin receptor (EPOR) and negatively regulates mitogenic signaling. To further elucidate the role of SHP1 in erythropoietin (EPO)-induced cellular responses we employed J2E erythroleukemic cells as a model for erythroid maturation and cytokine-triggered suppression of apoptosis. Our data indicate that overexpressed SHP1 inhibits both EPO-induced differentiation as well as prevention of apoptosis. The specific signaling pathways responsible are not unraveled so far. Therefore, we analyzed the involvement of SHP1 in two established EPO-stimulated pathways, the JAK/STAT and the MAP kinase cascades, by transient coexpression of reporter constructs containing binding sites for transcription factors targeted by these pathways and a SHP1 cDNA. Both pathways are inhibited by SHP1 as indicated by the lower induction of reporter gene activity. In conclusion, SHP1 regulates the transcriptional activity stimulated by the EPO-induced JAK/STAT and MAPK pathways and is involved in the signaling machinery responsible for erythroid differentiation and suppression of apoptosis.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Survival; DNA-Binding Proteins; Erythropoietin; Genes, Reporter; Intracellular Signaling Peptides and Proteins; Leukemia, Erythroblastic, Acute; Mice; Milk Proteins; Mitogen-Activated Protein Kinases; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Recombinant Proteins; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Hexamethylenebisacetamide (HMBA) is a potent differentiation inducer of murine erythroleukemia cells. Immunoprecipitation followed by Western blotting with an anti-phosphotyrosine (P-Tyr) antibody revealed that HMBA increased P-Tyr levels and/or amounts of several proteins containing P-Tyr in F5-5, a murine erythroleukemia cell line. Among these proteins, we identified a Mr 130,000 protein to be Janus-activated kinase 2 (JAK2). HMBA induced tyrosine phosphorylation of JAK2 and signal transducers and activators of transcription 5 (STAT5) but not other JAKs or STATs. This phosphorylation was apparent 12 h after treatment, maximal at 24 h, and persisted for at least 96 h. Consistently, HMBA increased STAT5 DNA-binding activities. Other chemical inducers, DMSO and butyrate, also induced a sustained activation of JAK2/STAT5, whereas fetal calf serum and erythropoietin induced transient activation but not differentiation. Furthermore, overexpression of a dominant-negative form of STAT5 inhibited the chemically induced differentiation. These results suggest that persistent activation of the signaling pathway plays a significant role in the inducer-mediated differentiation. Our data also suggest that molecular mechanisms for the inducer-mediated activation of JAK2 are independent of cytokine receptor-mediated activation mechanisms. We tentatively conclude that cytokine signaling is an important target of chemical inducers in these cells.

Topics: Acetamides; Animals; Antineoplastic Agents; Cattle; Cell Differentiation; Cytokines; DNA-Binding Proteins; DNA, Neoplasm; Erythropoietin; Fetal Blood; Friend murine leukemia virus; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Milk Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Defining how the stromal requirements of hematopoietic progenitors change during leukemia progression is an important topic that is not well understood at present. The murine ELM erythroleukemia is an interesting model because the erythroid progenitors retain dependence on bone marrow-derived stromal cells for long-term growth in vitro, and they also undergo erythroid differentiation in the presence of erythropoietin (EPO) and interleukin-3 (IL-3). In this report, we have shown using neutralizing antibodies that stem cell factor (SCF), insulin-like growth factor (IGF)-1, and integrin signaling pathways are all involved. We then determined whether ELM cells can be maintained long-term without stroma in various combinations of growth factors produced by stroma cells or growth factors for which ELM cells have receptors. This showed that ELM cells could be maintained with high efficiency in SCF alone; furthermore, the cells remained absolutely SCF-dependent and did not become more tumorigenic than cells maintained on stroma. In contrast, ELM cells underwent clonal extinction when serially cloned in IGF1; any cells that survived long-term growth in IGF-1 were found to be IGF1-independent. One important difference between maintaining ELM cells on stroma and growth in SCF is that stroma reversibly inhibits their differentiation in response to EPO and IL-3, whereas SCF does not.

Topics: Animals; Antibodies; Cell Differentiation; Cell Division; Cell Line; Culture Media; Erythropoietin; Extracellular Matrix; Humans; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Integrins; Interleukin-3; Leukemia, Erythroblastic, Acute; Mice; Signal Transduction; Stem Cell Factor; Stromal Cells; Tumor Cells, Cultured

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Topics: Adult; Antigens, CD34; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cobalt; Culture Media, Serum-Free; Enzyme-Linked Immunosorbent Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liver Neoplasms; Molecular Sequence Data; Peptides; Receptors, Erythropoietin; RNA, Messenger; Tumor Cells, Cultured

In the erythroleukemia cell line TF-1, recombinant human erythropoietin (rHEpo), but not c-kit ligand, enhanced the number of cells expressing the erythropoietin receptor (EpoR), as measured by flow-cytometric analysis of binding of the biotin-labeled Epo. Moreover, 125I-Epo binding and Scatchard analyses, indicated that TF-1 cells, maintained in standard conditions with IL-3, and those stimulated with c-kit ligand, bear a single class of EpoR. On the other hand, cells cultured in the presence of rHEpo had a higher number of receptors than IL-3 or c-kit ligand-stimulated cells, and had two binding sites with different affinities for the ligand. EpoR mRNA expression was higher in cells exposed to rHEpo than in IL-3 or c-kit-stimulated cells. This difference may have been dependent on either a higher level of transcription or an increased stability of mRNA. The observed changes of EpoR in rHEpo-stimulated TF-1 cell line could cooperate, together with the alteration of the gene (3' end deletion), in the occurrence of the erythroleukemic process. Changes induced in EpoR by rHEpo were not accompanied by an increase in the expression of glycophorin A or globin chain mRNAs. This may suggest that rHEpo is unable to induce erythroid differentiation in TF-1 cells. The results also indicate that this cell line could be a model for the investigation of the role of transcription factor(s) in the expression of EpoR, and for the study of the mechanism(s) underlying the changes in the number and affinity of the cell receptors.

Topics: Erythropoietin; Flow Cytometry; Humans; Leukemia, Erythroblastic, Acute; Polymerase Chain Reaction; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Stem Cell Factor; Tumor Cells, Cultured; Up-Regulation

Erythropoietin (EPO) is the major regulator of mammalian erythropoisis, which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor (EPO-R). Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells. Our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase (Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells. Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the binding of EPO to HEL cells. Furthermore, the binding of both STAT1 and STAT5 proteins to specific DNA elements (SIE and PIE elements) is revealed in an EPO-dependent manner. Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature erythroid cell from the spleen of mice infected with anemia strain of Friend virus.

Topics: Blotting, Western; Cell Nucleus; Consensus Sequence; Cytoplasm; DNA-Binding Proteins; Electrophoresis, Agar Gel; Erythropoietin; Humans; Janus Kinase 1; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Milk Proteins; Nuclear Proteins; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Regulatory Sequences, Nucleic Acid; Signal Transduction; STAT1 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

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.

Topics: B-Cell Lymphoma 3 Protein; Biological Transport; Cell Division; Cell Nucleus; Cells, Cultured; Cytoplasm; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Genes, Reporter; Granulocyte-Macrophage Colony-Stimulating Factor; HIV-1; Humans; Leukemia, Erythroblastic, Acute; Luciferases; NF-kappa B; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Mas; Proto-Oncogene Proteins; Recombinant Fusion Proteins; RNA, Messenger; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Insulin-like growth factor (IGF-I) is known to synergistically stimulate the proliferation of hematopoietic cells in combination with other hematopoietic growth factors. However, the precise mechanism underlying the cooperative effects of IGF-I is unknown. In a human interleukin-3 or erythropoietin (EPO)-dependent cell line, F-36P, IGF-I alone failed to stimulate DNA synthesis but did augment the EPO-dependent DNA synthesis of F-36P cells. The treatment of F-36P cells with a combination of EPO and IGF-I (EPO/IGF-I) was found to enhance EPO-induced tyrosine phosphorylation of STAT5, whereas IGF-I alone did not. Furthermore, c-CIS mRNA expression, one of the target molecules of STAT5, was more effectively induced by EPO/IGF-I than by EPO alone. To examine the mechanisms of the EPO- and EPO/IGF-I-induced proliferation of F-36P cells, we expressed dominant negative (dn) mutants of STAT5 and Ras in an inducible system. The EPO-induced DNA synthesis and the cooperative effect of EPO/IGF-I were significantly inhibited by the inducible expression of dn-STAT5 or dn-Ras. In addition, the inducible expression of dn-Ras abolished the IGF-I-enhanced tyrosine phosphorylation of STAT5. These results suggest that IGF-I may augment EPO-induced proliferation by enhancing tyrosine phosphorylation of STAT5 and raise the possibility that Ras may be involved in the augmentation of STAT5 tyrosyl phosphorylation.

Topics: Cell Division; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Insulin-Like Growth Factor I; Interleukin-3; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Milk Proteins; Mutation; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; ras Proteins; Recombinant Proteins; Signal Transduction; STAT5 Transcription Factor; Stem Cell Factor; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Nucleus; Cell Survival; DNA-Binding Proteins; DNA, Antisense; Erythropoietin; Globins; Hemoglobins; Humans; Kruppel-Like Transcription Factors; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Recombinant Proteins; Signal Transduction; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Zinc Fingers

p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Enzyme Activation; Erythrocytes; Erythropoietin; Hemoglobins; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Erythroblastic, Acute; Mice; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Protein Kinases; Tumor Cells, Cultured

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Differentiation; Cell Division; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dactinomycin; Disease Progression; DNA Damage; Erythroblasts; Erythropoietin; Female; Friend murine leukemia virus; G1 Phase; Gene Expression; Genes, p53; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mutation; Transcriptional Activation; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Although Friend murine erythroleukemia (MEL) cells express the erythropoietin receptor (EpoR), they are insensitive to erythropoietin (Epo). The nonresponsiveness to Epo presumably results from gp55, the product of the env gene encoded by the Friend spleen focus-forming virus (F-SFFV), acting as a pseudoligand and constitutively activating the receptor. Dimethyl sulfoxide (DMSO) induced the differentiation of MEL cells and partially restored responsiveness to Epo, with both increased proliferation and further hemoglobin synthesis. Treatment of MEL cells with DMSO caused a decrease in the cellular content of gp55 as measured by Western analysis and an increase in the level of the EpoR as measured by [125I]Epo binding. These changes were produced at least in part at the transcriptional level, because DMSO treatment caused a decrease and an increase in the levels of the mRNAs for gp55 and EpoR, respectively. To ascertain the role of gp55 in the restoration of the sensitivity of MEL cells to Epo by exposure to DMSO, expression vectors containing gp55 DNA in the sense and antisense orientations were transfected into MEL cells to increase or decrease, respectively, the amount of cellular gp55. An increase in the level of gp55 interfered with the ability of DMSO to restore sensitivity to Epo, whereas a decrease in the level of gp55 increased the Epo-sensitizing effects of DMSO. [125I]Epo was chemically cross-linked to a component with a calculated molecular weight of 65 kDa. DMSO treatment caused an increase in the level of [125I]Epo cross-linking. The protein cross-linked to Epo was immunoprecipitated with anti-EpoR serum but not with anti-gp55 serum, suggesting that Epo was cross-linked to its receptor. The finding of a decrease in the cellular content of gp55, an increase in the level of the EpoR, and an increase in the formation of the Epo/EpoR complex is consistent with the acquisition of sensitivity to Epo by MEL cells following treatment with DMSO.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Dimethyl Sulfoxide; Drug Screening Assays, Antitumor; Drug Synergism; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Recombinant Proteins; Tumor Cells, Cultured; Viral Envelope Proteins

Homeobox gene expression was examined in the erythroleukaemic cell line TF-1. Expression of a number of HOX A, B and C genes, including HOX A7 was detected. Expression of this gene has not previously been reported in erythroleukaemic cell lines. A 2.1 kb full length cDNA of the HOX A7 gene was cloned. The predicted amino acid sequence C-terminal to the homeodomain consists of an alanine-rich region and a strongly negatively charged domain consisting entirely of aspartic and glutamic acid residues.

Topics: Amino Acid Sequence; Base Sequence; DNA, Complementary; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Granulocyte-Macrophage Colony-Stimulating Factor; Homeodomain Proteins; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; Tumor Cells, Cultured

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Cell Differentiation; Cell Division; Cell Line; DNA-Binding Proteins; Erythropoietin; Gene Expression; Leukemia, Erythroblastic, Acute; Mice; Phosphorylation; Point Mutation; Protein Biosynthesis; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Stem Cell Factor; Stromal Cells; Trans-Activators; Tumor Cells, Cultured; Tumor Stem Cell Assay

Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

Topics: Animals; Erythropoietin; Humans; Iron; Iron Regulatory Protein 1; Iron-Regulatory Proteins; Iron-Sulfur Proteins; Leukemia, Erythroblastic, Acute; Mice; Receptors, Transferrin; RNA-Binding Proteins; Signal Transduction; Transferrin; Tumor Cells, Cultured; Up-Regulation

A recent report (Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246) documents the interaction of the erythropoietin (EPO) receptor (EPOR) with the stem cell factor (SCF) receptor (c-KIT) and suggests that SCF acts through the EPOR. To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. However, SCF-dependent phosphorylation of the EPOR did not initiate an EPO-like intracellular signal. SCF induced proliferation, SHC phosphorylation, and activation of ERK-1 but did not activate the JAK/STAT pathway. SCF stimulated SHC phosphorylation and ERK-1 activation independent of the EPOR in cells where the EPOR was down-regulated; the presence of the EPOR appeared to facilitate SCF activation of SHC and ERK-1. Furthermore, treatment of HCD57 cells with SCF increased cell number over a 3-day treatment, but apoptosis was observed in these cells. These data may illustrate two distinct pathways for erythroid cell proliferation and prevention of apoptosis in response to EPO, thereby providing a system to discriminate these intracellular signals.

Topics: Apoptosis; Cell Division; Erythropoietin; Leukemia, Erythroblastic, Acute; Phosphorylation; Signal Transduction; Stem Cell Factor; Tumor Cells, Cultured; Tyrosine

We have previously shown that, in HCD-57 cells, erythropoietin (EPO) induces a biphasic activation of the ribosomal S6 kinase p70S6k, an enzyme playing a key role in the regulation of cell cycle progression. Here we present evidence that p70S6k is activated through both phosphatidylinositol (PI) 3-kinase-dependent and independent pathways: whereas the early phase of EPO-dependent stimulation of p70S6k activity was strongly suppressed by the potent PI 3-kinase inhibitor wortmannin, late phase was much less affected. The dose-dependent inhibition of cell growth by wortmannin indicates an important role of PI 3-kinase in the mediation of EPO-induced cell proliferation. Furthermore, our data suggest that the EPO-receptor-associated tyrosine kinase JAK2 is not essentially involved in the mediation of EPO-induced p70S6k activation.

Topics: Androstadienes; Animals; Cell Division; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Oligonucleotides, Antisense; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Ribosomal Protein S6 Kinases; Signal Transduction; Tumor Cells, Cultured; Wortmannin

We have shown previously that treatment of isolated erythroid cell plasma membranes with erythropoietin leads to a rapid decrease in pp43, an erythropoietinsensitive membrane phosphoprotein (Choi, H. S., Wojchowski, D. M., and Sytkowski, A. J., J. Biol. Chem. 262, 2933, 1987; Choi, H. S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J., J. Biol. Chem. 265, 4143, 1990). We have now demonstrated this effect in intact cells and have obtained further information regarding pp43 function during erythropoietin stimulation. 32P-phosphorylated membranes were subjected to conditions of increasing pH. [32P]pp43 dissociated readily into solution, reaching half-maximal dissociation at pH approximately 9. This dissociation was enhanced markedly by increasing the ionic strength up to a maximum of 0.5 M KCl. These biochemical properties characterize pp43 as a membrane-associated protein. Addition of [gamma-32P]ATP to an aqueous supernatant prepared from unlabeled membranes resulted in the 32P-phosphorylation of pp43 in solution, after dissociation from the plasma membrane. Furthermore, erythropoietin treatment of unlabeled, intact cells followed by fractionation and 32P-phosphorylation resulted in a striking erythropoietin- and time-dependent increase in [32P]pp43 found in the supernatant and a concomitant decrease in [32P]pp43 found in the membrane pellet. This strongly suggests that erythropoietin stimulates the dissociation of pp43 from the plasma membrane and promotes translocation into the supernatant (cytoplasm). Using a renaturation kinase assay, we demonstrated that pp43 is capable of autophosphorylation on serine and threonine, thus identifying it as a new protein serine/threonine kinase. The results suggest a role for pp43 in transmembrane signaling.

Topics: Animals; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Erythropoietin; Hydrogen-Ion Concentration; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Osmolar Concentration; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Potassium Chloride; Protein Serine-Threonine Kinases; Recombinant Proteins; Tumor Cells, Cultured

Friend leukemia virus complex (FLV) consists of replication-defective, Friend spleen focus-forming virus (F-SFFV) and replication-competent, Friend murine leukemia virus (F-MuLV). We produced transgenic mice possessing F-SFFV gp55 gene and clarified that the gp55 glycoprotein encoded by F-SFFV env-related gene is, by itself, responsible for the initiation of erythroleukemia. The occurrence of erythroleukemia, however, is sporadic in these mice. Erythroleukemia cell lines established from these mice possessed mutations in the p53 allele. One had a temperature-sensitive mutant p53 allele, p53Val-135 and showed induction of apoptosis by expressing a wild-type p53 protein at 32 degrees C. Superinfection of the mice with Moloney murine leukemia virus (Mo-MuLV) conferred 100% induction of erythroleukemia, mutating p53 gene or activating Spfi-1 gene by insertional events. Activation of the JAK/STAT pathway, which is involved in cytokine signaling, was investigated in the gp55 signaling mediated by the erythropoietin receptor. JAK1 and STAT5 were constitutively tyrosine-phosphorylated but the DNA binding activity of STAT5 was not induced.

Topics: Animals; Cell Line; Cytokines; DNA-Binding Proteins; Erythropoietin; Exons; Friend murine leukemia virus; Genes, env; Janus Kinase 1; Leukemia, Erythroblastic, Acute; Mice; Mice, Transgenic; Milk Proteins; Moloney murine leukemia virus; Mutation; Protein-Tyrosine Kinases; Receptors, Erythropoietin; Retroviridae Infections; Signal Transduction; Spleen Focus-Forming Viruses; STAT4 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Tumor Suppressor Protein p53; Tumor Virus Infections

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of the erythroid hormone erythropoietin (Epo). In an attempt to understand how the virus alters the growth of erythroid cells, studies were carried out to determine if virus infection leads to the constitutive activation of the Jak-Stat pathway, one of the signal transduction pathways activated by Epo. Our data indicates that expression of SFFV in erythroid cells leads to the constitutive activation of the same Stat proteins that are transiently activated by Epo. While constitutive activation of Stat proteins by SFFV is associated with Epo-independent proliferation of splenic erythroid progenitor cells from Fv-2-sensitive mice and Epo-dependent HCD-57 cells, it is not sufficient to induce their differentiation. Although constitutive activation of the same Stat proteins is detected in erythroid cells from SFFV-infected Fv-2-resistant mice, it does not lead to their Epo-independent growth. It is also not required for transformation of erythroid cells by SFFV. Studies are in progress to identify the mechanism by which Stat proteins are phosphorylated in SFFV-infected cells in the absence of Epo. Although it has been shown that Epo activates Stat proteins through Jak2 kinase, our results suggest that the SFFV-induced Stat protein activation is Jak2-independent.

Topics: Animals; Cell Differentiation; Cell Line; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Genes, fos; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Mice; Protein Kinases; Signal Transduction; Spleen Focus-Forming Viruses; Virus Replication

We have generated a series of murine erythroleukemia clones that ectopically express a temperature sensitive mutant p53 allele. In many clones, activation of p53 at low temperature resulted in the accumulation of cells in G1 and in apoptosis. Several cytokines including erythropoietin, IL-3 and the ligand for the Kit receptor blocked p53-dependent apoptosis in p53ts-expressing cells at 32 degrees C. Cytokine-treated cells were reversibly arrested in G1 and resumed growth upon return to 37 degrees C. Certain clones exhibited only a G1 arrest in response to p53 activation at 32 degrees C. One of the these clones secreted erythropoietin and another secreted IL-3. We tested the possibility that autocrine secretion of IL-3 played a role in preventing apoptosis and showed that disruption of the autocrine loop by cell dilution or with neutralizing antibodies to IL-3 restored p53-dependent apoptosis at 32 degrees C. Thus, two properties of p53 protein, namely, its ability to arrest cells in G1 and its ability to promote apoptosis could be uncoupled by cytokines acting as survival factors.

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; Clone Cells; Cytokines; Erythropoietin; Interleukin-3; Leukemia, Erythroblastic, Acute; Mice; Mutagenesis; Recombinant Proteins; Stem Cell Factor; Temperature; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Interaction of erythropoietin with its type 1 receptor is essential to the development of late erythroid progenitor cells. Through the ectopic expression of receptor mutants in lymphoid and myeloid cell lines, insight has been gained regarding effectors that regulate Epo-induced proliferation. In contrast, effectors that regulate Epo-induced differentiation events (e.g. globin gene expression) are largely undefined. For in vitro studies of this pathway, erythroleukemic SKT6 cell sublines have been isolated which stably and efficiently hemoglobinize in response to Epo. Epo rapidly activated Jak2, STAT5 and detectably STATs 1 and 3, while no effects on GATA-1, EKLF or STAT5 expression were observed. Finally, efficient hemoglobinization of SKT6 cells was shown to be mediated by chimeric receptors comprised of the EGF receptor extracellular domain and truncated cytoplasmic subdomains of either the Epo receptor or the prolactin Nb2 receptor. This work further establishes SKT6 cells as an important model for studies of Epo-stimulated differentiation, and shows that this signaling pathway is promoted by a limited set of membrane-proximal receptor domains and effectors.

