losartan-potassium and Leukemia--Megakaryoblastic--Acute

Human umbilical cord blood (CB) is a recognized source of hematopoietic tissues for transplantation, the treatment of malignancies and gene therapy, among other potential clinical applications. A rich network of hematopoietic cytokines and growth factors possessing stimulatory effects on primitive hematopoietic stem/progenitor cells further characterizes fetal CB. To better elucidate these complex interactions and properties, we compared the hematopoietic activities of CB and normal human peripheral blood (PB), by examining growth/survival of normal hematopoietic progenitors and erythropoietin-dependent UT-7/EPO cells. Colony-forming activity assays of normal bone marrow (BM) BFU-E and CFU-GM showed that CB significantly enhanced progenitor cell growth in comparison to PB. Apoptosis was determined by enumerating APO 2.7 mAb stained cells using flow cytometry. UT-7/EPO cell cultures subjected to PB exhibited a four-fold higher rate of apoptosis than CB exposed cultures, indicating that CB markedly suppressed apoptosis in this human leukemic cell line. Immunoprecipitation of UT-7/EPO cell lysates and immunodetection of the anti-phosphotyrosine Ab (4G10), revealed that CB induced tyrosine phosphorylation of three proteins, with approximate molecular masses of 160, 117.5 and 80 kDa. The 80 kDa protein corresponds to the previously reported molecular mass for the Epo receptor, suggesting that erythropoietin is enriched in CB compared with adult PB.

Topics: Adult; Apoptosis; Cell Division; Cell Survival; Erythroid Precursor Cells; Erythropoietin; Fetal Blood; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Megakaryoblastic, Acute; Molecular Weight; Phosphorylation; Tumor Cells, Cultured; Tumor Stem Cell Assay

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet alpha-granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34(+)/CD41(-) cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-type c-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34(+)/CD41(-) cells to CD34(-)/CD41(+) cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

Topics: Biomarkers; Cell Differentiation; Colony-Forming Units Assay; DNA, Complementary; Erythropoietin; Fetal Blood; Gene Expression Regulation, Developmental; Humans; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Thrombopoietin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetradecanoylphorbol Acetate; Thrombopoietin; Transfection; Tumor Cells, Cultured

HML/SE is a cytokine-dependent cell line established from childhood acute megakaryoblastic leukemia. Granulocyte-macrophage colony-stimulating factor or stem cell factor (SCF) alone could stimulate proliferation of HML/SE cells, however interleukin-3, interleukin-6, granulocyte colony-stimulating factor and thrombopoietin could not. Although erythropoietin (EPO) alone stimulated neither proliferation nor differentiation of HML/SE cells, it did stimulate proliferation of HML/SE cells and production of hemoglobin in the presence of SCF. SCF activated the human EPO receptor promoter and induced EPO receptor gene expression. Given these results, we speculate that HML/SE cells acquired responsiveness to EPO via the EPO receptor induced by SCF. Mutation analysis of putative transcription factor binding sites in the human EPO receptor promoter suggested that Sp1, rather than the GATA-1 binding site, contributed to the induction of the hEPOR gene. Although it is well documented that hematopoietic stem cells and primitive progenitors require both an early-acting cytokine and a lineage-specific cytokine to differentiate to a certain lineage, related mechanisms are not well understood. HML/SE may serve as an excellent model system to analyze functions of early-acting cytokine SCF and lineage-specific cytokine EPO related to proliferation and differentiation of hematopoietic stem cells.

Topics: Antigens, Surface; Cell Differentiation; Cell Division; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Megakaryoblastic, Acute; Promoter Regions, Genetic; Receptors, Erythropoietin; RNA, Messenger; Stem Cell Factor; Tumor Cells, Cultured

To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

Topics: Cell Cycle; Cell Division; Cell Line; Cell Separation; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; GATA2 Transcription Factor; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Megakaryoblastic, Acute; Models, Genetic; Neoplasm Proteins; Receptors, Erythropoietin; Recombinant Proteins; Resting Phase, Cell Cycle; RNA, Messenger; Transcription Factors

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Base Sequence; Cell Transformation, Neoplastic; Cloning, Molecular; DNA, Complementary; Erythropoietin; Fusion Proteins, bcr-abl; Genes; Granulocyte-Macrophage Colony-Stimulating Factor; GRB2 Adaptor Protein; Humans; Leukemia, Megakaryoblastic, Acute; Molecular Sequence Data; Neoplasm Proteins; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Recombinant Fusion Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; src Homology Domains; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

