scholarly journals Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 2860-2871 ◽  
Author(s):  
DA Morgan ◽  
DL Gumucio ◽  
I Brodsky
Keyword(s):  
Cell Line ◽  
Transforming Growth Factor ◽  
Beta Globin ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Gamma Globin ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Dependent Growth ◽  
Stimulating Factor

Abstract Peripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin- 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins Ib and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF- deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nuclei-extruding normoblasts were seen. Transforming growth factor-beta inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain- specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells. beta-globin was induced and gamma-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of beta-like globin transcripts showed very low levels of epsilon- and beta-globin expression and high levels of gamma-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, epsilon message decreased to barely detectable levels while gamma and beta transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.

scholarly journals Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 2860-2871 ◽  
Author(s):  
DA Morgan ◽  
DL Gumucio ◽  
I Brodsky
Keyword(s):  
Cell Line ◽  
Transforming Growth Factor ◽  
Beta Globin ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Gamma Globin ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Dependent Growth ◽  
Stimulating Factor

Peripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin- 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins Ib and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF- deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nuclei-extruding normoblasts were seen. Transforming growth factor-beta inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain- specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells. beta-globin was induced and gamma-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of beta-like globin transcripts showed very low levels of epsilon- and beta-globin expression and high levels of gamma-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, epsilon message decreased to barely detectable levels while gamma and beta transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.

scholarly journals Growth and differentiation of a human megakaryoblastic cell line, CMK

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 42-48 ◽  
Author(s):  
N Komatsu ◽  
T Suda ◽  
M Moroi ◽  
N Tokuyama ◽  
Y Sakata ◽  
...  
Keyword(s):  
Cell Line ◽  
Colony Formation ◽  
Culture System ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Hematopoietic Factors ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract 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.

scholarly journals Inhibition of purinergic P2X receptor 7 (P2X7R) decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in U251 glioblastoma cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew Drill ◽  
Kim L. Powell ◽  
Liyen Katrina Kan ◽  
Nigel C. Jones ◽  
Terence J. O’Brien ◽  
...  
Keyword(s):  
Cell Line ◽  
P2x Receptor ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Colony Stimulating Factor ◽  
U251 Cell ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12–15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.

scholarly journals Establishment and erythroid differentiation of a cytokine-dependent human leukemic cell line F-36: a parental line requiring granulocyte- macrophage colony-stimulating factor or interleukin-3, and a subline requiring erythropoietin

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2261-2268 ◽  
Author(s):  
S Chiba ◽  
F Takaku ◽  
T Tange ◽  
K Shibuya ◽  
C Misawa ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemic Cell ◽  
Parental Line ◽  
Colony Stimulating Factor ◽  
Leukemic Cell Line ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Human Leukemic Cell Line ◽  
Stimulating Factor

We have established a new nonlymphoid leukemic cell ine from a patient with myelodysplastic syndrome (MDS), which progressed to overt leukemia. The parental cell line and a subline derived from this line have absolute dependency on several cytokines for their long-term survival and growth. The parental line designated F-36P requires granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for continuous growth, while a subline designated F-36E can be maintained in the presence of erythropoietin (Epo) alone. When these cytokines are depleted, both the parental and the subline cells die within several days, even in medium supplemented with fetal calf serum (FCS). F-36E, maintained in the presence of Epo, constitutively synthesizes hemoglobin at a significant level. F-36P, which is usually maintained in the presence of GM-CSF or IL-3, can be induced to synthesize hemoglobin when GM-CSF or IL-3 is substituted by Epo. The surface marker profile shows that the F-36P cells are positive for the leukocyte common antigen (CD45) and some common multilineage markers such as CD13, CD33, and CD34, and negative for T- and B-cell antigens and mature myelomonocytic antigens. However, some monoclonal antibodies recognizing erythroid and platelet glycoproteins react with these cells. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in a multipotent stem cell. It is also evident that the F-36 cells can be induced to differentiate into the erythroid lineage in the presence of Epo. This, to our knowledge, is the first description of a human leukemic cell line that can be stimulated to synthesize hemoglobin by Epo.

scholarly journals Hck expression correlates with granulocyte-macrophage colony- stimulating factor-induced proliferation in HL-60 cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 94-103
Author(s):  
D Linnekin ◽  
OM Howard ◽  
L Park ◽  
W Farrar ◽  
D Ferris ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The human myeloid cell line HL-60 expresses approximately 300 high- affinity granulocyte-macrophage colony-stimulating factor receptors (GM- CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL- 60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.

scholarly journals Hck expression correlates with granulocyte-macrophage colony- stimulating factor-induced proliferation in HL-60 cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 94-103 ◽  
Author(s):  
D Linnekin ◽  
OM Howard ◽  
L Park ◽  
W Farrar ◽  
D Ferris ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The human myeloid cell line HL-60 expresses approximately 300 high- affinity granulocyte-macrophage colony-stimulating factor receptors (GM- CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL- 60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Y Kanakura ◽  
B Druker ◽  
KW Wood ◽  
HJ Mamon ◽  
K Okuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Human Factor ◽  
Myeloid Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

scholarly journals The role of interleukin-1 and granulocyte-macrophage colony-stimulating factor in the paracrine stimulation of an in vivo-derived murine myeloid leukemia

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1301-1310
Author(s):  
KB Leslie ◽  
HJ Ziltener ◽  
JW Schrader
Keyword(s):  
Cell Line ◽  
Host Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulatory Activity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

WEHI-274.3 is a cell line isolated from an in vivo-derived, murine myelomonocytic leukemia. Although the survival and growth of WEHI-274.3 cells in vitro is absolutely dependent on the addition of exogenous growth factors such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or colony-stimulating factor-1, when injected into syngeneic mice the cell line is tumorigenic. Sera from normal mice contain low levels of an activity that sustains survival of WEHI-274.3 but does not stimulate growth. In contrast, sera from mice bearing the WEHI-274.3 leukemia contained levels of CSF-1 and GM-CSF that stimulated the growth of WEHI-274.3 cells. Supernatants of cultures of WEHI-274.3 cells contained an activity that stimulated 3T3 fibroblasts to release an activity that stimulated the growth of the WEHI-274.3 cells. The 3T3-stimulatory activity released by the WEHI- 274.3 cells was neutralized completely with an antiserum specific for murine IL-1 alpha, but not with antiserum specific for IL-1 beta. Moreover, WEHI-274.3 cells both in vitro and in vivo contained high levels of IL-1 alpha and IL-1 beta mRNAs. The leukemia-stimulatory activity released by the 3T3 cells was neutralized by an antiserum specific for GM-CSF. We postulate that the IL-1 alpha constitutively released by the WEHI-274.3 cells stimulates the production of GM-CSF from host cells such as fibroblasts or endothelial cells. A similar paracrine mechanism of growth stimulation may occur in acute myeloid leukemias in humans.

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