Topics: Amino Acid Sequence; Animals; DNA-Binding Proteins; ErbB Receptors; Erythroid Precursor Cells; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation, Developmental; Hemoglobins; Janus Kinase 2; Kruppel-Like Transcription Factors; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred Strains; Milk Proteins; Molecular Sequence Data; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Receptors, Prolactin; Recombinant Fusion Proteins; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

The erythroleukaemic cell line TF-1, infected with either the pBabe neo retrovirus or the retrovirus bearing the human erythropoietin (hEpo) gene, developed three growth factor-independent clones. Erythropoietin (Epo), interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) accelerated the proliferation of these clones. Autonomous growth of the clones was independent of Epo because it was not altered by Epo anti-sense oligonucleotides, nor was Epo detectable in culture supernatants. Cells from the mutant clones could not be induced by Epo to express glycophorin A and haemoglobin synthesis was markedly reduced. Haemin reversed the block in Epo-induced haemoglobin synthesis. Acquisition of growth factor-independence appears to be linked with the selective loss of differentiation capacity. These cells may provide a useful model for the study of the mechanisms involved in leukaemic transformation.

Topics: Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; DNA Primers; Erythropoietin; Glycophorins; Granulocyte-Macrophage Colony-Stimulating Factor; Hemoglobins; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Oligonucleotides, Antisense; Polymerase Chain Reaction; Retroviridae; Tumor Cells, Cultured

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.

Topics: Animals; Blotting, Western; Cell Differentiation; DNA-Binding Proteins; ErbB Receptors; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; GATA1 Transcription Factor; Globins; Hemoglobins; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Milk Proteins; Oligonucleotides, Antisense; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Recombinant Fusion Proteins; STAT5 Transcription Factor; Trans-Activators; Transcription Factors; Transfection; Tumor Cells, Cultured

Spi-1/PU.1 is a myeloid- and B-cell specific transcription factor which is also involved in Friend virus-induced murine erythroleukemia. The pre-leukemic phase of Friend erythroleukemia results from activation of the erythropoietin receptor (EpoR) by the spleen focus forming virus (SFFV) envelope glycoprotein, followed by the emergence of leukemic clones characterized by overexpression of Spi-1 and mutation of the p53 tumor suppressor gene. We developed a heterologous system to analyze the contribution of these alterations to the induction of primary erythroblast transformation. Avian erythroblasts expressing the activated mouse EpoR(R129C) differentiated into erythrocytes in response to hEpo. Expression of Spi-1 in these cells inhibited this ability to differentiate and rescued the cells from the apoptotic cell death program normally induced upon hEpo withdrawal. Although devoid of any effect by itself, a mutant p53 cooperated with Spi-1 and EpoR(R129C) to reinforce both phenotypes. Analysis of erythroblasts co-expressing Spi-1 and the wild-type mouse EpoR showed that differentiation arrest and inhibition of apoptosis depended on specific cooperation between Spi-1 and EpoR(R129C). This cooperation was also required to induce the sustained proliferation of differentiation-blocked erythroblasts in response to ligand activation of the endogenous tyrosine kinase receptor c-Kit. These results show that Spi-1/PU.1 requires signals emanating from specific cytokine and growth factor receptors to affect the survival, proliferation and differentiation control of primary erythroblasts. They also suggest that the function of Spi-1/PU.1 in the late phase of Friend leukemia requires specific signaling from the gp55-modified EpoR generated during the early phase of the disease.

Topics: Animals; Apoptosis; Bone Marrow Cells; Cell Differentiation; Cell Division; Cell Survival; Chickens; Erythroblasts; Erythropoietin; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Mice; Proto-Oncogene Proteins; Receptors, Erythropoietin; Recombinant Proteins; Spleen Focus-Forming Viruses; Stem Cell Factor; Trans-Activators; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Viral Envelope Proteins

Abnormal production of erythropoietin (Epo) has been described in several human and murine erythroleukemia. The murine IW32 cell line is derived from an F-MuLV-induced erythroleukemia. An autocrine Epo production due to the rearrangement of one Epo allele has been previously described (Beru et al., 1989). However, the exact mechanism leading to the transcriptional activation of the abnormal Epo gene was unknown. In this study, we show that this deregulated expression results from a deletion within chromosome 5. The Epo gene in the abnormal allele is under the control of the G-protein beta2 subunit gene promoter and the expressed mRNA results from the fusion of the non coding exon 1 of the G-protein beta2 subunit gene to a truncated Epo exon 1 gene. This resulting abnormal cDNA allows the expression of a normal Epo protein.

Topics: Animals; Base Sequence; DNA, Complementary; Erythropoietin; Gene Expression Regulation, Neoplastic; Gene Rearrangement; GTP-Binding Proteins; Humans; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Tumor Cells, Cultured

The SCL gene (also called Tal-1 or TCL5) was identified because of its association with chromosomal translocations in childhood T-cell lymphoid leukemias. SCL codes for a basic helix-loop-helix (bHLH) factor that can function as a transcriptional activator or repressor. In the adult, SCL expression is restricted to hematopoietic cells and tissues, but its function in the process of lineage commitment is unknown. The present study was designed to address the role of SCL in hematopoietic cell differentiation. SCL expression was determined in primary hematopoietic cells through the screening of cDNA samples obtained by reverse transcription-polymerase chain reaction (RT-PCR) from single cells at different stages of differentiation. SCL RNA expression was highest in bipotential and committed erythroid precursors and diminished with subsequent maturation to proerythroblasts and normoblasts. In contrast, SCL mRNA was low to undetectable in precursors of granulocytes and monocytes and their maturing progeny. The same pattern of expression was observed after erythroid or monocytic differentiation of a bipotent cell line, TF-1, in that SCL mRNA levels remained elevated during erythroid differentiation and were downregulated with monocytic differentiation. Accordingly, TF-1 was chosen as a model to investigate the functional significance of this divergent pattern of SCL expression in the two lineages. Four independent clones stably transfected with an SCL expression vector exhibited enhanced spontaneous and delta-aminolevulinic acid-induced erythroid differentiation as measured by glycophorin expression and hemoglobinization, consistent with the view that SCL is a positive regulator of erythroid differentiation. Furthermore, constitutive SCL expression interfered with monocytic differentiation, as assessed by the generation of adherent cells and the expression of Fc gamma RII in response to TPA. These results suggest that the downregulation of SCL may be required for monocytic differentiation.

Topics: Adult; Aminolevulinic Acid; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Lineage; DNA-Binding Proteins; DNA, Complementary; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Helix-Loop-Helix Motifs; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Monocytes; Polymerase Chain Reaction; Proto-Oncogene Proteins; Recombinant Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tumor Cells, Cultured

To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation.

Topics: Androstadienes; Cell Differentiation; Cell Line; Enzyme Inhibitors; Erythropoietin; Gene Expression; Glycophorins; Heme; Hemoglobins; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Time Factors; Tumor Cells, Cultured; Wortmannin

Retroviral insertional activation of Fli-1 is the first detectable genetic alteration associated with F-MuLV-induced primary erythroleukemias, while mutations within p53 are only observed in Epo-dependent (ED) cell lines derived from syngeneic mice serially transplanted with F-MuLV-induced primary erythroleukemias. In this study we have determined the mechanism of growth factor independence in several Epo-independent (EI) cell lines established from adult mice previously injected with ED-erythroleukemia cell lines or serially transplanted primary tumor cells. Here we have shown constitutive expression of the Epo gene in 12 of 15 (80%) EI-erythroleukemia cell lines. Among these 12 cell lines, eight were shown to possess clonal rearrangement of the Epo gene which could be detected in the tumors used to establish the majority of these EI-cell lines. Analysis of the pattern of proviral integration revealed that the activation of the Epo gene in these cell lines is independent of retroviral insertional mutagenesis, but apparently the result of genomic rearrangements. Furthermore, the acquisition of growth factor independence by these leukemic cells confers a selective growth advantage in vivo and is associated with enhanced tumorigenicity. Together these observations suggest that the activation of the Epo gene in the large majority of these F-MuLV-induced erythroleukemic cell lines establishes an autocrine loop resulting in the constitutive activation of the Epo receptor signal transduction pathway, thereby conferring a growth and survival advantage in vito and in vitro.

Topics: Animals; Antibodies; Base Sequence; DNA Primers; Erythropoietin; Friend murine leukemia virus; Gene Expression; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Neutralization Tests

The murine erythropoietin-dependent erythroleukemia cell line, HCD-57, was employed to study the cell cycle-specific behavior of erythropoietin. Cell cycle duration for HCD-57 cells was approximately 12 hours and was uninfluenced by erythropoietin. Populations of HCD-57 cells synchronized in G1 by centrifugal elutriation were able to pass through one complete cell cycle in the absence of erythropoietin but, thereafter, arrested in G1 as identified by propidium iodide staining and flow cytometry. Analysis of cell cycle behavior using the metachromic dye acridine orange, however, revealed that HCD-57 cells pass through a G0 cell cycle phase and, like serum-deprived 3T3 cells, actually arrest in G0 when deprived of erythropoietin. Expression of the cell cycle regulatory protein p34cdc2 was invariant throughout the cell cycle in HCD-57 cells. p34cdc2 was constitutively phosphorylated in G0 cells, and this effect was not modified by erythropoietin. Erythropoietin receptor distribution was log normal in HCD-57 cells in each phase of the cell cycle. The affinity of these surface receptors for erythropoietin was essentially invariant throughout the cell cycle, but receptor expression was upregulated in G2M cells as compared with cells in G1 or S phase. Taken together, these data indicate that erythropoietin has an important role in the G0-G1 to S phase transition but, based on receptor expression, is involved in other phases of the cell cycle as well.

Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Erythropoietin; Fibroblasts; Flow Cytometry; G1 Phase; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Receptors, Erythropoietin; Resting Phase, Cell Cycle; S Phase; Tumor Cells, Cultured

Down-regulation of c-myb mRNA levels by [Ca2+]i-increasing agents (A23187, thapsigargin, cyclopiazonic acid) and erythropoietin was comparatively studied in the erythropoietin-responsive murine erythroleukemia cell line, ELM-I-1. The Ca2+-induced suppression of c-myb mRNA could be inhibited by the calmodulin antagonists trifluoperazine and calmidazolium, as well as by cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinases, did not antagonize the Ca2+-mediated decrease in c-myb mRNA. In cyclosporin A-treated ELM-I-1 cells, a close correlation could be demonstrated between the antagonization of the Ca2+ effect on c-myb mRNA levels and inhibition of the calcineurin phophatase activity. On the other hand, FK506, which did not inhibit calcineurin activity in ELM-I-1 cells, failed to prevent the Ca2+-mediated decrease in c-myb mRNA. The erythropoietin-induced down-regulation of c-myb mRNA levels could be demonstrated also in the presence of EGTA and was resistant to calmodulin antagonists and cyclosporin A. In addition, no increase in [Ca2+]i was observed in ELM-I-1 cells in response to erythropoietin. Cyclosporin A inhibited the Ca2+-induced hemoglobin production, while the erythropoietin-mediated increase in hemoglobin synthesis was not affected. The results indicate that the Ca2+-induced decrease in c-myb mRNA and increase in hemoglobin synthesis is mediated by calcineurin, while these effects of erythropoietin occur independently of Ca2+ in ELM-I-1 cells. Calcineurin may be involved in the regulation of c-myb expression in erythroid precursor cells and Ca2+ signals via calcineurin may positively modulate the differentiation inducing action of erythropoietin.

Topics: Animals; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cyclosporine; Down-Regulation; Enzyme Inhibitors; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, myc; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; RNA, Messenger; RNA, Neoplasm; Tacrolimus; Tumor Cells, Cultured

The human erythropoietin receptor (EpoR) gene has been cloned and characterized. Very few EpoR genetic abnormalities have been reported so far. Polycythemia vera (PV) is characterized by low/normal serum erythropoietin (Epo) levels with proposed Epo hypersensitivity. Myelodysplastic syndromes (MDS) are characterized by refractory anemia with variable serum Epo levels. Several reports have suggested EpoR abnormalities in both types of stem cell disorders. We analyzed DNA obtained from peripheral blood mononuclear cells of seven healthy controls, 20 patients with myeloproliferative disorders (MPD, 11 patients with PV, five agnogenic myeloid metaplasia with myelofibrosis, four essential thrombocytosis) and eight patients with refractory anemia with ringed sideroblasts (RARS), an MDS variant. The DNA was digested with four restriction enzymes (BamHI, Bgl II, Sacl and HindIII), followed by Southern blot, using a 32P radiolabeled probe, containing 1.5 kb of the human EpoR cDNA. All 20 MPD patients and seven out of the eight MDS patients demonstrated a restriction pattern which was identical to the seven normal controls, as well as to the erythroid cell line K562, and also consistent with the expected restriction map, for all four enzymes tested. One RARS patient had a normal pattern with three enzymes but a different one with HindIII. The HindIII 12 kb large band was replaced by a faint 12 kb band and a new (about 9 kb) band appeared. The EpoR restriction map and the normal pattern obtained with the other three enzymes suggest that this patient has a 3 kb upstream deletion in one allelic EpoR gene. The same molecular pattern was detected in the patient's sister, who suffers from anemia with mild bone marrow (BM) dyserythropoiesis and plasmacytosis. Northern blot analysis showed that the patient's BM RNA carried normal EpoR message. This familial pattern may represent polymorphism. However, the patient's very high serum Epo level, her resistance to treatment with recombinant Epo, and the abnormally low growth rate of in vitro erythroid cultures, suggesting poor response to Epo in this MDS patient as well as the hematological abnormalities in her sister, support the speculation that the different EpoR gene might serve as a genetic predisposing marker and potentially could be involved (probably via post-transcriptional mechanisms and by an interaction with other factors or cytokines) in the pathogenesis. Our data suggest that the EpoR is intact in MPD and in most

Topics: Aged; Blotting, Northern; DNA; DNA, Neoplasm; Erythroid Precursor Cells; Erythropoietin; Family Health; Female; Humans; Leukemia, Erythroblastic, Acute; Male; Myelodysplastic Syndromes; Myeloproliferative Disorders; Prognosis; Receptors, Erythropoietin; RNA; Tumor Cells, Cultured

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.

Topics: Animals; Apoptosis; Base Sequence; bcl-X Protein; Cell Differentiation; Cell Survival; Erythroid Precursor Cells; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

The TF-1 cell line has been established from a patient with erythroleukemia. While various cytokines induce TF-1 cell proliferation, erythropoietin (Epo) only sustains the short-term growth of these cells and induces their differentiation along the erythroid lineage. A truncated Epo receptor (EpoR) is overexpressed in these cells. The truncation removed the 96 C-terminal amino acids, including seven tyrosine residues. An additional single mutation at position +3 of Tyr344 led to the replacement of leucine 347 by proline. Stimulation by Epo induced an impaired activation of the STAT5 transcription factor in these cells. The same defect in STAT5 activation was found in the murine FDCP-1 cell line transfected with a chimeric EpoR containing the abnormal TF-1 EpoR cytoplasmic domain. Infection of TF-1 cells with a retrovirus containing a normal murine EpoR was able to restore both Epo-induced STAT5 activity and cellular proliferation. In contrast, Epo-induced differentiation was reduced strongly in infected TF-1ER cells. These results suggest that Epo-induced differentiation correlates with impaired Epo-induced STAT5 activation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Differentiation; Cloning, Molecular; DNA-Binding Proteins; DNA, Complementary; DNA, Neoplasm; Erythropoietin; Gene Expression Regulation, Leukemic; Humans; Leukemia, Erythroblastic, Acute; Mice; Milk Proteins; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Receptors, Erythropoietin; Recombinant Fusion Proteins; Sequence Deletion; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Antisense oligomers (18 mers) corresponding to the erythropoietin and erythropoietin receptor 5' coding sequences cause marked suppression of proliferation of several lines of erythroleukemic cells. In these systems, phosphorothioate protected sense oligomers are inhibitory, while the unmodified sense oligomers have no significant effect on cell growth. These data indicate that proliferation of some erythroleukemic cells is under internal autocrine regulation by erythropoietin and its receptor.

Topics: Animals; Cell Division; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Oligonucleotides, Antisense; Receptors, Erythropoietin; Thionucleotides; Tumor Cells, Cultured

The human erythroleukemia cell line TF-1 was employed for the determination of proliferative stimulation induced by recombinant human erythropoietin (rhEpo). Potencies of various intact and sugar-trimmed rhEpo preparations were estimated using the International Standard for Human r-DNA-derived Epo (87/684) as a reference for activity. The cellular response was measured in a multi-channel photometer using a colorimetric microassay, based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan, by viable cells. The linear part of the log dose-response relationship encompassed 2.5-90 pM and activity of rhEpo preparations was measured at doses between 3 and 60 pM. The assay was designed as a parallel line test, using three or four concentrations for potency determinations, which fulfills pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation varied between 4.1% and 12.6% and most assays revealed potencies with limits of error within 87-113%. In order to acquire an additional means for an efficient probing of physiologically relevant features of rhEpo, a luminiscence-dependent Western detection system, based on a combined isoelectric focusing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis separation, was established. As opposed to conventional electrophoresis the two dimensional approach enabled the disclosure of minor truncations in the rhEpo-attached glycan moieties using picomolar quantities of the hormone. Moreover, the separated isoforms of rhEpo were quantified by computer-assisted densitometry and compared with the 87/684 standard. Accordingly, results obtained by the cellular response were balanced against the general pattern observed and the relative amounts of separated rhEpo isomers as determined by the quantitative Western analysis. The method described should be suitable for potency assessments of pharmaceutical formulations of rhEpo.

Topics: Antibodies; Blotting, Western; Carbohydrates; Cell Division; Electrophoresis, Gel, Two-Dimensional; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Recombinant Proteins; Tumor Cells, Cultured

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.

Topics: Cell Division; Cell Line; Cloning, Molecular; DNA Restriction Enzymes; DNA, Complementary; Erythropoietin; Gene Transfer Techniques; Genetic Markers; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kanamycin Kinase; Leukemia, Erythroblastic, Acute; Phosphotransferases (Alcohol Group Acceptor); Radioimmunoassay; Tumor Cells, Cultured

Three different surface modifications were conducted on the membranes of double-layer alginate/poly(L-lysine) microcapsules with tosyl chloride-activated poly(ethylene glycol), cyanuric chloride-activated poly(ethylene glycol) and tosyl chloride-activated poly(vinyl alcohol), separately. All these surface modifications strengthen the microcapsular membranes. Of the three surface-modified microcapsules, those treated with cyanuric poly(ethylene glycol) and tosylated poly(vinyl alcohol) have mechanical strength higher than that treated with tosylated poly(ethylene glycol). The permeabilities of the proteins were only slightly affected by the surface modifications. When IW32 erythroleukaemia cells were entrapped inside these microcapsules, cells proliferated to a density of (1.0-1.5) x 10(7) cells cm-3 after 7 d culturing compared with 2 x 10(6) cells cm-3 attained by free cell culturing. The intracapsular concentration of erythropoietin which was secreted by the IW32 cells accumulated to concentrations of 5-7 U cm-3. In addition, 3T3 cells were found to have a very low tendency to attach to the microcapsular membranes of all three surface-modified preparations.

Topics: 3T3 Cells; Alginates; Animals; Biocompatible Materials; Biomechanical Phenomena; Capsules; Cell Adhesion; Cell Division; Drug Carriers; Erythropoietin; Fibroblasts; Friend murine leukemia virus; Glucuronic Acid; Hexuronic Acids; Leukemia, Erythroblastic, Acute; Membranes, Artificial; Mice; Polyethylene Glycols; Polylysine; Polyvinyl Alcohol; Surface Properties; Tosyl Compounds; Triazines; Tumor Cells, Cultured

Topics: Adsorption; Animals; Cell Membrane; Erythropoietin; Genetic Vectors; Humans; Leukemia, Erythroblastic, Acute; Mice; Moloney murine leukemia virus; Receptors, Erythropoietin; Receptors, Virus; Recombinant Fusion Proteins; Tumor Cells, Cultured; Viral Envelope Proteins

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.

Topics: Animals; Autoradiography; Base Sequence; Binding Sites; Brain; DNA Primers; Erythropoietin; Frontal Lobe; Gene Expression; Iodine Radioisotopes; Kidney; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Receptors, Erythropoietin; Tumor Cells, Cultured

Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells--an erythropoietin-responsive model progenitor cell line--to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17- to 18-fold and 14- to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11- to 48-fold and 20- to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Cell Differentiation; Cell Division; Cells, Cultured; Cytokines; Erythropoietin; Fibroblasts; Hematopoietic Stem Cells; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Models, Biological; Phenylhydrazines; Platelet-Derived Growth Factor; Recombinant Proteins; Spleen; Time Factors; Tumor Cells, Cultured

The J2E cell line is an immature erythroid line which terminally differentiates in response to erythropoietin (epo), producing mature, hemoglobin-synthesizing red blood cells. We have shown that when these cells were injected into mice a rapid and fatal erythroleukemia developed with symptoms of severe anemia and hepatosplenomegaly. Southern blotting demonstrated that the leukemic cells were the introduced J2E cells. In addition to spleen and liver, the bone marrow was a major site of leukemic cell infiltration, and when grown in vitro leukemic cells from bone marrow remained responsive to erythropoietin. We reasoned, therefore, that treatment of mice with this hormone should alleviate the erythroleukemia, but regular injections of epo in vivo failed to arrest the progress of the disease. However, when bone marrow from leukemic mice was exposed continuously to the hormone ex vivo, before reinfusion into naive recipients, a marked extension in life span was observed. It was concluded that ex vivo epo treatment could be used therapeutically for J2E cell erythroleukemias.