HU-3 is a bipotential cell line derived from the bone marrow of a patient with megakaryoblastic leukemia. Continuously proliferating cells evolved from cultures supplemented with nutrient medium containing human serum and granulocyte-macrophage (GM) colony-stimulating factor (CSF). Growth and viability of the HU-3 cell line was strictly dependent on the presence of GM-CSF, interleukin-3, or thrombopoietin (Tpo). Independent of the cytokine, the cells constitutively expressed a well-defined megakaryocyte phenotype, with 70-95% of the cells positive for CD4, CD34, and platelet glycoproteins Ib, IIb, and IIIa. Fewer than 10% of the cells had detectable erythroid glycophorin A. Erythropoiesis was induced in HU-3 parental cells and five clones harvested from culture medium containing GM-CSF by replacement of the growth-promoting cytokine with stem cell factor (SCF) and erythropoietin (Epo). During the first week of induction, the proliferating cells slowly acquired erythroid markers. Concomitant with a maturational growth arrest during the second week, there was a rapid accumulation of gamma and beta globin chains and benzidine reactive hemoglobin, as well as a distinct erythroid morphology. The culture declined after 12 days because of the transient effect of SCF in maintaining viability. Parental and cloned cells cultured for 7 days in Tpo-supplemented medium responded to the synergistic growth effect of SCF and Epo but were markedly suppressed in their yield of hemoglobinized cells. Recycling of the cells in GM-CSF for 4 days did not reverse the suppressive effect of Tpo. These results suggest a role for Tpo in the lineage commitment of erythromegakaryocytic progenitors by suppressing the erythroid potential. With its constitutive megakaryocyte phenotype and inducible erythroid potential, the self-renewing bipotential HU-3 cell line may represent one of the earliest stages in megakaryocytopoiesis before irreversible lineage commitment. The suppressive effect of Tpo on the erythroid potential of cloned HU-3 cells enhances the value of this cell line for deciphering the molecular and cellular events during lineage commitment of progenitor cells.

Topics: Cell Division; Cell Transformation, Neoplastic; Cytogenetics; Cytokines; Erythropoiesis; Erythropoietin; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Megakaryoblastic, Acute; Stem Cell Factor; Thrombopoietin; Tumor Cells, Cultured

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.

Topics: Cell Differentiation; Cell Division; Cell Survival; DNA-Binding Proteins; DNA, Neoplasm; Erythroid-Specific DNA-Binding Factors; Erythropoietin; GATA1 Transcription Factor; GATA2 Transcription Factor; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-3; Janus Kinase 2; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Milk Proteins; Neoplasm Proteins; NF-E2 Transcription Factor; NF-E2 Transcription Factor, p45 Subunit; Ploidies; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Thrombopoietin; STAT5 Transcription Factor; Thrombopoietin; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

The megakaryoblastic cell line, UT-7, is dependent for its growth upon interleukin-3 (IL-3), erythropoietin, or granulocyte-macrophage colony stimulating factor (GM-CSF). A subculture of this line can be maintained in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this subculture from SCF results in rapid loss of viability and decreased proliferation. Cells grown in SCF also can be maintained in GM-CSF but not vice versa. In this work, we have characterized the SCF dependence of this UT-7 subculture. Stem cell factor removal results in apoptosis and a decline in viability which can be restored partially by re-addition of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented by light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quantitate the degree of apoptosis in the cell populations, and to assess whether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exposure of previously factor-starved cells to stroma decreased the percentage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggesting contribution of other stromal cell factors. Experiments performed using trans-well inserts which do not allow cell passage, showed greatest viability of cells in contact with stroma, but viability was also improved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a role for soluble stroma-produced substances. These data demonstrate that SCF alone can prevent apoptosis in cells dependent upon its presence for proliferation. Also, marrow stromal cells can serve as a partial substitute for growth factor in the prevention of apoptosis in these cells, probably due to constitutive presentation of SCF and other hematopoietic growth factors in both soluble and surface-bound forms.

Topics: Antibodies; Apoptosis; Bone Marrow Cells; Bucladesine; Cell Division; Cell Line; Cell Survival; Coculture Techniques; DNA, Neoplasm; Erythropoietin; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Kinetics; Leukemia, Megakaryoblastic, Acute; Microscopy, Electron; Recombinant Proteins; Stem Cell Factor; Stromal Cells; Time Factors; Tumor Cells, Cultured

The actions of transforming growth factor beta (TGF beta) and erythropoietin (Epo) were studied using normal erythroid progenitors from fetal rat liver and spleen at 18, 19 and 20 days. rhTGF beta 1 inhibited the growth of late BFUe colonies significantly at each age and in both organs in methylcellulose cultures containing 2 U/ml rhEpo. There was no significant inhibition of CFUe proliferation, except for spleen CFUe at 18 days, suggesting different CFUe sensitivities to growth factors at a given fetal age, 18 days, in liver and spleen. The colorimetric MTT assay was used to examine the inhibition of the growth of human leukemic UT-7 cells by TGF beta 1. TGF beta 1 inhibited the proliferation of UT-7 cells in cultures without Epo at 24 h and in cultures with Epo at 24 and 72 h. The specific binding of [125I]Epo to UT-7 surface was decreased by TGF beta 1 without any change in non-specific binding. TGF beta 1 also inhibited the expression of Epo-receptors on UT-7 cells, without changing receptor affinity. The inhibition of hematopoietic progenitor cell growth by TGF beta could involve altering the cell surface expression of growth factor receptors.