Topics: Animals; Cell Differentiation; Dose-Response Relationship, Drug; Erythroblasts; Erythropoietin; Female; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Tumor Cells, Cultured

Molecular cloning of a cDNA for a mouse erythropoietin (Epo) receptor (EpoR) has facilitated the understanding of the structure of this receptor. However, there is, as yet, no explanation for the discrepancy between the protein recognized by specific antibodies against mouse EpoR and the unexpectedly larger species that can be cross-linked to labeled Epo. It is unclear whether the product of an unidentified gene is included in the EpoR complex. In the present study, we directly compared the cross-linking patterns for human EpoR that were endogenously expressed in three types of Epo-responsive cell, and that was artificially expressed in nonhematopoietic cells after transfection with cDNA for human EpoR. We observed that 85-kD and 105-kD proteins formed ligand-receptor complexes in all the human Epo-responsive cells and, furthermore, that the formation of a complex derived from the 70-kD protein was dependent on the level of expression of the cloned EpoR mRNA in these cells. By contrast, a prominent cross-linked band derived from the 70-kD protein and a weaker band derived from the 80- to 85-kD protein, but no band derived from the 105-kD protein, could be shown in the case of nonhematopoietic cells transfected with the human EpoR cDNA. These observations suggest that the cloned cDNA for human EpoR alone does not allow generation of the complete EpoR in nonhematopoietic cells and that the 105-kD Epo-binding protein may represent the product of an as yet unidentified gene that is expressed in hematopoietic cells.

Topics: Amnion; Animals; Blotting, Northern; Blotting, Southern; Cell Line; Chlorocebus aethiops; Cross-Linking Reagents; DNA, Complementary; Erythropoietin; Gene Expression; Humans; Immunosorbent Techniques; Iodine Radioisotopes; Kidney; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Receptors, Erythropoietin; RNA, Messenger; Transfection; Tumor Cells, Cultured

We previously identified a translocation breakpoint in exon 8 of the erythropoietin receptor (EpoR) gene in TF-1 cells, a cell line derived from a human erythroleukemia. To investigate the potential pathogenetic significance of this abnormality, we more precisely mapped the breakpoint within exon 8 and studied the expression of the translocated gene by S1 nuclease mapping of EpoR transcripts and chemical crosslinking of labeled erythropoietin (Epo) to TF-1 cell surface receptors. Transcripts from the abnormal gene were found to be highly expressed in relation to normal EpoR transcripts in TF-1 cells. The breakpoint predicted by S1 mapping of abnormal EpoR transcripts agreed closely with that determined by Southern analysis. Chemical cross-linking of 125I-Epo to TF-1 cells showed an abnormal, low-molecular-weight cross-linked species directly recognized by anti-EpoR antibodies and present in considerable excess over the normal EpoR. Karyotype analysis showed that each of 10 TF-1 cell metaphases had, in addition to multiple other alterations, one chromosome 19 with additional chromosomal material translocated onto the short arm at 19p13.3, the location of the EpoR gene. We conclude that the structurally abnormal EpoR gene in TF-1 cells is highly expressed and produces an abnormal protein. We speculate that the chromosomal material brought into the EpoR locus by translocation is responsible for the high level of expression. We hypothesize that this translocation participated in the evolution of the erythroleukemia from which TF-1 cells were derived.

Topics: Base Sequence; Chromosomes, Human, Pair 19; Cross-Linking Reagents; DNA Probes; DNA, Neoplasm; Erythropoietin; Exons; Humans; Immunosorbent Techniques; Karyotyping; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Nucleic Acid Hybridization; Receptors, Erythropoietin; Restriction Mapping; RNA, Messenger; Single-Strand Specific DNA and RNA Endonucleases; Translocation, Genetic; Tumor Cells, Cultured

The signal transduction mechanism of erythropoietin (Epo), which regulates growth and differentiation of erythroid cells, is still unclear. Recent studies showing the activation by various ligands of a group of proteins called Stat (signal transducers and activators of transcription) proteins raised the possibility that such proteins may also be involved in the Epo signal transduction pathway. In this report, we show that Epo induces factors that specifically bind to the sis-inducible element and the gamma response region of the Fc gamma receptor factor I gene in the Epo-dependent mouse erythroleukemia cell line HCD-57. These factors contain phosphotyrosine and antibodies against Stat1 and Stat3 proteins reacted with them. In HCD-57 cells infected with Friend spleen focus-forming virus, which now grow in an Epo-independent manner, the DNA-binding factors were constitutively activated even in the absence of Epo. These results suggest that the factors induced by Epo contain components identical or related to known Stat proteins. It is also suggested that continuous activation of these DNA-binding factors may be responsible for the ability of spleen focus-forming virus to abrogate the Epo-dependence of HCD-57 cells and cause erythroleukemia in susceptible mice.

Topics: Animals; Base Sequence; DNA; DNA-Binding Proteins; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Receptors, IgG; RNA, Messenger; Spleen Focus-Forming Viruses; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

The murine erythroleukaemia cell line HCD-57 proliferates in response to erythropoietin. Stimulation of erythropoietin-deprived cells with the cytokine induces the phosphorylation and biphasic activation of the 70,000 M(r) S6 kinase. Two peaks of enzyme activity were observed after 30 and 120 min, respectively. Early and late phase of activation differ in their sensitivity to the protein kinase C inhibitor staurosporine suggesting different regulatory mechanisms.

Topics: Alkaloids; Animals; Cell Survival; DNA, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Genistein; Isoflavones; Leukemia, Erythroblastic, Acute; Mice; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases; Signal Transduction; Staurosporine; Tumor Cells, Cultured

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.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Drug Resistance; Erythroid Precursor Cells; Erythropoietin; Female; Interferon-alpha; Leukemia, Erythroblastic, Acute; Mice; Necrosis; Spleen; Treatment Outcome

The survival and proliferation of the UT-7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte-macrophage colony-stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin-stimulated UT-7 cells by anion-exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50-kDa kinase, whereas the second peak was only present in erythropoietin-stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin-stimulated cells. The 44-kDa and 42-kDa forms corresponded to extracellular signal-regulated kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showing that the 45-kDa form is not a shifted form of ERK-1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK-4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin-stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin-induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF-1, was hyperphosphorylated in erythropoietin-stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT-7 erythropoietin-stimulated cells and associated strongly with a unidentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both unstimulated and erythropoietin-stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.

Topics: Adaptor Proteins, Signal Transducing; Biological Transport; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Membrane; Cyclic AMP; Enzyme Activation; Erythropoietin; GRB2 Adaptor Protein; Humans; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Proteins; Signal Transduction; Tumor Cells, Cultured; Tyrosine

Topics: Cell Line; Erythropoietin; Growth Substances; Hematopoietic Cell Growth Factors; Humans; Leukemia, Erythroblastic, Acute; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Phosphotyrosine; Receptors, Erythropoietin; Signal Transduction; Stem Cell Factor; Transfection; Tumor Cells, Cultured; Tyrosine

We constructed a recombinant plasmid which expresses antisense src RNA in human cells and used it as a tool for investigating the role of pp60c-src in proliferation and differentiation of K562 human leukemia cells. In erythropoietin (EPO)-responsive cells, EPO induces rapid tyrosine phosphorylation of several cellular proteins including EPO receptor (EPOR) although EPOR has no tyrosine kinase domain. Here we show that antisense src RNA expression suppresses pp60c-src synthesis in the recombinant plasmid-transfected K562 cells, reduces the proliferation and inhibits hemoglobin synthesis and glycophorin A expression promoted by EPO in K562 cells. These findings suggest that pp60c-src plays crucial roles in the proliferation and EPO-induced erythroid differentiation of K562 cells.

Topics: Cell Differentiation; Cell Division; Erythropoiesis; Erythropoietin; Genes, src; Glycophorins; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Proto-Oncogene Proteins pp60(c-src); RNA, Antisense; Tumor Cells, Cultured

MAP kinases are a family of serine/threonine specific protein kinases becoming activated in response to different proliferative stimuli by phosphorylation at both threonine and tyrosine residues. We report the involvement of MAP kinases in the signal transduction of the hematopoietic growth factors erythropoietin (EPO), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the factor dependent human erythroleukemic cell line TF-1, suggesting a crucial role of these enzymes in the regulation of proliferation of hematopoietic cells. Both time course and degree of MAP kinase activation were similar for all three cytokines. A slightly lower stimulation effect of EPO corresponds to the observation that EPO stimulated cells proliferate at a lower rate.

Topics: 3T3 Cells; Animals; Cell Division; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Mice; Mitogen-Activated Protein Kinase 1; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Recombinant Proteins; Signal Transduction

Protein phosphorylation is an early event that follows the interaction of erythropoietin (Epo) with its receptor, even though this receptor lacks a kinase domain. To further define the role of protein kinases in Epo-mediated signal transduction, the effect of Epo on serine-threonine kinase activity was examined in the Epo-dependent cell line, HCD-57, using a kinase renaturation assay. In HCD-57 cells synchronized in G0 phase by centrifugal elutriation, multiple serine-threonine kinases were constitutively active, and exposure to Epo was associated with an increase in the activity of kinases with apparent molecular masses of 170, 120, and 90-95 kD. Phosphoamino acid analysis established the covalent incorporation of 32P into serine and threonine for constitutively active kinases and into serine alone for the 90-95 kD kinase. Reelectrophoresis experiments established that 32P incorporation represented kinase autophosphorylation as opposed to protein substrate phosphorylation. Epo-associated serine kinase autophosphorylation was both hormone concentration and time dependent as well as restricted to the G0, G1, and S phases of the cell cycle. Cell fractionation studies localized the activity of the 90-95 kD serine kinase to the plasma membrane.

Topics: Animals; Cell Cycle; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Cells, Cultured

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.

Topics: Adenosine Diphosphate; Adenosine Diphosphate Ribose; Animals; Cell Differentiation; Cell Division; Cyclic AMP; Dimethyl Sulfoxide; Drug Synergism; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation, Leukemic; GTP-Binding Proteins; Humans; Isoproterenol; Leukemia, Erythroblastic, Acute; Mice; Neoplasm Proteins; Neoplastic Stem Cells; Pertussis Toxin; Receptors, Adrenergic, beta; Recombinant Proteins; Signal Transduction; Thrombin; Tumor Cells, Cultured; Virulence Factors, Bordetella

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacements at 51 conserved sites predicted to be on the surface of the molecule. Alanine replacements permitted preservation of alpha-helical structure. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type and mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell line, and the human UT7-EPO leukemia cell line. When Arg14 on predicted Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specific bioactivity. In a similar manner, the mutein Arg103-->Ala in Helix C was completely lacking in biological activity, whereas both Ser104-->Ala and Leu108-->Ala had decreased bioactivity. In Helix D, the mutein Gly151-->Ala had markedly decreased bioactivity, whereas that of the adjacent Lys152-->Ala mutein was moderately impaired. In contrast, Ala replacements at three nearby sites on Helix D (147, 146, and 143) resulted in muteins with increased bioactivity. In conclusion, our mutagenesis experiments have identified functionally important domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.

Topics: Amino Acid Sequence; Animals; Biological Assay; Cell Division; Cell Line; Chlorocebus aethiops; Erythropoietin; Humans; Leukemia; Leukemia, Erythroblastic, Acute; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Protein Structure, Secondary; Radioimmunoassay; Recombinant Proteins; Thymidine; Transfection; Tumor Cells, Cultured

Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5' flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid-specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5' flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.

Topics: Animals; Base Sequence; Binding Sites; Deoxyribonuclease I; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation; In Vitro Techniques; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Nucleosomes; Promoter Regions, Genetic; Restriction Mapping; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Erythropoietin (EPO) is a hematopoietic growth factor that stimulates the proliferation and differentiation of erythroid progenitor cells. Although the EPO receptor has no kinase domain, EPO rapidly induces tyrosine phosphorylation of several proteins in EPO-responsive cells. Therefore, the receptor activation by the ligand could induce tyrosine-kinase activity of unidentified cellular protein(s). Here we show that c-fps/fes proto-oncogene product (p92c-fes), nonreceptor tyrosine kinase, is tyrosine-phosphorylated on treatment with EPO in a human erythroleukemia cell line TF-1 that is responsive to granulocyte-macrophage colony-stimulating factor, interleukin-3, and EPO. In addition, the kinase activity of p92c-fes was shown to be enhanced by treatment with EPO. Therefore, p92c-fes could be implicated in a signaling pathway triggered by EPO in human EPO-responsive cells.

Topics: Amino Acid Sequence; Blotting, Western; Cell Differentiation; Cell Division; Erythropoietin; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunosorbent Techniques; Interleukin-3; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Tumor Cells, Cultured; Tyrosine

Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB-02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose-dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c-kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Division; Cell Survival; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Molecular Weight; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; RNA, Messenger; Stem Cell Factor; Tumor Cells, Cultured

Cell surface ligand-receptor systems provide favorable routes for DNA transfection due to target cell specificity, transfer efficiency, and low toxicity. Using the transferrin receptor system as a model, an approach to transfection is developed here within which involves the complexing of DNA to stable maleimido-transferrin/thio-poly-L-lysine conjugates. These studies establish the importance of precise stoichiometry for activity of ligand:poly-L-lysine conjugates, as well as a chemistry for their controlled conjugation. Also considered quantitatively are effects of the following related parameters on the efficiency of receptor-mediated transfection: lysine polymer length, conjugate concentration, DNA:conjugate ratio, and treatment of target cells with chloroquine and desferrioxamine. Compared directly to standard procedures (electroporation, modified DEAE-dextran, lipofection, and modified Ca2PO4 protocols), transfection via this transferrin receptor-mediated system was > or = 10-fold more efficient, and essentially nontoxic to erythroleukemic F-MEL and J2E cells. Following transfection these cells retained the physiological capacity to undergo induced differentiation in response to dimethyl sulfoxide (F-MEL cells), or to erythropoietin (J2E cells), the natural hormonal regulator of erythropoiesis. Thus, an optimized approach to transferrin receptor-mediated transfection is developed which should be widely applicable for alternate cells and ligand-receptor systems both in vitro and in vivo.

Topics: Animals; Cell Differentiation; DNA; Drug Stability; Endocytosis; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Maleimides; Mice; Polylysine; Receptors, Transferrin; Transfection; Transferrin; Tumor Cells, Cultured

Erythroid colony-stimulating factor (E-CSF) is a B cell-derived membrane protein that specifically affects the growth and development of human and murine committed erythroid progenitors. We report the development of a sensitive new bioassay for E-CSF, based on the ability of the growth factor to stimulate 3H-thymidine incorporation into cloned Rauscher murine erythroleukemia cells. The assay has among its advantages the ability to measure growth factor activity on a purified target cell population in the absence of endogenous growth factor-producing accessory cells. In addition, this assay measures E-CSF's proliferative effect on erythroid progenitors in the absence of erythropoietin (Epo) after 72 to 96 hours. In contrast, the standard bone marrow fibrin clot assay traditionally used to measure E-CSF requires the addition of Epo to promote the development of hemoglobinized erythroid colonies that are quantified after 7 days (for murine cells) to 12 days (for human cells). With the use of this new Rauscher cell bioassay, we have identified an E-CSF-producing human cell line and, further, have measured E-CSF activity derived from nonhuman splenic B lymphocytes.

Topics: Animals; B-Lymphocytes; Biological Assay; Cell Division; Cell Line, Transformed; Colony-Stimulating Factors; DNA; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured

The murine erythroleukemic cell lines NN10 and IW32 secrete erythropoietin (Epo) constitutively. Although both cell lines have Epo receptors (Epo-R), they do not respond to external Epo to undergo erythroid differentiation. In both cell lines, hemoglobin synthesis can be induced by hemin or butyrate. We report here on the effects of these two inducers on Epo production by the same cell lines. Both hemin and butyrate caused a three- to five-fold increase in Epo secretion on a percell basis. The effects were dose- and time-dependent, as was found previously for hemoglobin synthesis. The mechanism of the effect on Epo secretion is posttranscriptional, since the steady-state level of Epo mRNA was not altered by the inducers.

Topics: Animals; Butyrates; Cell Count; Erythropoietin; Hemin; Leukemia, Erythroblastic, Acute; Mice; Receptors, Erythropoietin; RNA Processing, Post-Transcriptional; RNA, Messenger; Time Factors; Tumor Cells, Cultured

An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hmm-EPOR are more highly glycosylated forms of the EPOR, appear on the cell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, correlating with a 10-fold increase in binding of 125I-labeled EPO. In addition, the EPO-dependent downregulation of 125I-EPO binding and disappearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells expressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.

Topics: Animals; Cell Compartmentation; Endocytosis; Erythropoietin; Glycoproteins; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Phosphorylation; Protein Processing, Post-Translational; Receptors, Erythropoietin; Recombinant Proteins; Tumor Cells, Cultured

Topics: Adolescent; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Male

The selection of differentiation lineages into either erythroid or megakaryocytic series was analyzed with human erythroleukemia cell lines K562, HEL, and cytokine-dependent TF1. A tumor promoter, TPA, induced a megakaryocyte marker, glycoprotein IIb/IIIa (GP IIb/IIIa) or IIIa (GP IIIa), but suppressed erythroid differentiation. On the other hand, aphidicolin, which is a potent inhibitor of DNA replication, inhibited GP IIb/IIIa or IIIa expression, but induced the expression of erythroid phenotypes. These phenomena were observed in all erythroleukemia cell lines tested. The bromodeoxyuridine labeling experiments indicated that de novo DNA synthesis was completely suppressed by aphidicolin treatment but was well preserved in TPA-treated cells. Among these three cell lines, erythropoietin (EPO) treatment induced erythroid differentiation of TF1 cells, which was dependent on GM-CSF or IL-3. In this case, EPO functioned as the survival factor and mild stimulator for cell proliferation as well as the inducer of erythroid differentiation. However, when either GM-CSF or IL-3 was depleted from the culture medium, TF1 ceased cell growth; concomitantly, hemoglobin-positive cells appeared, which is consistent with the results obtained with aphidicolin. The incubation of K562 cells for 48 h with either TPA or aphidicolin induced the irreversible commitment of cells to megakaryocytic and erythroid lineages, respectively. Our results using three different erythroleukemia cell lines suggest that a possible linkage between the DNA replication system and the selection of a differentiation lineage is the common feature of human erythroleukemia cell lines, and that these culture systems provide a suitable model for the analysis of the signal transduction system for differentiation lineage selection.

Topics: Aphidicolin; Cell Differentiation; DNA Replication; Erythroid Precursor Cells; Erythropoietin; Hematopoiesis; Humans; In Vitro Techniques; Leukemia, Erythroblastic, Acute; Megakaryocytes; Tumor Cells, Cultured

Topics: Animals; Cell Line, Transformed; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Receptors, Erythropoietin

The erythroid-potentiating activity (EPA) of the tissue inhibitor of metalloproteinase-1 (TIMP-1) was re-examined using ELM-I-1-3, a mouse erythroleukemia cell line, which responded well to erythropoietin. Depletion of pre-existing TIMP-1 from fetal calf serum in culture medium using monoclonal antibody suppressed erythropoietin-induced differentiation as measured by the induction of hemoglobin, commitment assay and globin mRNAs. The removal of TIMP-1 also suppressed the proliferation of ELM-I-1-3 as measured by cell number and de novo DNA synthesis. These changes were reversed by the addition of purified TIMP-1 to the culture medium. Anti-TIMP-1 antibody also blocked both hexamethylene bisacetamide (HMBA)-induced erythroid differentiation and the proliferation of both ELM-I-1-3 and Friend erythroleukemia cells. Considering previous reports analyzing the chemical induction of Friend mouse erythroleukemia cell differentiation, our results suggest that erythropoietin- or HMBA-induced erythroid differentiation might also be coupled with cell proliferation. Our 3H thymidine-uptake experiment shows that TIMP-1 removal was also effective in the inhibition of cell growth of various other cell lines in addition to erythroleukemia cell lines. These results suggest that EPA action of TIMP-1 on erythroid leukemia cell lines is closely related to its activity to promote the cell growth of various cell lines and cells including erythroleukemia cell lines.

Topics: Acetamides; Animals; Antibodies; Cell Differentiation; Cell Division; DNA, Neoplasm; Erythroid Precursor Cells; Erythropoietin; Friend murine leukemia virus; Gene Expression; Globins; Glycoproteins; Humans; Leukemia, Erythroblastic, Acute; Mice; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Erythropoietin; Friend murine leukemia virus; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred DBA; Mutation; Recombinant Proteins; Spleen; Spleen Focus-Forming Viruses; Tumor Suppressor Protein p53; Viral Envelope Proteins

To analyze the structure of the murine erythropoietin receptor (EpoR), wild-type or mutant EpoR cDNAs were expressed in cell lines, and the proteins that cross-linked with 125I-labeled erythropoietin (Epo) were analyzed by immunoprecipitation using an antibody against the intracellular region of the cloned EpoR. COS-7 cell transfectants expressing the wild-type EpoR showed two major cross-linked species of 145 and 110 Kd, both of which were recognized by the antibody against the cloned EpoR after denaturation under reducing conditions. Furthermore, a reduction in sizes of both cross-linked bands was observed in COS-7 transfectants expressing a mutant receptor with an internal deletion, thus indicating that both species contain the cloned EpoR. COS-7 cells expressing mutant receptors with carboxy-terminal deletions showed cross-linked bands corresponding to the smaller species of the two observed in cells expressing the wild-type receptor. In contrast to COS-7 cell transfectants, DA3 cells expressing wild-type or mutant EpoR cDNAs showed an additional cross-like species of 130 Kd. The size of this species was not altered by deletions in EpoR, showing that it did not contain EpoR. The 130-Kd cross-linked band, which would contain a 95-Kd protein, was also observed in a murine erythroleukemia cell line, D1B. These results suggest that Epo associates with a second component of 95 Kd, which is specifically expressed in hematopoietic cells.

Topics: Animals; Cell Line; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Mutation; Phosphorylation; Receptors, Erythropoietin; Transfection; Tumor Cells, Cultured

The human erythroleukaemic cell line K562, in response to various chemical agents, undergoes differentiation and exhibits exclusive production of fetal and embryonic haemoglobins. In this study we have compared the efficiency of natural growth factors interleukin-3 and erythropoietin and three chemical inducers such as dimethyl sulfoxide (DMSO, 1.9%), phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) and hemin (25 microM) on growth and differentiation of these cells. Erythropoietin significantly stimulated the growth of K562 cells (P<0.0001), while interleukin-3 did not (P = 0.2783). However, neither of these growth factors individually or together induced differentiation of K562 cells. Hemin appears to be more efficient than DMSO or PMA in differentiation of K562 cells as measured by benzidine positive cells (70% or more). The differentiation of K562 cells by hemin occurs independently of protein kinase-C activation and the arrest of DNA synthesis. In contrast, hemin significantly stimulated RNA and protein synthesis (P<0.0001) as measured by [3H]-uridine and [3H]-leucine incorporation respectively. Analysis of hemin-treated K562 nuclear extract on sodium dodecylsulphate gel electrophoresis showed that one protein band of molecular weight 70 kDa decreased after 48 h of incubation in the presence of 25 microM hemin. The disappearance of this protein can be prevented by cycloheximide (100 microg/ml) and actinomycin D (0.1 microg/ml) and thus indicating that the removal of 70 kDa protein seems to be dependent on RNA and protein synthesis. The regulatory role of 70 kDa protein in hemin-induced differentiation of K562 cells is discussed.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Erythropoietin; Hemin; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Restriction endonuclease mapping demonstrates a 3' end deletion of one erythropoietin receptor (EpoR) gene in TF-1 cells, a human erythroleukemia cell line that overexpresses the EpoR and proliferates in response to erythropoietin (Epo). EpoR mRNA transcripts are highly abundant and normal in size. These findings raise interesting questions about the possible role of this EpoR gene abnormality in the pathogenesis of the erythroleukemia from which this cell line was derived. This is the first report of an abnormal human erythropoietin receptor gene.

Topics: Blotting, Northern; Blotting, Southern; Chromosome Mapping; DNA Restriction Enzymes; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mutation; Receptors, Cell Surface; Receptors, Erythropoietin; Transcription, Genetic; Tumor Cells, Cultured

We have produced a series of monoclonal antibodies (MoAbs) to amino acid region 99-129 of human erythropoietin (Epo) that distinguish unique structural features within this putative receptor binding domain of the hormone. The MoAbs recognize denatured Epo with widely different sensitivities on a Western blot and differentially bind to native Epo in solution. In addition, three of the four MoAbs neutralize the biological activity of Epo in a concentration-dependent fashion in vitro. Neutralization was measured both by inhibition of Epo-induced differentiation in Rauscher murine erythroleukemia cells and by inhibition of Epo-induced proliferation in normal murine splenic erythroid precursors. Characterization of the structural epitopes recognized by each of these four reagent MoAbs should provide us with important information concerning the requirements for hormone-receptor interaction.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Cell Division; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Epitopes; Erythropoietin; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred BALB C; Precipitin Tests; Receptors, Cell Surface; Receptors, Erythropoietin; Spleen; Tumor Cells, Cultured

The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.

Topics: Animals; Cell Differentiation; Erythropoietin; Humans; Interleukin-2; Kinetics; Leukemia, Erythroblastic, Acute; Macromolecular Substances; Mice; Molecular Weight; Phosphoproteins; Phosphorylation; Phosphotyrosine; Receptors, Interleukin-2; Recombinant Proteins; Transfection; Tumor Cells, Cultured; Tyrosine

Human B-lymphocyte-derived erythroid burst-promoting activity (B-BPA) is a pleiotropic, lineage-specific regulator of erythropoiesis. Our present data indicate that B-BPA plays an important role as an erythroid colony-stimulating factor (E-CSF) in modulating progenitor growth and differentiation throughout erythropoiesis. E-CSF has discrete effects on both early (erythroid burst-forming units, BFU-E) and late (erythroid colony-forming units, CFU-E) progenitors from normal bone marrow. In serum-substituted fibrin clot cultures, E-CSF stimulates the proliferation of BFU-E, resulting in an increase in the number of erythroid bursts over a wide range of erythropoietin (Epo) concentrations. We now have shown that E-CSF also acts on CFU-E by increasing their sensitivity to Epo markedly, resulting in a tenfold left-shift in the Epo dose-response curve. Using purified target-cell populations of human and murine erythroleukemia cells that are Epo-independent for growth, we have found that E-CSF stimulates cell proliferation directly, increasing the plating efficiency of these cells in suspension culture by 50%-165%. B-BPA also increased proliferation of these cells in semi-solid medium. Importantly, the combination of E-CSF and Epo resulted in a profound increase in the growth and maturation of the resultant colonies. Therefore, the data indicate that E-CSF can regulate the growth of cells independently of added Epo and, in addition, can synergize with Epo in regulating the growth and differentiation of erythroid progenitors.

Topics: Animals; B-Lymphocytes; Bone Marrow; Bone Marrow Cells; Cell Division; Cells, Cultured; Chromatography, DEAE-Cellulose; Colony-Stimulating Factors; Culture Media, Conditioned; Dose-Response Relationship, Drug; Drug Synergism; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Lymphokines; Mice; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured

A proportion of fetal liver hemopoietic blast cells express Fc gamma RII, and addition of the anti-Fc gamma RII monoclonal antibody CIKM5 induces a rise in calcium in these cells in suspension. Although these cells are thus capable of mobilizing intracellular calcium in response to surface receptor mediated events, neither granulocyte-macrophage colony-stimulating factor (GM-CSF) nor erythropoietin produced detectable changes in intracellular calcium ion concentration in these cells.

Topics: Antibodies, Monoclonal; Antigens, Differentiation; Calcium; Cell Line; Erythropoietin; Fetus; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Immunoenzyme Techniques; Immunophenotyping; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Receptors, Fc; Receptors, IgG; Tumor Cells, Cultured

ME26 virus is a recombinant mouse retrovirus construct homologous to the avian E26 virus. Both encode a 135-kDa gag-myb-ets fusion protein which is localized in the nucleus. We have recently shown that ME26 virus can induce erythropoietin (Epo) responsiveness in hematopoietic cells. Mice infected with ME26 virus develop a hyperplasia of Epo-dependent hematopoietic precursor cells from which permanent cell lines can be established. In vitro, ME26 virus specifically induces Epo responsiveness in the interleukin-3-dependent myeloid cell line FDC-P2 by enhancing expression of the Epo receptor (EpoR). In the present study we demonstrate that ME26 virus infection of FDC-P2 cells also results in enhanced expression of beta-globin and the erythroid-specific transcription factor GATA-1, a protein which can transactivate both the EpoR promoter and globin genes. In addition, these cells exhibit a down-regulation of c-myb expression similar to that seen in differentiating erythroid cells. To determine the molecular basis for activation of erythroid genes in ME26 virus-infected cells, we carried out transient expression assays with DNA constructs of either the EpoR promoter of the GATA-1 promoter linked to reporter genes. Our results indicate that while ME26 virus did not directly enhance expression from the EpoR promoter, both it and its avian parent, E26, transactivated the GATA-1 promoter. Furthermore, ME26 virus cooperates with the GATA-1 protein to enhance expression of the EpoR gene. We propose that the mechanism by which ME26 virus induces erythroleukemia involves transactivation of the GATA-1 gene, thus positively regulating the expression of the EpoR and leading to the proliferation of a unique population of Epo-responsive cells. By specifically inducing Epo responsiveness in hematopoietic cells via transactivation of a transcription factor, ME26 virus utilizes a novel mechanism for retrovirus pathogenesis.

Topics: Animals; DNA Mutational Analysis; DNA-Binding Proteins; Down-Regulation; Erythroid Precursor Cells; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; GATA1 Transcription Factor; Globins; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Promoter Regions, Genetic; Retroviridae; Transcription Factors; Transcriptional Activation

Erythropoietin is the major regulator of the proliferation and differentiation of erythroid precursors, but little is known about its molecular mechanism of action. Using a human erythroleukemic cell line (HEL), we investigated whether p21ras is involved in erythropoietin signal transduction. We found that stimulation of HEL cells with erythropoietin induces a 5-fold increase in the amount of GTP bound to the endogenous p21ras. This effect is dose-dependent and occurs very rapidly. We also observed that erythropoietin causes tyrosine phosphorylation of several proteins in a time-dependent manner that correlates with the p21ras activation. Moreover, inhibition of tyrosine kinases by genistein totally prevents the erythropoietin-induced accumulation of a p21ras.GTP complex. By using an antiserum against the GTPase-activating protein, we found that p120GAP is rapidly phosphorylated in tyrosine in response to erythropoietin. Furthermore, the ability of a lysate from erythropoietin-stimulated HEL cells to induce in vitro hydrolysis of GTP bound to p21ras was strongly reduced. These results demonstrate that activation of p21ras is an early event in the erythropoietin signal transduction pathway, and they suggest that accumulation of the p21ras.GTP complex may be triggered by inhibition of GTPase-activating protein activity.

Topics: Electrophoresis, Polyacrylamide Gel; Erythropoietin; Genistein; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Isoflavones; Leukemia, Erythroblastic, Acute; Oncogene Protein p21(ras); Phosphorylation; Proteins; ras GTPase-Activating Proteins; Recombinant Proteins; Signal Transduction; Tyrosine

Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd +/- 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding.

Topics: Animals; Erythrocyte Membrane; Erythropoietin; Humans; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Tumor Cells, Cultured

Erythropoietin is a cytokine which specifically regulates the proliferation and differentiation of erythroid progenitor cells. The expression of erythropoietin receptor on the cell membrane of the progenitor cells is a critical event during the erythroid differentiation process. In order to clarify the tissue-specific and differentiation stage-specific expression of the erythropoietin receptor gene, its transcriptional regulation was examined by transient expression assay, gel mobility shift assay and DNase I footprinting. The results clearly showed that GATA-1 transactivates the gene expression through a single GATA motif located around -200 bp upstream from the ATG codon in a dose dependent manner. Furthermore, Northern blot analysis revealed that erythropoietin receptor-mediated signals strongly enhanced GATA-1 gene expression in accordance with the appearance of hemoglobin-positive cells. Taken together with other observations, these results suggested the following scheme of erythroid differentiation: 1)GATA-1 is expressed in the early stage of blood cell development; 2) GATA-1 transactivates the erythropoietin receptor gene; 3) erythropoietin binds its receptor and the receptor-mediated signals enhance GATA-1 gene expression in erythroid progenitor cells; and 4) GATA-1 finally transactivates hemoglobin synthesis-related genes and globin genes in relatively matured erythroid cells.

Topics: Animals; Base Sequence; Cloning, Molecular; Deoxyribonuclease I; DNA; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation; Hematopoiesis; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Erythropoietin; Restriction Mapping; Signal Transduction; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Autonomous colony formation is a frequent event in erythroleukemia. In 13 cases of early erythroid leukemias, we investigated whether erythropoietin (Epo) autocrine stimulation was responsible for the growth factor autonomy. Epo transcripts were detected by Northern blotting in cells from one patient. These cells also expressed an Epo receptor (1,000 receptors per cell) with a 420-pM affinity and Epo was detected in the supernatant of cultured cells. In 8 of the 13 cases, Epo transcripts were revealed by the polymerase chain reaction ranging from 0.5 to 500 copies per cell. In situ hybridization proved that these Epo transcripts were present in the blast cells. No Epo gene abnormalities were detected by Southern blotting. In two cases, leukemic cells were grown in the presence of Epo-neutralizing antibodies or Epo antisense oligomers. In one case, the antibody significantly reduced autonomous growth. In contrast, the antibody had no effect in the second case in which blast cells transcribed the Epo gene at a low level. However, Epo antisense oligomers partially inhibited autonomous growth. This inhibition was reversed by addition of exogenous Epo. Overall, these results suggest that an extracellular or intracellular autocrine Epo stimulation occurs in some cases of erythroid malignancies.

Topics: Base Sequence; Blotting, Northern; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Nucleic Acid Hybridization; Oligonucleotides, Antisense; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Erythropoietin; Tumor Cells, Cultured

A murine erythroleukemia (MEL) cell line, F5-5, expressed 10,000 binding sites for erythropoietin (EPO) per cell, 10-fold more than was expressed by other murine erythroleukemia cell lines and normal erythroid progenitors. Northern (RNA) and Southern blot analyses revealed overexpression of mRNA for the EPO receptor (EPOR) and rearrangement of one of the EPOR gene alleles in F5-5 cells, respectively. Molecular cloning of F5-5-derived cDNA encoding EPOR revealed that the 5' noncoding region of the EPOR cDNA corresponds to the 3' long terminal repeat sequence of the polycythemic strain of Friend spleen focus-forming virus (F-SFFVP). The aberrant EPOR transcripts containing the 3' long terminal repeat sequence were mainly expressed in F5-5 cells. The same integration upstream of the EPOR gene was also observed in other subclones and the parent cell line. It is possible that overexpression of EPOR by viral promoter insertion will confer growth advantage to an F-SFFVP-infected erythroid progenitor cell, leading to positive clonal selection through further leukemogenic steps.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; DNA, Neoplasm; Erythropoietin; Gene Expression Regulation, Viral; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Oligonucleotides; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Erythropoietin; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Spleen Focus-Forming Viruses

Erythropoietin, a glycoprotein produced by the kidneys in response to anemia and hypoxia, is a major growth factor for cells of the erythroid lineage. Erythropoietin interacts with high-affinity cell surface receptors (EpoR) present on developing progenitors and is required for their survival. Previously we characterized the gene for EpoR and demonstrated that its promoter acts in a cell-specific manner. Here we show that the hematopoietic-specific transcription factor GATA-1 is necessary, and indeed is sufficient as the sole cell-restricted regulator, for activation of the EpoR promoter in fibroblast transfection assays. Hence, GATA-1, which participates in transcriptional control of the majority of erythroid-expressed genes, also acts on the promoter of an essential lineage-restricted receptor (EpoR). This central contribution of GATA-1 to EpoR promoter function provides a mechanism whereby a cell-restricted regulator may ensure the viability and subsequent maturation of progenitor cells during hematopoietic differentiation.

Topics: 3T3 Cells; Animals; Blotting, Northern; Cell Line; Cell Nucleus; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; Gene Expression; Leukemia, Erythroblastic, Acute; Mice; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Erythropoietin; Restriction Mapping; RNA, Neoplasm; Transcription Factors; Transcription, Genetic; Transfection; Zinc Fingers

Induction of carbonic anhydrase isozyme I (CA-I) by erythropoietin or hemin was investigated using erythroleukemia (K562) cells. Immunological estimation and purification of carbonic anhydrases showed that untreated K562 cells contained only carbonic anhydrase isozyme II(CA-II), while incubation of the cells with 2 units of erythropoietin (EP) per ml of the incubation medium or with 50 microM hemin resulted in the induction of CA-I. The purified CA-I induced in K562 cells was enzymatically and immunologically identical to that from mature erythrocytes. Flow cytometric analysis showed that incubation of K562 cells with EP as well as hemin induced CA-I at the 3rd h, while alpha-globin was detected at the 8th h. Northern blot analysis of CA-I mRNA using a cloned genomic DNA as a probe showed that mRNA of CA-I was induced by EP. These results suggest that induction of CA-I is regulated at the transcriptional level during developmental changes of erythroid cells, and that CA-I may play a physiologically important role during erythroid differentiation.

Topics: Blotting, Northern; Blotting, Western; Carbonic Anhydrases; Enzyme Induction; Erythropoiesis; Erythropoietin; Globins; Humans; Leukemia, Erythroblastic, Acute; Staining and Labeling; Tumor Cells, Cultured

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.

Topics: Animals; Cell Line; Cytosol; Erythropoietin; Female; Hematopoietic Stem Cells; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred Strains; Molecular Weight; Peptide Fragments; Phosphates; Phosphopeptides; Phosphoproteins; Phosphorylation; Spleen; Tetradecanoylphorbol Acetate

Erythropoietin (Epo) is a glycoprotein hormone that specifically regulates the proliferation and differentiation of erythroid progenitor cells. The effect of Epo on the phosphorylation of cellular proteins was investigated in a murine erythroleukemia cell line ELM-I-1 which differentiates to produce hemoglobin in response to Epo. The phosphorylation of a prominent 98 kDa phosphoprotein (pp98) (pI approximately 6.5) in the particulate fraction of ELM-I-1 cells decreased markedly upon exposure of the cells to recombinant human Epo. The effect was rapid and transient, occurring within 1 min after Epo exposure, and disappearing within 15-30 min. Interleukin 3, another hematopoietic factor which is known to act also on erythroid progenitors, did not induce this effect. Phosphoamino acid analysis revealed that pp98 is phosphorylated on serine residues. The results suggest that this rapid and transient alteration in protein phosphorylation may serve as critical trans-membrane signal for Epo.

Topics: Animals; Erythropoietin; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphoserine; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Recombinant Proteins; Signal Transduction; Tumor Cells, Cultured

Alterations in the expression of two proto-oncogenes, c-myb and c-myc, have been implicated in the differentiation of transformed erythroid cells induced by chemical inducers, such as dimethyl sulfoxide (Me2SO). In the present study, we compared the expression of c-myb and c-myc during erythropoietin (Epo) and Me2SO induction of Rauscher erythroleukemia cells, which differentiate in response to both inducers, and Friend erythroleukemia cells, in which Epo-induced differentiation is blocked. Our results demonstrate that Epo induces specific changes in expression of c-myb and c-myc in both Rauscher and Friend cells. Epo increases c-myc transcript, in contrast to a decreased caused Me2SO, indicating that the biphasic mode of c-myc regulation seen with Me2SO is not required for erythropoiesis. The Epo-induced changes in c-myb and c-myc do not require new protein synthesis, thus identifying these proto-oncogenes as early response genes for Epo. Both cell types also exhibit rapid changes in membrane protein phosphorylation in response to Epo. Since the signal pathway from Epo receptor activation to the nucleus appears equally functional in both Rauscher and Friend cells, the data suggest that the inability of Friend cells to differentiate in response to Epo is due to a block at a later step in the induction process.

Topics: Animals; Cell Differentiation; Cell Membrane; Cycloheximide; Dimethyl Sulfoxide; Erythropoietin; Friend murine leukemia virus; Gene Expression Regulation; Genes, myc; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Phosphorylation; Proto-Oncogenes; Rauscher Virus; Receptors, Cell Surface; Receptors, Erythropoietin; Transcription, Genetic; Tumor Cells, Cultured

Human K562 leukemia cells have been induced to differentiate along the erythroid lineage by aclacinomycin (ACM), an anthracyclic antitumor drug. During differentiation over 3 days in culture, the expression and the nature of erythropoietin (EPO) receptors have been analyzed using 125I-labeled bioactive recombinant human EPO. Aclacinomycin at 20 nM, the concentration inducing optimum differentiation, progressively increased EPO-specific binding. On day 3, EPO binding was nine-fold higher than that of the controls (1031 +/- 101 cpm/5 x 10(6) cells versus 112 +/- 15 cpm); with various concentrations of ACM, the increase in EPO binding appeared to parallel the recruitment of hemoglobin-producing cells. However, at 95% of growth inhibition, EPO binding remained constant while the percentage of differentiated cells decreased. Specific binding was reversible, saturable, and proportional to cell number; bound EPO was displaced by unlabeled EPO. Scatchard analysis of the equilibrium binding data suggested the existence of a single class of EPO receptors with an apparent Kd of 867 +/- 458 pM, corresponding to 400 +/- 142 receptors per cell. Affinity cross-linking of 125I-EPO using disuccinimidyl suberate followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions demonstrated that EPO was preferentially cross-linked to a protein of approximately 116 kD. Taken together, these results demonstrate that, in addition to cytostatic properties, antitumor drugs such as ACM can modulate the expression of differentiation factor receptors on the surface of leukemic cells.

Topics: Aclarubicin; Adjuvants, Immunologic; Binding Sites; Cell Differentiation; Erythroid Precursor Cells; Erythropoietin; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Receptors, Cell Surface; Receptors, Erythropoietin

Erythropoietin triggers the differentiation of erythrocyte progenitors by binding to receptors on their plasma membrane. We report here that pretreatment of erythropoietin-responsive murine erythroleukemia cells with chemical inducers resulted in a striking increase in erythropoietin-specific hemoglobinization. This amplification of the erythropoietin biologic response was accompanied by the induction of a new population of high-density receptors (approximately 20,000 per cell) exhibiting marked positive cooperativity. Erythropoietin binding to new receptors displayed a convex upward Scatchard plot and a Hill coefficient (nH) of 6.75. Measurement of erythropoietin receptor mRNA demonstrated an initial decrease in receptor transcript followed by an approximately 2- to 3-fold increase after 24-48 hr. This increase in receptor message does not appear to account for the magnitude of the receptor up-regulation by dimethyl sulfoxide. We propose that this positive cooperativity reflects the interaction (clustering) of receptors, presumably through the formation of homooligomers or heterooligomers, and that this receptor interaction may amplify the erythropoietin signal transduction pathway.

Topics: Animals; Blotting, Northern; Cell Line; Dimethyl Sulfoxide; Erythropoietin; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Models, Biological; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; RNA, Neoplasm

Reduced expression of the proto-oncogene c-myb appears necessary for erythroid differentiation induced by chemical agents and by the natural regulator, erythropoietin (Epo). Treatment of Epo-responsive Rauscher erythroleukemia cells with an anti-sense oligodeoxynucleotide to c-myb in the absence of other inducers downregulated myb protein markedly and caused hemoglobinization of the cells within 48 hours. Epo treatment, which downregulates c-myb in these cells, induced hemoglobinization to the same degree. Epo also induced the appearance of anion transport protein on the plasma membrane, consistent with terminal differentiation. In contrast, antisense c-myb did not induce this erythroid marker. The results are consistent with a role for myb protein in the regulation of hemoglobin synthesis.