Topics: Animals; Cell Division; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Gestational Age; Humans; Leukemia, Megakaryoblastic, Acute; Liver; Neoplasm Proteins; Neoplastic Stem Cells; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Spleen; Transforming Growth Factor beta

Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF.

Topics: Animals; Base Sequence; Cell Division; DNA-Binding Proteins; DNA, Neoplasm; Erythropoietin; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon Regulatory Factor-1; Janus Kinase 2; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Mice; Milk Proteins; Molecular Sequence Data; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Immunologic; Receptors, Thrombopoietin; STAT5 Transcription Factor; Thrombopoietin; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT-7/Epo showed a greater level of heme content and ratio of benzidine-positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cell Division; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoietin; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Hemoglobins; Humans; Interleukin-3; Leukemia, Megakaryoblastic, Acute; Nucleic Acid Hybridization; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Tumor Cells, Cultured

Erythropoietin (Epo) affects not only erythrocyte production but, in vitro, also promotes megakaryocyte maturation. However, the mechanism of action of Epo on megakaryocytic development remains to be determined. Recently, we reported the establishment of a human Epo-dependent megakaryoblastic leukemic cell line UT-7. Exposure of UT-7 to the tumor promoter, phorbol myristate acetate (PMA), resulted in the appearance of mature megakaryocytic properties, including the expression of platelet factor 4 and beta-thromboglobulin. With exposure to PMA, however, UT-7 cells lost their responsiveness to Epo and Scatchard analysis showed an 85% decrease in the number of Epo receptors after 24 h. While the number of binding sites declined, the affinity of Epo binding was unchanged. Associated with the decline in the number of Epo receptors was a profound decrease (> 95%) in the level of Epo receptor (Epo-R) mRNA. To determine the level of regulation of the Epo-R gene, its rate of transcription was measured by nuclear run-off assay in untreated cells and in cells exposed to PMA for 6, 12, and 24 h. The rate of transcription was nearly identical at all time points in control and PMA-treated cells. Stability of Epo-R mRNA also was measured in the presence of actinomycin D, an inhibitor of transcription. The half-life of Epo-R mRNA in untreated and PMA-treated cells was 90 and 30 min, respectively. These results indicate that the down-modulation of the expression of the Epo-R gene is mainly caused by increased instability of mature mRNA of Epo-R. Posttranscriptional regulation may be an important mechanism in the regulation of hematopoietic growth factor receptor genes and one of the mechanisms by which lineage restriction is achieved.

Topics: Cell Differentiation; Cycloheximide; Down-Regulation; Erythropoietin; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Receptors, Erythropoietin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In this study, specific binding sites were examined for erythropoietin (EPO) on the mouse leukemic cell line, L8057. This cell line is megakaryoblastic in origin as evidenced by an enlargement of cell size, multinuclearity, intense activity of acetylcholinesterase, more expression of glycoprotein IIb and IIIa antigen, and higher ploidy distribution after the treatment with 12-o-tetradecanoylphorbor-13-acetate (TPA). The original undifferentiated cells possessed a single class of low affinity binding sites for recombinant human (rh) EPO with a Kd of 3.5 nM. Following the treatment with TPA, high affinity binding sites (Kd; 440 pM) were expressed in addition to the low affinity sites. EPO stimulated the incorporation of 3H-leucine into TPA-treated L8057 cells, and the maximal effect of EPO was observed at the same order as the Kd value of high affinity sites. The present data demonstrates that the expression of high affinity binding sites for EPO is associated with the differentiation of L8057 cells which have megakaryocytic characteristics. Furthermore, protein synthesis stimulated by EPO may be mediated through the high affinity sites.

Topics: Acetylcholinesterase; Animals; Antigens; Cell Differentiation; Cell Transformation, Neoplastic; DNA, Neoplasm; Erythropoietin; Iodine Radioisotopes; Leucine; Leukemia, Experimental; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Mice; Platelet Membrane Glycoproteins; Protein Binding; Receptors, Cell Surface; Receptors, Erythropoietin; Recombinant Proteins; Tetradecanoylphorbol Acetate; Thymidine; Tumor Cells, Cultured