Topics: Animals; Anion Transport Proteins; Carrier Proteins; Cell Membrane; Erythropoietin; Gene Expression Regulation; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Mice; Oligonucleotides, Antisense; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Tumor Cells, Cultured

Friend erythroleukemia cells (FLC) passaged in mice are highly tumorigenic and multiply extensively in the livers of suckling DBA/2 mice without differentiating. In contrast, in vitro passaged FLCs injected intravenously were of low tumorigenicity, multiplied to a limited extent in the livers of suckling mice, and underwent marked differentiation from the proerythroblast to the orthochromatic erythroblast stage in the liver. The presence of characteristic C-type virions budding from the cell surface in various stages of erythroid differentiation served as a marker of the injected FLCs. When the same in vitro passaged FLCs that differentiated in the liver were injected subcutaneously in suckling mice, they formed large subcutaneous tumors consisting of sheets of undifferentiated tumor cells. It is concluded that the tumorigenicity of FLCs depended on the site of tumor growth and that there is an inverse correlation between the tumorigenic capacity and the capacity to differentiate.

Topics: Animals; Animals, Suckling; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Erythropoietin; Friend murine leukemia virus; Injections, Intravenous; Injections, Subcutaneous; Interleukin-3; Leukemia, Erythroblastic, Acute; Liver; Mice

We have shown previously that purified human erythropoietin rapidly alters the phosphorylation of an integral erythroid membrane protein, pp43 (Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936). We have now purified pp43 to apparent homogeneity and have prepared antibodies to it. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer of membrane proteins to nitrocellulose, the antibodies identified pp43 and a series of higher molecular weight antigenically related proteins, up to 50 kDa, in erythropoietin-responsive Rauscher murine erythroleukemia cells and in normal murine erythroid cells. Examination of purified subcellular fractions confirmed the localization of pp43 and the related proteins to the plasma membrane. Phosphorylation with [gamma-32P]ATP demonstrated that, in contrast to pp43, these higher molecular weight proteins were not phosphorylated. Marked differences in both the abundance of pp43 and related proteins and the degree of erythropoietin-sensitive pp43 phosphorylation were found between the plasma membranes of Rauscher cells and those of "non-responsive" Friend murine erythroleukemia cells. In addition only trace amounts of a 50-kDa antigenically related protein and no phosphorylated pp43 were detected in the plasma membranes of two erythropoietin-insensitive human erythroid cells lines, K562 and HEL. The results suggest that the abundance and degree of phosphorylation of pp43 and the antigenically related proteins is strongly correlated with the erythropoietin responsiveness of the particular erythroid cell types.

Topics: Animals; Cell Fractionation; Cell Line; Cell Membrane; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Epitopes; Erythropoietin; Immunoassay; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Membrane Proteins; Mice; Molecular Weight; Phosphoproteins; Phosphorylation

The transcriptional binding protein NFE-1 (also called GF-1 and Ery-f1) is thought to play a necessary, but not sufficient, role in the regulation of differentiation-related gene expression in a subset of hematopoietic lineages (erythroid, megakaryocytic, and basophil-mast cell). In order to clarify the mechanism which underlies the lineage-specificity of the NFE-1 expression, as well as the relationship between the expression of this factor and growth factor responsiveness, we have evaluated the capacity of erythropoietin (Epo)-, granulomonocytic (GM)-colony stimulating factor (CSF)-, and granulocyte (G)-CSF-dependent subclones derived from the interleukin 3 (IL-3)-dependent cell line 32D, to express 1) NFE-1 mRNA, 2) NFE-1-related nuclear proteins, and 3) chloramphenicol acetyl transferase (CAT) activity when transfected with a CAT gene under the control of NFE-1 cognate sequences. NFE-1 mRNA was found to be expressed not only in cells with mast cell (IL-3-dependent 32D) and erythroid (Epo-dependent 32D Epo1) phenotypes, but also in cells with predominantly granulocyte/macrophage properties, such as the GM-CSF- (early myelomonocytic) and G-CSF- (myelocytic) dependent subclones of 32D. However, a gradient of expression, correlating with the lineage, the stage of differentiation, and the growth factor responsiveness of the cell lines, was found among the different subclones: Epo greater than or equal to IL-3 greater than GM-CSF greater than G-CSF. Binding experiments demonstrated NFE-1 activity in all cell lines except the G-CSF-dependent line. Function of the NFE-1 protein was assessed by the expression of the CAT gene linked to the SV40 promoter and a mutant (-175 T----C) HPFH gamma-globin promoter. High level CAT expression was seen only in the Epo1 cells although low level expression was also seen in the parent 32D. These results demonstrate that the specificity of the expression of NFE-1 for the erythroid--megakaryocytic--mast cell lineages is obtained by progressive inactivation of its expression in alternative lineages.

Topics: Animals; Blotting, Northern; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; Gene Expression Regulation; Globins; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-3; Leukemia, Erythroblastic, Acute; Mice; Phenotype; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured

Topics: Animals; Cell Line, Transformed; Dimethyl Sulfoxide; Erythropoietin; Leukemia, Erythroblastic, Acute; Osmolar Concentration; Receptors, Cell Surface; Receptors, Erythropoietin

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. These belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.

Topics: Cell Differentiation; Cell Line, Transformed; Cell Separation; Cell Transformation, Viral; Centrifugation; Dimethyl Sulfoxide; Erythropoietin; Friend murine leukemia virus; Humans; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Receptors, Cell Surface; Receptors, Erythropoietin; Species Specificity; Time Factors

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N-terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO-dependent cell line. Only Group II MoAbs inhibit a newly developed EPO-dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Membrane; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Neutralization Tests; Precipitin Tests; Receptors, Cell Surface; Receptors, Erythropoietin

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.

Topics: Animals; Blotting, Northern; Cell Division; Cell Line; Cell Transformation, Viral; DNA Replication; Erythropoietin; Friend murine leukemia virus; Genes, Viral; Kinetics; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Mice; Nucleic Acid Hybridization; Receptors, Cell Surface; Receptors, Erythropoietin; Spleen Focus-Forming Viruses; Viral Envelope Proteins

Topics: Animals; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Drug Synergism; Erythropoietin; Gene Expression Regulation, Neoplastic; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Neoplasm Proteins; Rauscher Virus; Receptors, Cell Surface; Receptors, Erythropoietin; Signal Transduction; Up-Regulation

Erythropoietin (Epo) and dimethyl sulfoxide (DMSO) are believed to induce the differentiation of transformed erythroid cells by different signal transduction pathways. We have now obtained evidence for the interaction of these pathways. We used a Rauscher murine erythroleukemia cell line with a relatively low (8% to 10%) hemoglobinization response to Epo alone. Pretreatment of these cells for 1 day with DMSO followed by its removal and the addition of Epo resulted in a marked enhancement of the Epo specific hemoglobinization. We have designated this effect "DMSO priming." This priming effect of DMSO on the Epo response was both time-dependent and DMSO concentration-dependent. DMSO priming potentiated the Epo response in three ways. Firstly, DMSO priming increased the total number of Epo responsive cells from 8% to 10% to 40% to 60%. Secondly, DMSO priming reduced the time required to reach the optimal Epo-induced response from 4 days to 2 days. Thirdly, the Epo dose-response curve was left-shifted approximately 20-fold. DMSO priming was also associated with a marked increase in Epo receptor density characterized by an apparently new receptor population and by the appearance of positive cooperativity between receptors. Our results suggest that the DMSO priming effect is due to potentiation of the Epo signaling pathway, thus resulting in a much more rapid and dramatic Epo-induced hemoglobinization response.

Topics: Animals; Dimethyl Sulfoxide; Drug Synergism; Erythropoietin; Hemoglobins; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Receptors, Cell Surface; Receptors, Erythropoietin; Signal Transduction; Tumor Cells, Cultured

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Cell Differentiation; Cloning, Molecular; Erythropoietin; Gene Expression Regulation; Genomic Library; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Receptors, Cell Surface; Receptors, Erythropoietin; Transcription, Genetic

Here we describe the characterization of Epo-responsive mouse erythroleukemia cell line ELM-I-1. ELM-I-1 cells possess Epo binding sites on their membranes and differentiate into hemoglobin-positive cells when cultured in the presence of Epo. About 20% of the cells were hemoglobin-positive after a 3- to 4-day exposure to recombinant human Epo in liquid culture. Supplementation of recombinant mouse IL-3 during culture had an augmentative effect on Epo-mediated differentiation, although IL-3 alone did not induce differentiation. Both Epo and IL-3 stimulated the growth of ELM-I-1 cells, and their effects were a slightly additive. These findings indicate that ELM-I-1 cells are suitable for studying the interaction between Epo and IL-3 in erythroid differentiation at a subcellular level. ELM-I-1 may also offer a valuable bioassay system for Epo.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Dose-Response Relationship, Drug; Erythropoietin; Interleukin-3; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Radiation-Induced; Mice; Mice, Inbred C3H; Recombinant Proteins; Tumor Cells, Cultured

The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with [3H]thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.

Topics: Animals; Cell Differentiation; DNA; DNA Repair; Erythrocyte Aging; Erythroid Precursor Cells; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight

Macrophages have been shown to directly influence the growth and development of mature erythroid progenitors (CFU-E) in normal and erythroleukemic mice. We examined the mechanism by which macrophages mediate their effect on in vivo erythropoiesis. As reported for whole macrophages, serum-free supernatants (SN) from normal resident peritoneal macrophages suppressed in vivo normal and conventional Friend virus (CFV)-infected CFU-E and caused clinical regression of CFV-induced leukemia in mice. Macrophage SN had no effect on the erythropoietin (EPO)-independent CFU-E characteristic of infection with the polycythemia-inducing strain of Friend virus (FVP), or progression of FVP leukemia. Using biochemical, immunologic, and functional assays, the erythrosuppressive factor in macrophage SN was identified as interleukin-1 alpha (IL-1 alpha). The in vivo erythroid suppressive effects of macrophages, macrophage SN, and IL-1 alpha were reversed by simultaneous treatment with EPO. IL-1 alpha itself had no effect on CFU-E colony formation in vitro. Pretreatment of animals with antibodies to murine tumor necrosis factor-alpha (TNF-alpha) completely abrogated the suppression of CFU-E by macrophages, macrophage SN, or human recombinant IL-1 alpha. These results suggest that macrophages regulate erythropoiesis by production of IL-1 alpha, which in turn mediates its in vivo suppressive effects on CFU-E through TNF.

Topics: Animals; Endotoxins; Erythropoiesis; Erythropoietin; Interleukin-1; Leukemia, Erythroblastic, Acute; Macrophages; Mice; Mice, Inbred Strains; Tumor Necrosis Factor-alpha

We isolated from the Rauscher erythroleukemia cell line (Red 5), a subclone (Red 5-1.5), which contains erythropoietin (epo) binding sites and demonstrates an epo-dependent erythroid differentiation. One class of high affinity binding sites was detected with a Kd (+/- S.D.) of 0.43 +/- 0.09 nM and a mean density/cell of 1200 +/- 311. The cell-associated 125I-epo was displaced by nonlabeled epo but not by other hormones or factors. The 125I-epo binding to Red 5-1.5 cells was maximal within 3 h at 15 degrees C and 1 h at 37 degrees C and proportional to cell number. The addition of epo increased [3H] uridine incorporation into RNA by 6 h and [3H]thymidine incorporation into DNA by 60 h followed by 59Fe incorporation into protein, cell proliferation, and formation of hemoglobin-containing colonies. The incorporation of 59Fe into protein demonstrated a linear dose response (from 0.002 to 1.5 units of epo/ml) beginning 60 h after addition of the hormone to the cultures, and there was a dose-dependent increase (from 0.1 to 1.0 unit of epo/ml) in the formation of hemoglobin-containing colonies. We concluded that the binding of 125I-epo to Red 5-1.5 suggests the presence of specific epo receptors. The sequence of the epo-induced proliferation and differentiation events is similar to primary erythroid cultures but requires longer epo exposure. Receptor occupancy correlates with the induced biological response.

Topics: Animals; Cell Differentiation; Cell Line; DNA; Erythropoietin; Leukemia, Erythroblastic, Acute; Microscopy, Electron; Receptors, Cell Surface; Receptors, Erythropoietin; RNA; Thymidine; Uridine

The murine erythroleukemia (MEL) cell line, TSA8, becomes responsive to erythropoietin after induction with dimethyl sulfoxide (DMSO). We examined the signalling pathways involved in the commitment of TSA8 cells to become the erythroid progenitor cells responsive to erythropoietin, comparing them with the pathway used in an erythropoietin-induced change of the progenitor cells. Amiloride, an inhibitor of the Na+/H+ antiporter, completely blocked the commitment of TSA8 cells to become responsive to erythropoietin at a concentration that did not affect cell proliferation, while it showed no effect on the differentiation or proliferation of the erythroid progenitor cells derived from TSA8 cells by erythropoietin. Ethyleneglycol-bis (beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA) inhibited the commitment of TSA8 cells to CFU-E-like cells without affecting colony formation. In contrast, EGTA did not inhibit erythropoietin-induced differentiation of the progenitor cells, but did inhibit their proliferation. These results indicate that erythropoietin uses different signalling pathways from those used in the induction of the commitment of TSA8 cells.

Topics: Amiloride; Animals; Cell Differentiation; Colony-Forming Units Assay; Cycloheximide; Egtazic Acid; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Mice; Signal Transduction; Tumor Cells, Cultured

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.

Topics: Cell Division; Cell Line; Colony-Stimulating Factors; Cross-Linking Reagents; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.

Topics: Animals; Base Sequence; Cloning, Molecular; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Receptors, Cell Surface; Receptors, Erythropoietin; Restriction Mapping

Erythropoietin (epo) is the physiologic regulator of red blood cell development. We have demonstrated the presence of epo receptors on two erythroleukemia cell lines, IW32 and IW201. IW32 produces epo constitutively while IW201 is a nonproducing cell line. Neither cell lines respond to epo to differentiate. In this study, we have shown that these cells bound 125I-epo specifically. Binding could be displaced by the presence of unlabeled epo but not by insulin or epidermal growth factor. In contrast to that found in epo responsive cells isolated from Friend virus infected mouse spleen, which were reported to contain both "high" and "low" affinity receptors, our finding indicated only a single class of "low" affinity receptors with apparent dissociation constants of 1.7 nM and 2.0 nM for IW32 and IW201 cell lines, respectively. It is suggested that the absence of "high" affinity receptors may account for the lack of responsiveness of these cells to epo.

Topics: Animals; Cell Line; Erythropoietin; Iodine Radioisotopes; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Receptors, Cell Surface; Receptors, Erythropoietin

Functional erythropoietin (Ep) receptor (Ep-R) on transplantable mouse leukemic cells was solubilized by 3[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfate (CHAPS). An assay of the solubilized Ep-R was established: (1) radioiodinated Ep was bound to the solubilized Ep-R, (2) Ep.Ep-R complexes were covalently cross-linked, (3) the cross-linked products were selectively precipitated by polyethylene glycol, (4) the products were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (5) the Ep.Ep-R cross-linked products were measured. Optimal conditions to yield the cross-linked products and to stabilize the solubilized Ep-R were found. The solubilized Ep-R had properties similar to those of Ep-R on the membrane with respect to binding with Ep and the cross-linked products. These results would be exploited to allow purification of Ep-R.

Topics: Animals; Cell Membrane; Chemical Precipitation; Cholic Acids; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Polyethylene Glycols; Receptors, Cell Surface; Receptors, Erythropoietin; Solubility; Succinimides

The effects of tunicamycin and neuraminidase treatment on the specific binding of 125I-erythropoietin to a murine erythroleukemia cell clone, B8, were investigated. Neuraminidase treatment of B8 cells did not affect the specific binding of erythropoietin, but tunicamycin treatment caused a 2.5 to 4-fold increase in the amount of 125I-erythropoietin binding. Scatchard analysis of the binding data showed that the increase in the amount of binding resulted from increased affinity of the receptor. These results suggest that N-linked sugars of the erythropoietin receptor protein are involved in the interaction of erythropoietin with the cell-surface receptors on B8 cells.

Topics: Animals; Cells, Cultured; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Protein Binding; Receptors, Cell Surface; Receptors, Erythropoietin; Tunicamycin

High and lower affinity receptors for erythropoietin (EP) were initially identified on a very pure population of EP-responsive erythroblasts obtained from the spleens of mice infected with anemia strain of Friend virus (FVA). The structure of the receptor for EP in these cells was determined to be proteins of 100 and 85 Kd by cross-linking 125I-EP. In this investigation, studies on the receptors for EP were extended to other mouse erythroid cells and human erythroid cells as well as to the placentas of mice and rats. Only lower affinity receptors for EP were detected on erythroblasts purified from the spleens of mice infected with the polycythemia strain of Friend virus and a murine erythroleukemia cell line, both of which are not responsive to EP in culture. Internalization of 125I-EP was observed in both groups of cells. The structure of the receptor determined by cross-linking 125I-EP was two equally labeled proteins of 100 Kd and 85 Kd molecular mass in all these mouse erythroid cells. The structure of the receptor was found to be very similar in human erythroid colony forming cells cultured from normal blood. These cells respond to EP with erythroid maturation and were previously shown to have high and lower affinity receptors. Placentas from mice and rats were found to have only lower affinity receptors for EP, and when placental membranes were cross-linked to 125I-EP, the same 100 Kd and 85 Kd bands were found as seen in mouse and human erythroid cells. The structure of the receptor was similar in cells that have high affinity receptors (FVA-infected and human erythroid colony-forming cells) and nonresponsive erythroid cells and placenta that have lower affinity receptors, but only the cells with the high affinity receptors respond to the addition of EP with erythroid maturation.

Topics: Animals; Cross-Linking Reagents; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Humans; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Placenta; Rats; Receptors, Cell Surface; Receptors, Erythropoietin; Spleen

Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

Topics: Animals; Bacterial Proteins; Biotin; Cell Line; Erythropoietin; Indicators and Reagents; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Mice; Radioligand Assay; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Streptavidin; Tumor Cells, Cultured

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.

Topics: Adenylyl Cyclases; Animals; Cell Differentiation; Cell Line; Cells, Cultured; Cyclic AMP; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Stem Cells

A variety of regimens were utilized on KMOE cells to maximally raise globin mRNA levels for the purpose of improving the usefullness of this line for globin gene studies. Steady-state mRNA levels of embryonic (epsilon), fetal (gamma) and adult (beta) globin genes were assayed by the S1-nuclease protection method before and after exposure to inducing compounds. Exposure of KMOE cells to cytosine arabinoside and hemin leads to over 20-fold increases in beta- and gamma-globin mRNA steady-state levels, and an over 60-fold increase in epsilon-globin mRNA level. Exposure to cytosine arabinoside alone induced beta- and epsilon-globin but not gamma-globin gene expression. The alpha-like globin genes (zeta and alpha) were also monitored but found to be poorly expressed and not significantly inducible. The presence of epsilon-globin mRNA and the lack of alpha-globin mRNA distinguishes this line, KMOE-EL, from the KMOE sublines previously described.

Topics: Aminolevulinic Acid; Cytarabine; Dimethyl Sulfoxide; Drug Synergism; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Globins; Heme; Hemin; Humans; Leukemia, Erythroblastic, Acute; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured

Topics: Animals; Cell Line, Transformed; Erythropoietin; Gene Expression Regulation, Leukemic; Immunoblotting; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured

Human acute erythroleukaemia arises from the inability of the haemopoietic stem cell to differentiate. K 562 cell line provides a homogeneous population of primitive erythroleukaemic cells that are at the same point of differentiation. The effect of human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant erythropoietin on the differentiation of K 562 clonogenic cells was studied. Cells were cultured in methylcellulose culture for 5 days at 37 degrees C in humidified atmosphere containing 5% CO2 in air and scored for erythroid differentiation by benzidine staining. A combination of both growth factors induced erythroid differentiation in more than 80% of K 562 clonogenic cells. This combination may be useful in the treatment of patients with erythroleukaemia.

Topics: Cell Differentiation; Cell Line; Colony-Stimulating Factors; Erythropoiesis; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Recombinant Proteins; Tumor Stem Cell Assay

We have established a novel cell line, designated as TF-1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion, and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate, TF-1 entered two different pathways. Specifically, hemin and delta-aminolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary, TF-1 is a cell line of immature erythroid origin that requires GM-CSF, IL-3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors.

Topics: Adult; Antigens, Surface; Bone Marrow; Cell Line; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Leukemia, Erythroblastic, Acute; Male; Mitosis; Stem Cells; Tumor Cells, Cultured

The IW32 and NN10 cell lines are erythroleukemic cell lines which secrete erythropoietin (epo) into the culture medium constitutively. IW32 cells have a rearranged and amplified epo gene in addition to the normal gene. NN10 cells contain only the normal epo gene. DNase I hypersensitivity studies suggested that, in IW32 cells, it is the rearranged gene which is transcriptionally active. Sequence analysis of the upstream region of the rearranged epo gene suggests that the rearrangement has served to introduce a transcriptionally active gene close to the epo gene. This juxtaposition may explain the transcriptional activation of the epo gene at the rearranged locus.

Topics: Animals; Base Sequence; Blotting, Southern; Cell Line; Cloning, Molecular; DNA; Erythropoietin; Gene Amplification; Gene Expression Regulation; Gene Rearrangement; Leukemia, Erythroblastic, Acute; Mice; Repetitive Sequences, Nucleic Acid; Software; Transcription, Genetic

Erythroleukemic mouse spleen cells were analyzed by flow cytometry at three fluorescence wavelengths, two emissions for the calcium indicator indo 1 and immunofluorescence with an antiserum directed against murine leukemia retrovirus antigen. The antigen-positive subpopulation of cells responded to EPO by transiently changing indo 1 fluorescence emissions, indicating a change in cytoplasmic calcium concentration. EGTA blocked the EPO response, suggesting that Ca2+ influx may have followed EPO exposure in these cells. Antigen-negative cells showed no Ca2+ response to EPO. Infected mice all contained antigen-positive spleen cells, but, as measured by changes in indo 1 fluorescence, these subpopulations varied in their ability to respond to EPO.

Topics: Animals; Antigens, Viral; Calcium; Erythropoietin; Flow Cytometry; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured

A microassay for erythropoietin (Ep) activity in serum using [3H]thymidine uptake by K562 cells is presented. The method is similar to that of Krystal except that cells of the K562 human pluripotent leukemia cell line replace spleen cells from phenylhydrazine-treated anemic mice. Response to the hormone by K562 cells and spleen cells was colinear. Using the Krystal bioassay, 14 young hemoglobin S homozygotes had Ep activity levels of 17.9-113.8 mU/ml serum, whereas the new method with K562 cells gave a range of 19.2-115.3 mU/ml. The correlation coefficient between the two sets of data (r) was 0.999 (p less than 0.001). With the modified technique we have assayed 34 sickle cell patients, whose sera ranged from 19.2 to 1400 mU of Ep/ml with corresponding hemoglobin concentrations of 10.7 g % to 3.0 g %. Values for normal subjects were 22.1 +/- 2.1 mU/ml (n = 7). The stimulation of [3H]thymidine uptake is significantly inhibited by an anti-Ep antiserum. The assay permits quantification of stimulatory activities in a large number of samples with relative ease and is also suitable to explore the interactions of erythropoietic factors with their appropriate receptors on stem cells.

Topics: Adolescent; Adult; Animals; Cell Line; Child; Child, Preschool; Erythropoietin; Female; Humans; Immune Sera; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred C57BL; Neutralization Tests; Spleen; Thymidine

Transplantable erythroblastic leukemia was induced by 300-rad irradiation of C3H mice. Conditions for in vitro growth of the leukemic cells were studied. None of interleukin-3, granulocyte/macrophage colony-stimulating factor and erythropoietin could support the growth of the cells in vitro. In contrast, the leukemic cells grew into a stroma-dependent cell line, ELM-D, in close contact with the stromal cell layer of 900-rad-irradiated long-term bone marrow culture. A stroma-independent cell line, termed ELM-I-1, was further established from the non-adherent population in the co-culture of the leukemic cells, ELM-D, with stromal cells. Reverse transcriptase activity was not detectable in ELM-D or ELM-I-1 cells. Studies on binding and cross-linking of 125I-erythropoietin showed that ELM-I-1 cells had erythropoietin receptors, and two major radiolabeled protein products with molecular weights of 120 kDa and 140 kDa were detected on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions.

Topics: Animals; Cell Division; Colony-Stimulating Factors; Cross-Linking Reagents; DNA-Directed DNA Polymerase; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred C3H; RNA-Directed DNA Polymerase; Tumor Cells, Cultured

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor-potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.

Topics: Culture Media; Erythropoietin; Gene Expression Regulation; Humans; Leukemia, Erythroblastic, Acute; Lymphokines; Lymphoma, Large B-Cell, Diffuse; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Biologically active 125I-labeled human recombinant erythropoietin (EPO) was used to demonstrate specific receptors for this erythroid-specific hemopoietic growth factor on the cell surface of murine erythroleukemia cell clone B8. The binding of radioiodinated EPO to these cells was time and temperature dependent, specific, saturable, and reversible. During erythroid differentiation by dimethyl sulfoxide, B8 cells displayed a rapid and marked increase in the amount of specific 125I-EPO binding before the appearance of hemoglobin-containing cells. Scatchard analysis of the saturation binding data revealed that B8 cells had a single class and low number (350 to 650) of EPO receptors per cell with an apparent Kd of 1.2 to 1.4 nM. In addition, the number of EPO receptors on B8 cells was increased twice by induction with DMSO for 1 day, but the binding affinity of EPO toward its receptors did not change significantly. Affinity cross-linking experiments with disuccinimidyl suberate demonstrated two radiolabeled components with apparent molecular weights of 145,000 and 130,000 under both reducing and nonreducing conditions. Labeling of the two components was inhibited by incubation of cells with unlabeled EPO. These results suggest that some murine erythroleukemia cells potentially express EPO receptors as a differentiation marker of erythroid lineage, which contain two polypeptides with molecular weights of 109,000 and 94,000.

Topics: Affinity Labels; Animals; Cell Differentiation; Dimethyl Sulfoxide; Erythropoietin; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Time Factors; Tumor Cells, Cultured

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.

Topics: Animals; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Hematopoietic Stem Cells; Humans; Iodine Radioisotopes; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Succinimides; Tumor Cells, Cultured

A new cell line of human erythroleukemia cells differentiates spontaneously so that 30% of the cells are always hemoglobinized. Erythropoietin did not affect the percentage of such cells but stimulated the cell growth, indicating that the cells have functioning receptors. Binding of human recombinant radioiodinated erythropoietin to the receptors was specific. The bound ligand was internalized into cells at 37 degrees C but not at 15 degrees C. Scatchard analysis showed two classes of binding sites. Covalent binding of erythropoietin to its receptors yielded two products detected on sodium dodecyl sulfate-polyacrylamide gels electrophoresed under reducing conditions. Under non-reducing conditions, these species disappeared and larger products appeared.

Topics: Cell Differentiation; Cell Division; Cell Line; Chronic Disease; Erythropoietin; Humans; Kinetics; Leukemia; Leukemia, Erythroblastic, Acute; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins

Erythropoietin, a glycoprotein that regulates erythropoiesis, initiates its biological effects by binding to a cell-surface receptor. Little is known about the structure of the erythropoietin receptor and the events that follow binding of erythropoietin to its receptor, in part because of the difficulty of obtaining sufficient quantities of cells that express the erythropoietin receptor. We used both iodinated and metabolically labeled erythropoietin to characterize the receptor on a variety of erythroleukemia cell lines not previously tested, and we have identified both human and murine cell lines that display large numbers of erythropoietin receptors. Both erythropoietin-responsive and -nonresponsive cell lines exhibit a single class of binding sites. The human erythroleukemia cell line OCIM1 exhibits approximately 3000 erythropoietin receptors per cell with a Kd of 280 pM. The erythropoietin-responsive Rauscher red 5-1.5 murine erythroleukemia cell line displays approximately 1700 receptors per cell with a Kd of 440 pM. The GM979 murine erythroleukemia cell line has approximately 1600 receptors per cell with a Kd of 660 pM. Induction of the erythroid phenotype by dimethyl sulfoxide or its suppression by phorbol 12-myristate 13-acetate was accompanied by an increase or decrease, respectively, in erythropoietin receptor number. Affinity crosslinking of labeled erythropoietin to the receptor identified two proteins corresponding to estimated molecular masses of 95 and 105 kDa. The OCIM1, Rauscher, and GM979 erythroleukemia cell lines provide a useful model for the study of postreceptor signaling events, as well as a convenient source for purification of the erythropoietin receptor.

Topics: Animals; Cell Line; Dimethyl Sulfoxide; Erythropoietin; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Tetradecanoylphorbol Acetate

Topics: Animals; Erythropoietin; Friend murine leukemia virus; Kinetics; Leukemia, Erythroblastic, Acute; Receptors, Cell Surface; Receptors, Erythropoietin; Tumor Cells, Cultured

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

Topics: Animals; Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Cell Division; Cells, Cultured; Erythropoietin; Hematopoietic Stem Cells; Lactams, Macrocyclic; Leukemia, Erythroblastic, Acute; Liver; Mice; Quinones; Rifabutin

Immature murine erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) differentiate in vitro under the influence of erythropoietin (EP). These cells were used as a model for the examination of morphological changes occurring during terminal erythroid differentiation. FVA cells differentiate more completely in vitro in response to EP than continuous erythroleukemia cell lines do in response to chemical induction. Because they can be isolated in much greater numbers and in much higher purity than bone marrow or spleen cells explanted from anemic mice, FVA cells are an attractive alternative for studies of mammalian terminal erythroid differentiation. FVA cells cultured with EP followed a sequence of differentiation events that included a progressive decrease in cell size, disappearance of nucleoli, condensation of nuclei, and accumulation of hemoglobin. After 45 h of culture most FVA cells enucleated, giving rise to vacuolated reticulocytes and free nuclei that were surrounded by a thin layer of cytoplasm and a plasma membrane. The ratio of nuclear to cytoplasmic volumes increased significantly by 24 h of culture but did not change significantly from 24 through 36 h of culture. Variation in the morphology of enucleating FVA cells indicated that not all cells proceeded through a rigorously defined series of morphological stages prior to enucleation. These results are discussed in terms of previous studies of erythroblast maturation.

Topics: Animals; Cell Differentiation; Cell Line; Cell Nucleus; Cytoplasm; Erythroblasts; Erythrocyte Count; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice

The ability of non-thyroidal stimuli to affect the binding affinity and capacity of solubilized nuclear receptors for thyroid hormones was studied in a normal homeostatic system (erythropoiesis) and a pathobiologic one (lung-ozone interaction). No significant effects on affinity were found, as Kd control values for receptors derived from rat bone marrow averaged 57 (+/- 28) pM while experimental (hypoxic) values averaged 89 (+/- 55) pM. Kd control values in rat lung were found to average 142 (+/- 22) pM while average values derived from experimental protocols with ozone and methimazole were 267 (+/- 44) pM and 161 (+/- 35) pM respectively. Finally, Kd control values for receptors derived from cultured MEL cells averaged 19 (+/- 2.6) pM while experimental values during exposure to DMSO or IGF1 were 23 (+/- 3.6) pM and 26 (+/- 11) pM respectively. In contrast, binding capacity (expressed as fmoles of hormone bound per unit protein of solubilized receptor) was markedly perturbed in several tissues by various agents: ozone effects on lung were shown by an average control value of 3.3 (+/- 0.4) as opposed to an experimental average of 28 (+/- 1.9); and hypoxia effects on erythroid tissue were displayed by an average control value of 0.7 (+/- 0.07) as opposed to the experimental figure of 1.8 (+/- 0.03). In cultured MEL cells, binding capacity was seen to be increased from control values of 388 (+/- 15) sites/cell to 1243 (+/- 142) sites/cell after DMSO exposure and 2002 (+/- 10) sites/cell after IGF1 exposure. Parallel experiments done with receptors derived from rat liver yielded values similar to those reported by other investigators and were unaffected by the experimental agents. These data are consistent with the hypothesis that some thyroid target tissues may vary their response to homeostatic concentrations of hormone by modulating their production of receptors as a consequence of exposure to non-thyroidal stimuli.

Topics: Animals; Cell Line; Erythropoietin; Hypoxia; Leukemia, Erythroblastic, Acute; Lung; Male; Ozone; Rats; Rats, Inbred Strains; Receptors, Thyroid Hormone

The role of nuclear protooncogenes during erythroid cell differentiation was examined by transfecting exogenous c-fos and c-myb genes into mouse erythroleukemia cells, which can be induced to differentiate either with erythropoietin (Epo) or dimethyl sulfoxide. Expression of exogenous c-myb or c-fos oncogene completely inhibited Epo-induced erythroid differentiation but only partially inhibited dimethyl sulfoxide-induced differentiation. Normally Epo-induced differentiation leads to a drastic decline of c-myb mRNA levels and an increase of c-myc transcripts in the early stage of differentiation. Cells expressing exogenous c-fos gene, however, maintained high levels of c-myb mRNA after Epo treatment. This high level of c-myb transcripts was found to be due to block of transcription shutoff (or transcriptional activation) rather than to mRNA stabilization. It is concluded that the down-regulation of endogenous c-myb gene expression is a prerequisite for commitment of Epo-induced erythroid differentiation and that expression of c-myb gene may be indirectly regulated by c-fos gene product. We also concluded that early down-regulation of c-myc gene expression is not essential for erythroid differentiation and that gene regulation of chemically induced erythroid differentiation may differ from that of Epo-induced differentiation.

Topics: Animals; Cell Differentiation; Cell Line; DNA Probes; DNA, Neoplasm; Erythropoietin; Gene Expression Regulation; Half-Life; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Proto-Oncogenes; RNA, Messenger; Transcription, Genetic; Transfection

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Cross-Linking Reagents; Erythropoietin; Hematopoietic Stem Cells; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Liver; Rats; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Succinimides

Radioiodinated or biologically tritiated recombinant human erythropoietin was used to characterize receptors for this hormone on the surface of Friend erythroleukemic cells (745A and TSA8) and cells from mouse erythropoietic tissues (liver from fetus and spleen from animals made anemic by injection of Friend virus or phenylhydrazine). Specific binding of erythropoietin to these cells was time-dependent and dose-dependent. Binding studies at 37 degrees C showed that dissociation constants of erythropoietin-receptor complexes were in the range of 100-300 pM. The number of receptors on erythroleukemic cells increased after treatment with dimethylsulfoxide. Covalent binding of 125I-erythropoietin to its receptors with a cross-linking reagent, disuccinimidyl suberate or glutaraldehyde, resulted in the formation of two major radiolabeled products that migrated as 120-kDa and 140-kDa species on sodium dodecyl sulfate/polyacrylamide electrophoresis gels under reducing conditions. Under non-reducing conditions, both 120-kDa and 140-kDa species disappeared and two cross-linked products, a minor product with a molecular mass of 250 kDa and a major product of high molecular mass that kept it from migration into the separating gels, appeared. The relationship of the cross-linked products found under non-reducing conditions with those under reducing conditions remains to be clarified.

Topics: Animals; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Erythropoietin; Leukemia, Erythroblastic, Acute; Liver; Mice; Protein Binding; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Spleen

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.

Topics: Animals; Cell Differentiation; Cell Line; Colony-Forming Units Assay; Dimethyl Sulfoxide; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Receptors, Cell Surface; Receptors, Erythropoietin

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.

Topics: Ammonium Chloride; Animals; Cell Line; Cell Transformation, Neoplastic; Chloroquine; Endocytosis; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred Strains; Receptors, Cell Surface; Receptors, Erythropoietin

Two different strains of Friend spleen focus-forming virus, SFFVP and SFFVA, are known to cause a rapid erythroleukemia. The SFFVP-infected cells can proliferate and differentiate maximally without the addition of the erythroid-specific hormone erythropoietin, whereas the SFFVA-infected cells require erythropoietin for differentiation and for maximum proliferation. We previously reported that a recombinant virus containing sequences from the 3' half of the SFFVP envelope gene and the SFFVP long terminal repeat on an SFFVA background has all of the biological and biochemical characteristics of SFFVP. We are now presenting data on a new recombinant virus to show that only the 3' half of the SFFVP envelope gene is responsible for the differences observed between the two strains.

Topics: Animals; Cell Division; Cell Transformation, Viral; Chromosome Mapping; DNA, Recombinant; Erythropoietin; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Mice; Phenotype; Spleen Focus-Forming Viruses; Viral Envelope Proteins

Erythropoietin (Epo) is a glycoprotein factor that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Here we describe the isolation of Epo-responsive mouse erythroleukemia cell line SKT6, the characterization of the specific binding of biologically active 125I-labeled human Epo (125I-Epo) to its membrane receptor, and, finally, report information concerning the molecular structure of the receptor. About 75% of erythroid colony-forming precursor cell-like colonies derived from SKT6 cells were hemoglobin-positive after 3- to 4-day exposure to Epo in methylcellulose culture. Radioiodinated Epo bound specifically to SKT6 cells, and Scatchard analysis of the data showed a high affinity for 125I-Epo (Kd = 0.15 nM) but displayed only a small number of specific receptors (approximately equal to 470 per cell). Membrane components that specifically interact with 125I-Epo were identified by covalent crosslinking with disuccinimidyl suberate, and three receptor species with apparent Mr 63,000, 94,000, and 119,000 were found in membrane from SKT6 cells, suggesting the complex structure of the receptor molecules. Specific bindings were also detected in all of the Epo-unresponsive Friend erythroleukemia cells examined, and cross-linking study revealed the presence of only the 63,000 species as a binding site.

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Erythropoietin; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Receptors, Cell Surface; Receptors, Erythropoietin

A monoclonal antibody (designated K:1-6F) generated by hybridization of mouse myeloma cells with spleen cells from mice immunized with the erythroleukemic cell line K562 was found by fluorescence-activated cell sorter analysis, dot-blot assay and electroimmunoblotting to bind to a majority of cells in the K562 and HEL erythroleukemic cell lines, to a subset of cells of the erythroid lineage from normal bone marrow, to a subset of cells in all analysed cases (total 10) of erythroleukemia, and weakly to cells from patients with myeloid leukemia. The antibody did not bind to normal erythrocytes, monocytes, T- and B lymphocytes or granulocytes, as well as a panel of human malignant cell lines of hemopoietic origin (HL60, U937, Daudi, Molt-3, RH-L4 and U266). Biochemical characterization of the antigen defined by the antibody suggests that eht epitope is defined by a carbohydrate structure alone or in combination with proteins. Four molecules with Mr 100 kD, 65 kD, 45 kD and 18 kD respectively were immunoprecipitated from Triton X-100 extract of K562 erythroleukemia cells. Neuraminidase did not affect the binding of the antibody, whereas tunicamycin reduced the K:1-6F expression. The K:1-6F Mab was in normal bone marrow found to be specific for erythroid precursor cells and may therefore be useful in examination of normal and leukemic erythropoiesis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation; Colony-Forming Units Assay; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Hematopoietic Stem Cells; Humans; Immunoassay; Immunoenzyme Techniques; Immunosorbent Techniques; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred BALB C; Neuraminidase; Tumor Cells, Cultured; Tunicamycin

Rauscher murine erythroleukemia cells grow continuously in vitro and undergo terminal differentiation in response to the physiological inducer erythropoietin. In the course of this developmental process they express many erythroid-specific markers. In order to investigate the expression of cell surface determinants during Rauscher cell differentiation we generated monoclonal antibodies to uninduced cells. Using an anti-Rauscher cell monoclonal antibody, we have identified a cell surface determinant, designated ERY-1, that is present on normal murine erythroid cells. This determinant is apparently absent from the early progenitor BFU-E, but is present on the more mature progenitors CFU-E and CFC-E. It disappears during erythroid maturation and is absent from the mature erythrocyte. This pattern of ERY-1 expression is exhibited with remarkable fidelity during the erythropoietin-induced differentiation of Rauscher cells. Such differentiation-specific expression of the ERY-1 determinant suggests that it may play a functional role in erythropoiesis.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Surface; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Erythrocytes; Erythropoiesis; Erythropoietin; Flow Cytometry; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Mice; Phenylhydrazines; Spleen; Time Factors

Murine erythroleukaemia cells represent erythroid precursors blocked near the CFU-E or proerythroblast stage. In contrast to their non-leukaemic equivalents, neither their proliferation nor their differentiation seems to be affected by erythropoietin. However, we show in this paper that both uncommitted and committed, benzidine-positive, cells bind iodinated erythropoietin. The binding is of high affinity (Kd = 490 +/- 160 pM) and reversible with a half-life of the complex of 77 +/- 19 min. The number of binding sites is low (300-600 per cell). In contrast the haematopoietic non-erythroid cell lines HL 60 and L 1210 and the myeloid-erythroid human cell line K 562 do not exhibit specific binding. If these binding sites represent true hormone receptors, their presence on a permanent cell line should facilitate erythropoietin receptor purification.

Topics: Animals; Binding Sites; Cell Line; Erythropoietin; Friend murine leukemia virus; Kinetics; Leukemia, Erythroblastic, Acute; Mice

The hydrophobicity, ionic charges, degrees of glycosylation, and Western blot patterns of murine plasma erythropoietin and erythropoietin produced by clones of two murine erythroleukemic cell lines (IW32 and NN10) were compared. Erythropoietins from these different sources exhibited a number of similarities in their biochemical properties: identical retention on DEAE Affigel blue column and similar apparent molecular weights (30-36 kDa) in Western blot analysis. However, differences were also observed: heterogeneous binding to different lectin columns and varied retention on a phenyl Sepharose column. The data thus confirm that these murine plasma erythropoietins have biochemical properties in common with previously studied erythropoietins from humans or sheep. The major difference between erythropoietins secreted by erythroleukemic cell lines and other molecules belonging to the same family appears to be related to their glycosylation.

Topics: Animals; Chromatography, Affinity; Chromatography, Ion Exchange; Clone Cells; Collodion; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Lectins; Leukemia, Erythroblastic, Acute; Mice; Paper

The phosphorylation of a prominent 43-kDa phosphoprotein (pp43) in the membranes of normal murine erythroid cells was reduced markedly by exposure of the membranes to highly purified erythropoietin. A virtually identical reduction of pp43 phosphorylation was seen when erythropoietin-responsive Rauscher murine erythroleukemia cell membranes were exposed to the hormone. This effect was both time-dependent, occurring within 30 min after erythropoietin exposure, and concentration-dependent. Phosphoamino acid analysis revealed that pp43 is phosphorylated on serine residues. The results provide the first evidence that rapid alterations in membrane protein phosphorylation may serve as a trans-membrane signal for erythropoietin.

Topics: Adenosine Triphosphate; Animals; Erythrocyte Membrane; Erythropoietin; Kinetics; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Phosphoproteins; Phosphorylation; Phosphoserine; Spleen

LSCC HD3 chicken erythroleukemia cells, transformed by a temperature-sensitive avian erythroblastosis virus (tsAEV), secreted into the medium several transforming factors which after separation by Bio-Cel P-60 chromatography, stimulated quiescent (G0) chicken embryo fibroblasts and NIH 3T3 mouse cells to replicate DNA in serum-free medium and to form colonies in soft agar. Most of these factors were also mitogenic for the LSCC HD3 cells themselves when they were rendered phenotypically untransformed by incubation at 42 degrees C to inactivate the ts AEV. The transformed LSCC HD3 cells also secreted a non-mitogenic 40 kDa factor which blocked the erythropoietin-induced differentiation of untransformed LSCC HD3 (at 42 degrees C) and the DMSO-induced differentiation of Friend murine erythroleukemia cells into hemoglobin-synthetizing erythroid cells.

Topics: Alpharetrovirus; Animals; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Viral; Chickens; Culture Media; Dimethyl Sulfoxide; Erythropoietin; Growth Substances; Leukemia, Erythroblastic, Acute; Mice; Oncogenes

We have studied the internalization of 125I-erythropoietin (Epo) and regulation of Epo receptors by the ligand in a murine erythroleukemia cell clone, TSA8. To determine internalization, a high-salt acid wash was performed. Internalization of 125I-Epo was found in TSA8 cells as well as in fetal mouse liver cells (FMLC), although the percentage of internalized radioactivity reached 40% after incubation at 37 degrees C for 150 min and was lower than that in FMLC. Exposure of TSA8 cells to unlabeled Epo resulted in a rapid, time-dependent reduction in 125I-Epo binding activity. The net loss of the activity was related to the ambient Epo concentration and 5 X 10(-8)M Epo induced approximately 80% loss of total binding capacity. Scatchard analysis of the binding data revealed that the high-affinity receptor number was decreased but the affinity was increased in the Epo-treated cells. In low-affinity receptors, however, the receptor affinity was decreased and the receptor number was not changed much by preincubation with Epo. These results suggest that the decrease in 125I-Epo binding activity after preincubation with unlabeled Epo is mainly accounted for by a decrease in the number of high-affinity receptors, and the high-affinity receptors play an important role in the biological response to Epo.

Topics: Animals; Biological Assay; Cell Line; Endocytosis; Erythropoietin; Hydrogen-Ion Concentration; Leukemia, Erythroblastic, Acute; Ligands; Mice

Cultured murine erythroleukemia (MEL) cells can be induced to differentiate into erythrocytes. During this induced differentiation, a certain type of sequentially programmed gene expression and repression appears to occur in addition to the induction of globin mRNA. This system provides a commitment model for erythrodifferentiation and decancerization. By transfection of a beta-globin/TK chimeric gene into a B8/3 cell line, we examined the regulatory factors controlling beta-globin gene expression. Our results suggested that the timing of the appearance of inducible, positive trans-acting factor (s) and activation of chromatin conformation occur during induction. We demonstrated that a novel MEL cell line, TSA 8, could be induced to be committed to CFU-E, an erythropoietin-mediated progenitor cell, with the addition of DMSO. In the commitment process, we observed an asymmetric cell division, which could explain the self-renewal and the commitment of multipotential hemopoietic stem cells. For this commitment, the receptor for erythropoietin is required, but is insufficient and the other factor (s) are induced in the earlier phase of induction. Finally, we found that erythropoietin induced two signals for proliferation and differentiation of the progenitor cells and that these two signals are not coupled.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Colony-Forming Units Assay; Erythropoietin; Gene Expression Regulation; Globins; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Mice; RNA, Messenger

Using the spleen cells of mice infected with the anemia-inducing strain of Friend leukemia virus, an in vitro model system of erythropoiesis has been developed in which a homogeneous population of murine proerythroblasts terminally differentiates in response to erythropoietin (EP). The biochemical events involved in EP's capacity to maintain viability, induce hemoglobin production, and promote the development of the specialized erythrocyte membrane were studied during the 48-72 hour period required for proerythroblasts to differentiate into reticulocytes. The results show that EP increases glucose uptake and the syntheses of RNA and protein in the first few hours after exposure of the erythroblasts to the hormone. A coordinated production of heme, alpha and beta globin occurs later and peaks at about 48 hours. This peak corresponds to the time at which the majority of cells are undergoing enucleation and becoming reticulocytes. The syntheses of the erythrocyte membrane and membrane skeletal proteins are not coordinated, and multiple patterns of synthesis are found with respect to the time of EP exposure. A number of proteins are lost from the membrane fraction while the characteristic proteins of the mature erythrocyte become prominent in the membrane fraction of erythroid cells as they develop from reticulocytes into erythrocytes.

Topics: Animals; Cell Differentiation; Erythroblasts; Erythrocyte Aging; Erythrocyte Membrane; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Hemoglobins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Reticulocytes; Spleen; Tumor Cells, Cultured

Comparison of maps of the human and mouse erythropoietin (EPO) genes shows overall general conservation. A cloned mouse EPO gene was used to study EPO gene expression by the Northern blot method. Ten hours after bleeding to induce stress erythropoiesis in the rat, the only tissue to show detectable message was the kidney. Other preliminary studies on EPO expression in cells transformed by FMuLV that secrete EPO constitutively showed no detectable viral sequences close to the gene regardless of high levels of EPO expression. Further studies on regulation of EPO gene expression are now possible with the probes developed from the cloned gene.

Topics: Animals; Erythropoietin; Gene Amplification; Gene Expression Regulation; Genes; Humans; Kidney; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Organ Specificity; Rats; Sequence Homology, Nucleic Acid

Topics: Animals; Biological Assay; Chickens; Chromatography, High Pressure Liquid; Erythropoiesis; Erythropoietin; Leukemia, Erythroblastic, Acute; Oncogenes; Transformation, Genetic

Avian leukosis viruses induce erythroblastosis in chicks by integrating into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous-associated virus 1-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both differentiate and proliferate extensively in vitro. All 14 leukemias studied expressed high levels of erbB-related proteins that were 5 to 10 kilodaltons larger but otherwise very similar to the gp74erbB protein of avian erythroblastosis virus ES4 with respect to biosynthesis, glycosylation, and cell surface expression. Two leukemias contained and released retroviruses that transduced erbB. Chicken embryo fibroblasts fully infected with these viruses expressed high levels of erbB RNA and protein but retained a normal phenotype. Our results suggest that certain forms of c-erbB, activated by long terminal repeat insertion or viral transduction, are capable of inducing erythroleukemia but unable to transform fibroblasts.

Topics: Animals; Avian Leukosis Virus; Base Sequence; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chickens; ErbB Receptors; Erythroblasts; Erythropoietin; Fibroblasts; Glycoproteins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Oncogene Proteins, Viral; Oncogenes; Phenotype; Receptors, Cell Surface; Retroviridae; RNA, Viral; Transcription, Genetic; Transduction, Genetic

Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 37 degrees C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells.

Topics: Animals; Cell Line; Endocytosis; Erythrocyte Aging; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Iron Radioisotopes; Leukemia, Erythroblastic, Acute; Mice; Receptors, Cell Surface; Receptors, Transferrin; Spleen; Transferrin

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Blood Protein Electrophoresis; Blood Proteins; Carbonic Anhydrases; Cell Differentiation; Cytoskeletal Proteins; Erythroblasts; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Membrane Proteins; Mice; Mice, Inbred BALB C; Neuropeptides; Spectrin; Spleen

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colony-forming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.

Topics: Animals; Cell Division; Cell Line; Colony-Forming Units Assay; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Time Factors

The in vitro growth of primary erythroleukemia cells has been examined in the presence and absence of the hormone erythropoietin (EPO). Although these leukemic cells had previously been considered to be hormone-independent, addition of EPO was found to be essential for maximum growth in culture. Erythroid colonies that grew in the presence of EPO were leukemogenic when returned to mice. Influence of EPO on the in vivo growth of leukemic cells was indicated by our findings that administration of the hormone caused a more severe leukemia and rapid death, and transfusion of red blood cells, which lowers endogenous EPO, led to decreased spleen size and increased survival of leukemic mice. We suggest from our results that hormone-associated therapy might be efficacious in the treatment of this and, perhaps, other leukemias.

Topics: Animals; Blood Transfusion; Cell Division; Cell Line; Erythrocyte Transfusion; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Transplantation, Isogeneic

Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.

Topics: Animals; Blood; Cell Line; Chromatography, Gel; Clone Cells; Culture Media; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice

We previously documented that several erythroleukemia cell lines released factors that stimulated erythropoiesis in vivo and in vitro. A simple five-step scheme has been devised that allows purification of this erythropoietic activity to apparent homogeneity. The methods employed included lectin affinity chromatography (wheat germ agglutinin), gel filtration (ultro gel ACA44), ion exchange, hydroxylapatite, and high performance liquid chromatography. Following polyacrylamide gel electrophoresis, biologic activity was recovered in an area corresponding to a molecular weight of 35,000 daltons. Silver staining of a polyacrylamide gel after electrophoresis of our most purified preparation revealed a single band at 35,000 daltons.

Topics: Animals; Blood; Cell Line; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Culture Media; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice

Spleen cells taken from mice infected as adults with two different variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, as well as spleen cells taken from mice infected as newborns with Friend murine leukemia virus (F-MuLV) were assayed in a proliferation assay in the presence or absence of the erythroid hormone erythropoietin (Epo). Infection of NIH Swiss mice with SFFV resulted in excessive proliferation of erythroid cells that could still differentiate, and spleen cells taken from these mice were able to incorporate high levels of tritiated thymidine ([3H]dThd) in the absence of Epo, even in the presence of antibodies to Epo. In contrast, the level of proliferation of spleen cells from SFFVA-infected mice, but not those from SFFVP-infected mice, could be greatly enhanced by the addition of Epo to the cultures. Infection of newborn mice with F-MuLV resulted in the generation of Friend mink cell focus-inducing virus, which caused excessive proliferation of erythroid cells that appeared to be blocked in differentiation, resulting in severe anemia. Spleen cells from these mice were unable to proliferate in the absence of Epo. However, when increasing doses of Epo were added to the cultures, the cells proliferated at levels equivalent to the levels seen with SFFV. These results indicate that a proliferation assay based on the incorporation of [3H]dThd into spleen cells in response to Epo can be used as a quantitative means of assessing and comparing the effects of erythroleukemia-inducing retroviruses on the proliferation of their target cells.

Topics: Animals; Animals, Newborn; Cell Differentiation; Cell Division; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mink Cell Focus-Inducing Viruses; Mitotic Index; Neutralization Tests; Spleen; Thymidine; Time Factors; Tumor Virus Infections

The acute erythroleukemia induced in mice by the anemia-inducing strain of the Friend virus complex is caused to regress by normal macrophages. We examined the possibility that reversal of leukemia is related to a macrophage regulatory function in erythropoiesis. We found that the ability of macrophages to induce leukemia regression correlates with nonimmunological, in vivo suppression of normal and susceptible leukemic erythroid progenitors. The macrophage effect on erythropoiesis appears to be due to changes in a humoral regulator, related to but independent of erythropoietin. The results suggest a novel regulatory system for erythropoiesis, operative in vivo, and involving macrophages as accessory or suppressor cells. This regulation appears to be disrupted in erythroleukemic mice, but can be restored, and the disease can be made to regress by treatment with normal macrophages.

Topics: Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Macrophages; Mice; Mice, Inbred Strains

Injection of cobalt into rats resulted in erythropoietin (EPO) mRNA accumulation in the kidney. The same response was obtained upon bleeding. No EPO mRNA was detected in the spleen, salivary gland, or thymus following cobalt injection or bleeding. In some animals, but not in others, EPO mRNA was also expressed in the liver in response to cobalt injection. Time course studies showed that message appearance begins sometime between 3 and 6 h after cobalt injection. This correlated very well with the EPO concentration in the circulation; EPO levels in the circulation were the same as those of controls at 3 h but increased to six- to sevenfold that of controls by 6 h after cobalt injection. The mature EPO mRNA in the rat and mouse comigrated with the 18S rRNA, indicating that it is about 1,850 nucleotides in length.

Topics: Animals; Cobalt; Erythropoietin; Gene Expression Regulation; Hemorrhage; Kidney; Leukemia, Erythroblastic, Acute; Liver; Male; Rats; RNA, Messenger; Time Factors

Retroviruses that cause acute oncogenesis are generally complexes of a replication-competent helper virus and a replication-defective component. However, the pure defective components have not been previously available. We prepared the defective spleen focus-forming virus component of Rauscher erythroleukemia virus (R-SFFV) by transfecting a colinear R-SFFV DNA clone into a retroviral packaging cell line (psi 2 cells). The transfected cells released virus (psi 2/SFFV) that was free of helper virus and that induced erythropoietin-dependent erythroid burst formation in bone marrow cultures. When injected into normal adult NIH/Swiss mice in moderate doses, psi 2/SFFV caused a rapid splenic erythroblastosis that regressed. Extensive erythroblastosis could be maintained by repeated injections of psi 2/SFFV into anemic mice or by the addition of a helper virus. We conclude that R-SFFV alone causes proliferation but not immortalization of a population of erythroblasts that is normally replenished from a precursor stem cell pool. Because these precursor cells are inefficiently infected, a single moderate inoculum of psi 2/SFFV causes a wave of erythroblastosis. The properties of the proliferating erythroblasts are substantially determined by the R-SFFV viral component.

Topics: Animals; Cell Line; Erythropoiesis; Erythropoietin; Helper Viruses; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Mice; Rauscher Virus; Spleen; Spleen Focus-Forming Viruses; Viral Envelope Proteins; Virus Replication

The properties of clonogenic and leukemic cells, derived from mice infected with different helper virus pseudotypes of the polycythemic strain of Friend spleen focus-forming virus (SFFVp), have been analyzed. Four different replication-competent murine leukemia viruses (MuLVs) were used as helpers for the defective SFFVp genome: the Friend MuLVs, Moloney MuLV, and an amphotropic MuLV. Three different biological parameters were measured: (i) the kinetics of emergence of clonogenic cells characteristic of the late stages of Friend erythroleukemia; (ii) the ability of cells in these colonies to give rise to secondary colonies (self-renewal capacity); and (iii) the capacity of cell lines derived from these colonies to respond to inducers of erythroid differentiation. The properties of these cells was found to be independent of the helper virus used, suggesting that it is the SFFVp genome, not the helper virus, that plays a determinant role in the late stages of erythroleukemia.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Genes, Viral; Helper Viruses; Hematopoietic Stem Cells; Hemoglobins; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Mice; Moloney murine leukemia virus; Spleen; Spleen Focus-Forming Viruses

A specific immunological assay was used to demonstrate that the erythropoietic factor produced by the recently described FMuLV-induced murine erythroleukemic cell line IW32 is an authentic erythropoietin (epo). Several independent virus-induced erythroleukemic and myeloblastic cell lines were tested for epo production. Among six erythroleukemic cell lines induced by FMuLV, another (NN10) was shown to produce epo by biological and immunological assays. Four Friend-virus-induced erythroleukemias and four FMuLV-induced myeloblastic cells were negative. The amounts of epo produced were similar in IW32 and NN10 supernatants after 48 h in culture. The in vitro bioassay gave the highest levels (up to 1000 mU/ml), the in vivo bioassay the lowest, and the radioimmunoassay gave intermediate results. NN10 and IW32 cell lines have been induced by two different FMuLV and were shown to be independent by cytogenetic studies. The molecular weights of IW32 and NN10 epo were close to the molecular weight of mouse plasma epo but elution profiles suggested that some differences might exist between these epos. Cloned IW32 and NN10 cells were shown to retain both the ability for erythroid differentiation after incubation with chemical inducers and the ability to produce epo. This demonstrates that malignant erythroid cells were the source of epo production in these cell lines.

Topics: Animals; Biological Assay; Cell Line; Clone Cells; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Molecular Weight; Radioimmunoassay

Rauscher murine erythroleukemia cells, grown continuously in vitro, undergo erythroid differentiation in response to the hormone erythropoietin. Therefore, they serve as an important model system with which to examine critical biochemical aspects of this developmental process. Intact, uninduced Rauscher cells possess a functional beta-adrenergic receptor-adenylate cyclase complex. The adrenergic agonists, isoproterenol, epinephrine, and norepinephrine, exhibited activation constants (Kact) of 0.1, 0.5, and 20 mumol/L, respectively. Thus, the beta-receptor-cyclase complex of Rauscher cells is apparently one of the most sensitive of all erythroid cells reported thus far. The epinephrine-stimulated cyclic adenosine monophosphate (cAMP) response was inhibited by propranolol, alprenolol, and hydroxybenzylpindolol, with inhibition constants (KI) of 3.8, 2.2, and 0.1 nmol/L, respectively. Using [125I]-iodohydroxybenzylpindolol as ligand, uninduced Rauscher cells were shown to possess 1,100 receptors/cell, with an equilibrium dissociation constant (KD) of 400 pmol/L. Erythropoietin, but not dimethylsulfoxide, induction caused a specific increase in receptor density to 3,300/cell on differentiating Rauscher cells. This is the first demonstration of membrane receptor regulation by erythropoietin that may be important in the complex interplay of hormonal effects during erythropoiesis.

Topics: Adenylyl Cyclases; Animals; Cell Differentiation; Dimethyl Sulfoxide; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Receptors, Adrenergic, beta

The erythroleukemia induced in mice by the Friend virus complex is caused to regress by macrophages. To determine whether the effect of macrophages on leukemia is due to their role in the regulation of erythropoiesis, investigators examined the influence of macrophages from normal and leukemic mice on in vitro colony formation by erythroid progenitor cells [colony-forming units of erythroid progenitor cells (CFU-E)]. Plasma clot cultures of CFU-E were grown over monolayers of macrophages separated from the CFU-E cultures by a layer of agar. Macrophages from progressors (those leukemic mice that will not undergo leukemia regression) significantly stimulated CFU-E, whereas macrophages from regressors (those that will undergo leukemia regression) had no effect on colony formation. Monolayers of normal resident macrophages did not affect in vitro erythropoiesis, while less mature macrophages (peripheral blood mononuclear cells) or nonspecifically activated macrophages (exudate induced) in monolayer stimulated CFU-E. Stimulation by macrophages from leukemic mice was dependent on the presence of virus-producing cells. CFU-E from erythroleukemic mice were resistant to stimulation by leukemic macrophages. These results demonstrated that macrophages from progressively leukemic mice influenced normal colony formation of CFU-E in vitro and that these effects could be related to the ability of macrophages to cause leukemia regression.

Topics: Animals; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Leukemia, Erythroblastic, Acute; Macrophages; Mice; Mice, Inbred Strains; Stem Cells

IW32 is a recently described murine erythroleukemia cell line that produces an erythropoietic factor similar to erythropoietin by in vivo and in vitro bioassays and without species specificity. Biochemical characteristics of IW32 erythropoietic factor and sheep or mouse plasma erythropoietins were compared. Murine colonies derived from erythroid colony-forming units (CFU-E) in plasma clot culture were used as the bioassay system. Both IW32 erythropoietic factor and sheep plasma erythropoietin were stable in the pH range of 3 to 10, after exposure to denaturing agents (8 mol/L urea, 4 mol/L guanidine hydrochloride, 1% sodium dodecyl sulfate), to a reducing agent (0.1 mol/L 2-mercaptoethanol) and to an oxidizing agent (5 mmol/L sodium metaperiodate). Only the combination of 0.1 mol/L 2-mercaptoethanol and 1% sodium dodecyl sulfate resulted in a significant loss of activity. IW32 erythropoietic factor and murine plasma erythropoietin were similarly precipitated by ethanol and ammonium sulfate. IW32 erythropoietic factor eluted as a single major peak after gel exclusion chromatography, with an estimated molecular weight of 45,000 daltons. Results were identical using supernatants from cultures in the presence of and absence of fetal calf serum. The supernatant of IW32 cells cultured without serum induced erythroid colonies after seven days on normal human bone marrow nonadherent mononuclear cells cultured in serum-free conditions. All these results made it very likely that IW32 cells produce an authentic erythropoietin. This cell line would be very useful for the study of murine erythropoietin.

Topics: Animals; Blood Physiological Phenomena; Cell Line; Chemical Fractionation; Culture Media; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred Strains; Sheep

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Division; Cell Transformation, Viral; Erythrocytes; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Hormones; Leukemia, Erythroblastic, Acute; Mice; Retroviridae; Spleen

A Friend mink cell focus-inducing (Fr-MCF) virus isolated from a Friend tumor cell line was able to induce acute erythroleukemia associated with polycythemia when injected as a Friend murine ecotropic leukemia virus (F-MuLV) pseudotype into adult Swiss and ICFW mice. One virus isolate recovered from leukemic cells and designated as FV-F3 presented the following properties: (i) persistence of the same leukemogenic power when propagated in vivo and in vitro; (ii) in vivo spleen focus-forming (SFFV) capacity; (iii) presence of erythropoietin (EPO)-independent CFU-E in leukemic animals; (iv) expression of a 32 RNA specifically recognized by a SFFV probe, in FV-F3 infected cells; and (v) expression in FV-F3-infected cell of polypeptides in the range of gp52 SFFV. Peptide analysis of these products revealed close similarities with the parental MCF virus. These data suggest that a SFFV genome arose by genetic recombinational events involving MCF virus.

Topics: Animals; Cell Line; Erythropoietin; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Polycythemia; Recombination, Genetic; RNA, Viral; Spleen; Viral Envelope Proteins; Viral Proteins

We have shown morphologic and biochemical evidence for erythroid differentiation in the erythropoietin-responsive cell line Rauscher murine erythroleukemia. These cells synthesize adult and presumably embryonic hemoglobin in response to erythropoietin and dimethyl sulfoxide. Clear differences are observed in some clones between different inducers. The molecular basis for these differences, which may relate to differential modes of inducer action, must be clarified. These cells possess a functional beta-adrenergic receptor/adenylyl cyclase complex remarkably similar to that found on erythrocytes of several species. The receptor density is up-regulated specifically by erythropoietin and, as such, is the first description of membrane receptor regulation by this hormone. Thus, we now have the opportunity to investigate the development of the receptor-cyclase complex on the differentiating erythroid cell and to elucidate its role in the complex interplay of erythropoietin and other hormonal effects during erythropoiesis.

Topics: Animals; Cell Differentiation; Cell Transformation, Viral; Cyclic AMP; Erythropoiesis; Erythropoietin; Globins; Isoproterenol; Leukemia, Erythroblastic, Acute; Mice; Rauscher Virus; Receptors, Adrenergic, beta

A transplantable murine leukemia, primarily induced by a biologically cloned Friend helper virus, was shown to induce polycythemia in recipient ICFW mice. A leukemia cell line (IW.32) was established in vitro from this transplantable leukemia. Sodium butyrate and hemin induced erythroid differentiation in these leukemia cells as has already been shown with other erythroleukemia cells. The supernatant of this cell line was devoid of spleen focus-forming virus activity. However, it induced the incorporation of 59Fe in polycythemic mice and the in vitro differentiation of murine and human cfu-e into erythroid colonies. Therefore, these erythroleukemia cells produced a factor with all the biological properties of erythropoietin. The erythropoietic activity of IW.32 supernatant was higher in vitro [equivalent to 0.5-1 international unit (IU) of erythropoietin per ml] than in vivo (0.15-0.3 IU/ml). This erythropoietin-like activity was stable at 100 degrees C for 3 min, which ruled out the possibility that a virus was responsible for these effects. Preliminary studies demonstrated that the biochemical properties of the IW.32 factor are strongly similar to those of Connaught step 3 erythropoietin, thus supporting the hypothesis that the IW.32 factor is indeed an erythropoietin.

Topics: Animals; Biological Assay; Cell Line; Erythropoiesis; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice

Results reported herein describe the expansion of erythroid stem cells (CFU-e and BFU-e) from marrow and spleen cells from mice infected with Rauscher leukemia virus (RLV). Maximum colony numbers were seen by 5 weeks after viral inoculation. The increase in splenic CFU-e and BFU-e was related to development of splenomegaly, the latter being a characteristic feature of murine oncornavirus infection. Both splenic and marrow RLV-derived CFU-e were erythropoietin (Ep) dependent. However, splenic and marrow RLV-derived BFU-e proliferation occurred at Ep levels considered suboptimal for normal colony formation. This increased erythroid stem cell proliferation may be due in part to the increased demand on erythropoiesis as a result of the profound fatal anemia which occurs in RLV-induced erythroleukemia. These studies suggest that factors operating at the level of BFU-e may enhance erythropoiesis, and hence may be a direct result of murine oncornavirus infection.

Topics: Adult; Animals; Bone Marrow; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Rauscher Virus; Spleen

A Rauscher virus (RV)-transformed erythroid cell line, RA-1, was shown to be a non-producer cell line. RA-1 cells express not only gp51-54 env-related glycoprotein, but also gp70, which is more closely related to gp51-54 coded by a recombinant env gene than to the MuLV-R gp70. RA-1 cells could be infected by Friend, Moloney and Gross viruses, but not by the homologous Rauscher murine leukaemia virus. Rescue of spleen focus-forming activity was obtained on infection of these cells with MuLV-F or MuLV-Mol, but not with MuLV-Gross. The RNA of the RV complex resembles closely that of Friend virus (FV). It contains a 32S, presumably defective, genome, which most likely is responsible for spleen focus formation, and a 35S helper virus genome. Oligonucleotide fingerprint data suggest that RV has evolved independently of FV. Erythroid early BFU-E cells of mice infected with RV of Friend helper virus-infected RA-1 cells were shown to require no addition of conditioned medium to form large erythroid colonies (BFU-E) in the presence of only small amounts of erythropoietin.

Topics: Animals; Base Sequence; Cell Line; Defective Viruses; Erythropoiesis; Erythropoietin; Helper Viruses; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute; Mice; Oligoribonucleotides; Rauscher Virus; RNA, Viral; Viral Envelope Proteins; Viral Proteins

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Helper Viruses; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Spleen

Typical morphological features of surface structural alterations during Friend cells differentiation are described. Scanning electron microscopy (SEM) revealed that DMSO induction switched on cell alteration of the proerythroblast-like cells, possessing microvilli projections on cell membrane with some ruffles, to an advanced stage with a blebby surface. This was followed by the formation of a pear-like polarized cell separated into two zones by a narrow cytoplasmic bridge at the equatorial plane. The polarized cells showed a smooth surface and tended to disconnect into two unequal cells. The villous leukemic erythroblast has negatively charged sialic acid residues on the glycocalyx, available for latex hydrazide probe binding, while the blebby and polarized cells lack it. Tocopherol added to culture medium of DMSO-induced erythroleukemic cells prevented the formation of blebs and the polarization phenomena, without affecting hemoglobin synthesis. The tocopherol-treated cells contain available negative charges for latex hydrazide binding similar to uninduced Friend cells. Erythropoietin potentiated a repolarization ability and morphological alteration capacity to the tocopherol-treated cells and this was accompanied by a loss of glycocalyx-negative charges. At these growth conditions erythyropoietin induced a dose-dependent proliferation effect.

Topics: Animals; Cell Differentiation; Cell Membrane; Dimethyl Sulfoxide; Erythropoietin; Extracellular Space; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Microscopy, Electron, Scanning; Sialic Acids; Vitamin E

In vitro erythropoietin (Ep) responsiveness of human bone marrow mononuclear cells was determined in 12 normal human volunteers and four patients with erythroleukemia (EL), two patients with refractory anaemia with excess blasts (RAEB), and one patient with de novo acute myelogenous leukaemia (AML). The bone marrow cells were cultured in a microtitre methylcellulose system containing 30% human AB serum and human urinary Ep in concentrations ranging from 0 to 2 units/ml. Erythroid colony growth from normal marrow cultures was Ep-dependent. It was augmented by added Ep and inhibited by Ep antiserum. Marrow cells from one patient with EL and one patient with RAEB after transformation to AML had no erythroid colony formation with or without added Ep. All of the remaining patients formed 'spontaneous' or endogenous erythroid colonies (EEC) without the addition of Ep. In three of these (two with EL and one with de novo AML), the erythroid colony formation was augmented by added Ep. In three other patients (one with EL and two with RAEB), erythroid colony growth was unaffected by added Ep or Ep antiserum, and thus appeared to be Ep-independent.

Topics: Aged; Anemia; Bone Marrow; Cell Count; Cells, Cultured; Colony-Forming Units Assay; Erythropoietin; Female; Humans; Immune Sera; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute; Male; Middle Aged

Topics: Animals; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Spleen

In order to study the changes in erythroid precursor cells in erythroleukemia, bone marrow cells from 4 patients were cultured for erythroid colony-forming units (CFU-e). The bone marrow from 3 of the patients showed an excess of ringed sideroblasts, and the incidence of CFU-e was very low. The one patient without ringed sideroblasts showed abundant erythroid colony formation. Erythroid colonies of more than eight erythroblasts consisted of normal-appearing erythroblast, while ringed sideroblasts were observed in scattered erythroblasts or in small clusters. The number of granulocyte macrophage colony-forming units (CFU-GM) was markedly lower than normal in all 4 cases. In 2 cases investigated, blast colonies were formed from bone marrow cells under the stimulation of phytohemagglutinin/leukocyte-conditioned medium. These results show that the differentiation of hemopoietic stem cells to erythroid as well as to myeloid cells is affected in erythroleukemia.

Topics: Aged; Bone Marrow; Cells, Cultured; Erythroblasts; Erythropoiesis; Erythropoietin; Female; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Macrophages; Male; Middle Aged

Topics: Animals; Cell Cycle; Cell Differentiation; Erythropoiesis; Erythropoietin; Hemin; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Models, Biological; Spectrin

An erythropoietin-independent virus-induced murine erythroleukemia (FVP) is used to compare the effects of an erythropoiesis inhibitory factor (EIF) isolated from human urine with the effects of prostaglandin F2 alpha. The consequent inhibition of FVP-induced erythropoiesis suggests that EIF and PGF2 alpha have similar effects on the FVP-induced erythropoiesis in mice, and the effect of PGF2 alpha is indirect. The similarity of the actions of EIF and PGF2 alpha may indicate a potential role for prostaglandins in the physiological control of some types of erythrocytosis.

Topics: Animals; Bone Marrow Cells; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Hematocrit; Humans; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred AKR; Mice, Inbred DBA; Prostaglandins; Prostaglandins D; Prostaglandins F

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.

Topics: Animals; Bone Marrow Cells; Cell Transformation, Viral; Cells, Cultured; Defective Viruses; Erythropoietin; Fibroblasts; Helper Viruses; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Leukemia, Myeloid; Mice; Mice, Inbred Strains; Rats; Sarcoma Viruses, Murine; Spleen; Virus Cultivation

Topics: Abelson murine leukemia virus; Antigens, Surface; Antigens, Viral; Cell Differentiation; Cytotoxicity, Immunologic; Erythropoietin; Friend murine leukemia virus; Genes, Regulator; Hematopoiesis; Leukemia Virus, Murine; Leukemia, Erythroblastic, Acute

Clonal lines of Rauscher erythroleukemia cells exhibited selective responses to two inducers of differentiation, erythropoietin and dimethyl sulfoxide. There were substantial quantitiative differences between clones that reponded to both inducers. Several clones differentiated only in response to erythropoietin. Erythropoietin stimulated cell proliferation and differentiation whereas dimethyl sulfoxide inhibited proliferation, suggesting dissimilar modes of action.

Topics: Animals; Cell Differentiation; Cell Division; Clone Cells; Dimethyl Sulfoxide; Erythropoietin; Hemoglobins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Rauscher Virus

The transforming capabilities of FVA, RLV and FVP have been examined using an in vitro transformation assay. Treatment of bone marrow cells with FVP in vitro led to the formation of hemoglobinized erythroid bursts even when these cells were cultured in methylcellulose for 5 days without added erythropoietin (Epo). A variety of FVA and RLV preparations also produced erythroid bursts without Epo but these bursts contained significantly less hemoglobin than those induced by FVP. When very low levels of Epo were added to cultures of FVA- and RLV-infected cells, the bursts were hemoglobinized, that is, similar to FVP-induced bursts. The burst-inducing agent in FVA preparations was shown to be a virus and not Epo. Spleen focus-forming virus (SFFV) pseudotypes, derived from FVA or FVP, also produced erythroid bursts in vitro, whereas four helper murine leukemia viruses did not. These studies indicated that the SFFV component was essential for erythroid burst transformation and specified the degree of hemoglobinization in the bursts formed.

Topics: Anemia; Animals; Cell Transformation, Viral; Cells, Cultured; Defective Viruses; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Hot Temperature; Leukemia, Erythroblastic, Acute; Mice; Polycythemia; Rauscher Virus

In order to study the changes in erythroid precursor cells in primary acquired and secondary sideroblastic anemia, bone marrow cells from 4 patients with primary acquired sideroblastic anemia (PASA) and 3 patients with refractory anemia with excess of myeloblasts (RAEM) or erythroleukemia associated with an excess of ringed sideroblasts were cultured for erythroid colony-forming units (CFU-E). The number of CFU-E was markedly decreased in all 7 cases, and erythroid colonies formed consisted exclusively of normal-appearing erythroblasts, while ringed sideroblasts were observed in scattered single erythroblasts or in small aggregates of erythroblasts in primary as well as in secondary sideroblastic anemia. These findings may indicate the presence of 2 populations of erythroid progenitor cells in the bone marrow of patients with primary acquired and secondary sideroblastic anemia. A slight to moderate decrease in granulocyte-macrophage colony-forming units (GM-CFU) was observed in 3 cases of PASA. The decrease in GM-CFU, however, was marked in sideroblastic anemia associated with RAEM or erythroleukemia.

Topics: Aged; Anemia, Aplastic; Anemia, Sideroblastic; Colony-Forming Units Assay; Erythroblasts; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Iron; Leukemia, Erythroblastic, Acute; Male; Middle Aged

The elucidation of the mechanism of action of erythropoietin depends upon a detailed assessment of its effects at the molecular level. We have now begun to examine the effects of human erythropoietin and other inducers on clonal lines of Rauscher murine erythroleukemia cells. Over 100 clonal lines have been examined by assessing the hemglobinization of colonies grown in plasma clot culture in response to erythropoietin and dimethylsulfoxide. Many of the clones respond to both inducers. However, some clones respond only to erythropoietin. The cells also differentiate in suspension culture, exhibiting striking morphological changes characteristic of erythroid development. This system should serve as an excellent model for the study of control mechanisms in erythropoiesis.

Topics: Animals; Cell Line; Clone Cells; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Mice; Mitosis

The effect of various agents which are known to increase the differentiation of Friend erythroleukemia cells was investigated in cultures of mouse bone marrow cells. N,N-dimethylacetamide (5 and 15 mM) and acetamide (60 mM) significantly increased the number of erythroid colonies observed. Tetramethylurea, dimethylformamide, pyridine N-oxide, and butyric acid were ineffective. Dimethylsulfoxide at a concentration of 1% significantly increased colony number in cultures of marrow cells obtained from male mice, but had no effect in cultures of female bone marrow cells.

Topics: Acetamides; Animals; Cells, Cultured; Colony-Forming Units Assay; Dimethyl Sulfoxide; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Male; Mice; Tumor Virus Infections

Normal and transformed erythroid cell precursors provide the opportunity for study of a number of problems relevant to the regulation of proliferation and differentiation in a developmental system. Evidence is presented which suggests that the hormone, erythropoietin, has a primary role in regulating precursor cell proliferation. A wide variety of chemicals can modify the rate at which proliferating transformed precursors initiate expression of the genetic program characteristic of terminal erythroid differentiation. Several sites of inducer action, including the plasma membrane and chromatin, are suggested as part of the pathway which leads to the complex pattern of gene transcription responsible for differentiation.

Topics: Acetamides; Animals; Cell Differentiation; Cell Line; Clone Cells; Dactinomycin; DNA; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Hematopoietic Stem Cells; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Models, Biological; RNA, Messenger

Friend virus-transformed murine erythroleukemic cells (FL cells) have been used as an in vitro model for the study of the expression of the genetic program involved in the final stages of erythroid differentiation. Treatment of the FL cells with chemical inducers such as dimethylsulfoxide results in their differentiation from 'pro-erythroblasts' to orthochromatic normoblasts and the appearance of several erythroid markers including hemoglobin, enzymes of the heme pathway, heme, glycophorin, and spectrin. These markers appear in an ordered sequence, suggesting that two genetic programs are involved in the erythroid differentiation of the cells. Preliminary studies with erythropoietin-stimulated fetal liver cultures in vitro suggest that the same is true for normal erythroid differentiation.

Topics: Analysis of Variance; Animals; Cell Differentiation; Chromatin; Dimethyl Sulfoxide; Erythrocytes; Erythropoietin; Friend murine leukemia virus; Genes; Heme; Leukemia, Erythroblastic, Acute; Liver; Mice; Mice, Inbred DBA; Spectrin

We studied the growth of erythroid burst-forming units (BFU-E) and erythroid colony forming units (CFU-E) from bone marrow and blood in six patients with erythroleukemia. Five patients grew CFU-E, while BFU-E were found in the marrow of two and in the peripheral blood of only one patient. In all cases with colony growth, the numbers of colonies were markedly decreased with respect to normal controls. Patient BFU-E were composed of fewer clusters than those of controls. BFU-E and CFU-E growth was dependent on the addition of erythropoietin to the medium, and no growth was observed in absence of erythropoietin. At present it is not known if the growth obtained is derived from residual normal erythropoietic stem cells or from abnormal erythroid precursors of the leukemic cells.

Topics: Adult; Aged; Bone Marrow Cells; Cells, Cultured; Erythropoiesis; Erythropoietin; Female; Humans; Leukemia, Erythroblastic, Acute; Male; Middle Aged

Inoculation of complete Freund's adjuvant (CFA) into BALB/c mice either before or after infection with Rauscher murine leukemia virus (MuLV-R) led to an acceleration of the disease as determined by spleen weight. Treatment with CFA also induced a higher number of spleen erythroblast foci and, in the bone marrow, erythropoietin-independent cells that produced erythroid colonies in vitro. CFA induced in the bone marrow not only an increase in myeloid progenitor cells that can produce colonies in agar, but an ever larger increase in the number of erythroid colony-forming cells. Virus-induced erythroblastosis was probably enhanced by CFA due to the production of many target cells. The more primitive burst-forming cell, which produced large colonies of erythroid cells after 10 days in culture, was also physiologically transformed in MuLV-R-infected mice; bursts could be formed by cells of such animals in the absence of erythropoietin.

Topics: Animals; Antibody-Producing Cells; Antigen-Antibody Reactions; Bone Marrow; Bone Marrow Cells; Cell Division; Erythropoietin; Female; Freund's Adjuvant; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred BALB C; Rauscher Virus; Spleen; Splenomegaly

We have reviewed erythroid cell differentiation from two points of view: 1) differences between fetal and adult human red cells with particular reference to alterations which can occur in the normal pattern of erythroid cell development during the course of leukemia; 2) beochemical events which occur during erythroid cell maturation, as a model system for the study of the control of gene expression. During the course of many leukemias there is the synthesis of red cells containing fetal hemoglobin. In most cases this phenomenon is limited to a small population or clone of red cells and probably represents a nonspecific response of the bone marrow to a hematologic stress. However, in juvenile chronic myeloid leukemia and, in rare cases of erythroleukemia, there is a major reversion to fetal erythropoiesis, with progressive increase in fetal hemoglobin levels and synthesis of red cells which contain not only fetal hemoglobin but have a true fetal pattern of protein synthesis affecting proteins other than Hb F, namely Hb A2, carbonic anhydrase and the membrane antigens i and I. In this case, the fetal erythropoiesis may be a more specific manifestation of the leukemic process and may be related to the phenomenon of fetal protein synthesis (alpha-fetoprotein of carcinoembryonic antigen) observed in other types of neoplasia. Further information on the etiology and pathogenesis of abnormal cell proliferation and differentiation in the leukemias can be obtained by the study of experimental systems permitting the investigation of the regulation of gene expression in differentiating mammalian cells. Maturing erythroid cells provide a promising system for such investigations for many reasons: differentiating erythroid cells can be obtained relatively free of other cell types; a large amount of a well characterized product, hemoglobin, is synthesized; techniques are now available that permit isolation of erythroid precursors at different stages of differentiation (5-8); and finally, highly sensitive methods of measuring globin mRNA levels by DNA-RNA hybridization are currently available (13, 26, 27). We have used such techniques to measure levels of globin mRNA in separated populations of murine erythroid cells at different stages of maturation. These studies demonstrated a correlation between globin mRNA content and degree of morphological maturation. In the least well differentiated cells, however, there appeared to be a disproportionate amount of mRNA for the lev

Topics: Anemia, Hemolytic; Animals; Blood Cell Count; Cell Differentiation; Cell Division; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Fetal Hemoglobin; Genes; Globins; Hematopoietic Stem Cells; Heme; Hemoglobin H; Humans; Leukemia; Leukemia, Erythroblastic, Acute; Mice; Phenylhydrazines; RNA, Messenger; Time Factors

Topics: Alleles; Animals; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA; Enzyme Induction; Erythrocytes; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Genes; Globins; Hemoglobins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; RNA; RNA, Messenger; Transcription, Genetic

Infection of mice with the polycythemia-inducing strain of Friend leukemia virus caused a rapid emergence of new erythroid precursor cells. These cells which, in the absence of erythropoietin, proliferated in vitro to form colonies and even differnetiated, quickly out-numbered the usual erythropoietin-dependent hematopoietic elements in bone marrow and spleen. Ultimately, the marrow and spleen were probably totally repopulated with this erythropoietin-independent cell.

Topics: Animals; Bone Marrow; Cell Division; Erythropoiesis; Erythropoietin; Female; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Male; Mice; Spleen

An erythropoietin-independent murine erythroleukemia (FVp) has been used to evaluate the effects of an erythropoiesis-inhibitory factor (EIF) isolated from human urine. The consequent inhibition of FVp-induced erythropoiesis suggests that EIF exerts its effect independently of ESF. The inhibitory effect of EIF on FVp-induced erythropoiesis may reveal a potential for the physiological control of some types of erythroleukemia.

Topics: Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Friend murine leukemia virus; Hematocrit; Humans; Iron Radioisotopes; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred AKR; Organ Size; Spleen; Splenomegaly; Time Factors; Urine

Topics: Animals; Benzidines; Blood Coagulation; Cells, Cultured; Culture Media; Dimethyl Sulfoxide; Erythrocytes; Erythropoiesis; Erythropoietin; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Staining and Labeling

Topics: Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Depression, Chemical; Dimethyl Sulfoxide; DNA, Neoplasm; Erythropoietin; Friend murine leukemia virus; Heme; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Neoplasm Proteins; RNA, Neoplasm; Stimulation, Chemical

Topics: Animals; Carbon Radioisotopes; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Culture Media; Dimethyl Sulfoxide; Erythropoietin; Globins; Iron; Leucine; Leukemia, Erythroblastic, Acute; Lipids; Mice; Proteins; Stimulation, Chemical; Tritium

Topics: 5-Aminolevulinate Synthetase; Animals; Erythrocyte Count; Erythropoiesis; Erythropoietin; Hematocrit; Hemoglobins; Leukemia, Erythroblastic, Acute; Liver; Mice; Phenylhydrazines; Rauscher Virus; Reticulocytes; Spleen

Topics: Bone Marrow Cells; Bone Marrow Examination; Cell Count; Child; Erythropoiesis; Erythropoietin; Hemoglobins; Humans; Leukemia, Erythroblastic, Acute; Male

Topics: Blood Cell Count; Blood Platelets; Blood Transfusion; Bone Marrow Examination; Cell Nucleus; Erythrocytes; Erythrocytes, Abnormal; Erythropoiesis; Erythropoietin; Exchange Transfusion, Whole Blood; Hematocrit; Humans; Iron; Iron Isotopes; Leukemia, Erythroblastic, Acute; Lymphoma; Mitosis; Reticulocytes

Topics: Anemia; Bilirubin; Blood Transfusion; Bone Marrow; Bone Marrow Cells; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Leukemia, Erythroblastic, Acute; Leukocyte Count; Male; Middle Aged; Pneumonia; Reticulocytes; Vitamin B 12

Topics: Animals; Chick Embryo; Cobalt; Erythropoietin; Leukemia, Erythroblastic, Acute; Nandrolone; Poultry; Prednisone; Vitamin B 12