UT-7 is a human megakaryoblastic cell line capable of growing in interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (Epo) (Cancer Res 51:341, 1991). We used this cell line and a selected Epo-dependent subcell line (UT-7/Epo) to study the early signal transduction events induced by Epo. When UT-7 cells were exposed to Epo, tyrosine phosphorylation of several proteins (with molecular weight equivalent to that of p85, p110, and p145) was observed. Protein phosphorylation occurred in both a dose- and time-dependent manner. p85 showed a marked increase in phosphotyrosine content within 30 seconds; maximal phosphorylation was observed at 1 minute. Subsequently, tyrosine phosphorylation of p110 and p145 was observed, beginning at 1 minute and reaching plateau at 5 minutes. The degree of phosphorylation of these three proteins gradually decreased thereafter. In addition, in UT-7/Epo cells, Epo induced tyrosine phosphorylation of other proteins that were not observed in Epo-induced UT-7 cells. The concentration of Epo required to induce tyrosine phosphorylation was in the same range of concentration required to stimulate cell growth. Epo was also able to activate p21ras as measured by exchange of guanosine diphosphate for guanosine triphosphate. These data show that tyrosine phosphorylation and P21ras activation are early signals in the Epo-induced mitogenic pathway.

Topics: Erythropoietin; Guanosine Triphosphate; Hematopoiesis; Humans; In Vitro Techniques; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Phosphoproteins; Phosphorylation; Phosphotyrosine; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Signal Transduction; Tumor Cells, Cultured; Tyrosine

The UT-7 cell line was established from a patient with a megakaryoblastic leukemia (Komatsu et al, Cancer Res 51: 341, 1991). Its proliferation is strictly dependent on the presence of hematopoietic growth factors including erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). We investigated the differentiation capacities of this cell line under the action of several growth factors, using immunomarkers, flow cytometry, and ultrastructural techniques. In the presence of GM-CSF and IL-3, eosinophil and basophil promyelocytes were detected, as well as a few cells with erythroid and megakaryocytic (MK) differentiation features. In contrast, Epo induced a marked erythroid differentiation with an increase of glycophorin A expression, accompanied by a few hemoglobinized cells. Differentiation induced by the growth factors took 24 to 48 hours to begin, and increased with cell passages to a plateau at 2 weeks of culture. However, this was not only due to a cell selection because the differential effects of Epo and GM-CSF were observed from a single cell clone and the phenotype could be reversed by opposite growth factors, even after a long period of culture. We subsequently investigated the phenotype of UT-7 in the presence of combinations of Epo, IL-3, and GM-CSF, and showed that GM-CSF and IL-3 act predominantly over Epo. This effect was mediated by a rapid downmodulation of Epo receptors by GM-CSF at messenger RNA and binding sites levels, without a change in receptor affinities. On the other hand, Epo had no effect on number and affinity of GM-CSF receptors. This study shows that UT-7 is a growth factor-dependent pluripotent cell line in which commitment may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors.

Topics: Basophils; Cell Differentiation; Dose-Response Relationship, Drug; Eosinophils; Erythroblasts; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-3; Leukemia, Megakaryoblastic, Acute; Receptors, Erythropoietin; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The pathogenic human parvovirus B19 has extreme tropism for human erythroid progenitor cells and has resisted cultivation in conventional cell lines. We report first propagation of this virus in an erythropoietin-dependent strain of a megakaryoblastic leukemia cell line called UT-7. Virus protein was present in about 5% of cells after 1 week of culture. Appropriate ratios of major and minor capsid proteins were determined by immunoblot, and newly synthesized capsid protein was detected by immunoprecipitation of radioactively labeled cell lysates. High molecular weight monomer and dimer intermediates were detected by Southern analysis, indicating active viral replication. Approximately 1,000 genome copies were present per infected cell, and at the optimal multiplicity of infection 20- to 50-fold more virus was produced than inoculated. Virus propagation only occurred in UT-7 cells that were adapted to growth in erythropoietin; virus signal was not detected in UT-7 cells adapted for growth in granulocyte-macrophage colony-stimulating factor or interleukin-3, even with exposure to erythropoietin for several days. Infectious virus was detected in cultures as long as 3 months after inoculation. Despite persistence, there was no evidence of viral integration on Southern analysis. This cell line may prove useful for the production of infectious virus and in the analysis of B19 parvovirus persistence, cytotoxicity, and permissivity.

Topics: Blotting, Southern; Capsid; DNA, Viral; Erythropoietin; Humans; Immunoblotting; Immunosorbent Techniques; Leukemia, Megakaryoblastic, Acute; Nucleic Acid Hybridization; Parvovirus B19, Human; Tumor Cells, Cultured; Virus Cultivation

Topics: Blood Platelets; Cell Division; Drug Synergism; Erythropoietin; Hematopoiesis; Humans; Leukemia, Megakaryoblastic, Acute; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13-acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.

Topics: Blotting, Northern; Bone Marrow Cells; Cell Differentiation; Cell Division; Colony-Stimulating Factors; Erythropoietin; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Immunologic Techniques; Interleukin-1; Interleukin-3; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Platelet Membrane Glycoproteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured