scholarly journals Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells.

1995 ◽  
Vol 181 (6) ◽  
pp. 2189-2199 ◽  
Author(s):  
K W Muszynski ◽  
F W Ruscetti ◽  
G Heidecker ◽  
U Rapp ◽  
J Troppmair ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Growth Factor ◽  
Cell Lines ◽  
Colony Formation ◽  
Antisense Oligonucleotides ◽  
Tyrosine Kinase Receptor ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.

scholarly journals Erythropoietin-specific cell cycle progression in erythroid subclones of the interleukin-3-dependent cell line 32D [published erratum appears in Blood 1993 Jun 1;81(11):3168]

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 935-941 ◽  
Author(s):  
Y Shimada ◽  
G Migliaccio ◽  
H Ralph ◽  
AR Migliaccio ◽  
H] Shaw H$[corrected to Ralph
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Cell Lines ◽  
Suicide Rate ◽  
S Phase ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Dependent Cell ◽  
Stimulating Factor

Recently, a variety of growth factor-dependent subclones of the murine interleukin-3 (IL-3)-dependent cell line 32D have been isolated. These subclones include those dependent for growth on erythropoietin (Epo) (32D Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) (32D GM), or granulocyte colony-stimulating factor (G-CSF) (32D G). 32D Epo1.1 is a revertant of 32D Epo and is capable of growing in IL-3. These cell lines express the differentiation program appropriate to the specific growth factor and depend on the growth factors not only for proliferation but also for survival. To determine how the signal for proliferation is triggered by various growth factors, we examined the DNA histograms and the expression of cell cycle-specific genes in the different cell lines. The cell cycle-specific genes analyzed were myc (early G1), myb (late G1), and the structural genes for the calcium- binding protein 2A9 (middle G1) and histone H3 (G1-S boundary). The DNA histogram analysis of cells in the logarithmic phase of growth showed that approximately 40% of 32D, 32D GM, 32D G, and 32D Epo1.1 (growing in IL-3) were cells with a 2N DNA content (and therefore in G0/G1), and another 40% have a DNA content intermediate between 2N and 4N (in S phase). In contrast, 32D Epo and 32D Epo1.1 (growing in Epo) had fewer cells in the G0/G1 phase of the cell cycle compared with the number of cells that were in the S phase (19% to 31% v 69% to 78%, respectively). Because all the cell lines have comparable doubling times (15 to 18 hours), the cell distribution among the phases of the cell cycle is proportional to the length of the phase. Therefore, cells growing in IL- 3 (32D and 32D Epo1.1), GM-CSF (32D GM), or G-CSF (32D G) progress along the cycle in a manner typical of previously reported nontransformed cell lines. In contrast, cells growing in Epo (32D Epo or 32D Epo1.1) spend relatively less time in G0/G1 and correspondingly more time in S. These data were confirmed by the analysis of the tritiated thymidine (3H-TdR) suicide rate and of the expression of cell cycle-specific genes. The 32D and 32D Epo1.1 cells growing in IL-3 had a suicide rate of congruent to 50%, whereas the suicide rate of 32D Epo and 32D Epo1.1 growing in Epo was higher than 75%.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Erythropoietin-specific cell cycle progression in erythroid subclones of the interleukin-3-dependent cell line 32D [published erratum appears in Blood 1993 Jun 1;81(11):3168]

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 935-941 ◽  
Author(s):  
Y Shimada ◽  
G Migliaccio ◽  
H Ralph ◽  
AR Migliaccio ◽  
H] Shaw H$[corrected to Ralph
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Cell Lines ◽  
Suicide Rate ◽  
S Phase ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Dependent Cell ◽  
Stimulating Factor

Abstract Recently, a variety of growth factor-dependent subclones of the murine interleukin-3 (IL-3)-dependent cell line 32D have been isolated. These subclones include those dependent for growth on erythropoietin (Epo) (32D Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) (32D GM), or granulocyte colony-stimulating factor (G-CSF) (32D G). 32D Epo1.1 is a revertant of 32D Epo and is capable of growing in IL-3. These cell lines express the differentiation program appropriate to the specific growth factor and depend on the growth factors not only for proliferation but also for survival. To determine how the signal for proliferation is triggered by various growth factors, we examined the DNA histograms and the expression of cell cycle-specific genes in the different cell lines. The cell cycle-specific genes analyzed were myc (early G1), myb (late G1), and the structural genes for the calcium- binding protein 2A9 (middle G1) and histone H3 (G1-S boundary). The DNA histogram analysis of cells in the logarithmic phase of growth showed that approximately 40% of 32D, 32D GM, 32D G, and 32D Epo1.1 (growing in IL-3) were cells with a 2N DNA content (and therefore in G0/G1), and another 40% have a DNA content intermediate between 2N and 4N (in S phase). In contrast, 32D Epo and 32D Epo1.1 (growing in Epo) had fewer cells in the G0/G1 phase of the cell cycle compared with the number of cells that were in the S phase (19% to 31% v 69% to 78%, respectively). Because all the cell lines have comparable doubling times (15 to 18 hours), the cell distribution among the phases of the cell cycle is proportional to the length of the phase. Therefore, cells growing in IL- 3 (32D and 32D Epo1.1), GM-CSF (32D GM), or G-CSF (32D G) progress along the cycle in a manner typical of previously reported nontransformed cell lines. In contrast, cells growing in Epo (32D Epo or 32D Epo1.1) spend relatively less time in G0/G1 and correspondingly more time in S. These data were confirmed by the analysis of the tritiated thymidine (3H-TdR) suicide rate and of the expression of cell cycle-specific genes. The 32D and 32D Epo1.1 cells growing in IL-3 had a suicide rate of congruent to 50%, whereas the suicide rate of 32D Epo and 32D Epo1.1 growing in Epo was higher than 75%.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 1870-1874 ◽  
Author(s):  
YQ Zhou ◽  
ER Stanley ◽  
SC Clark ◽  
JA Hatzfeld ◽  
JP Levesque ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Colony Stimulating Factor ◽  
Cooperative Effects ◽  
Gm Csf ◽  
Low Concentrations ◽  
Low Cell Density ◽  
Stimulating Factor

Abstract By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

scholarly journals Recombinant human macrophage colony-stimulating factor (M-CSF) requires subliminal concentrations of granulocyte/macrophage (GM)-CSF for optimal stimulation of human macrophage colony formation in vitro.

1987 ◽  
Vol 166 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
D Caracciolo ◽  
N Shirsat ◽  
G G Wong ◽  
B Lange ◽  
S Clark ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Human Macrophage ◽  
Colony Stimulating Factor ◽  
Colony Assay ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human macrophage colony-stimulating factor (M-CSF or CSF-1), either in purified or in recombinant form, is able to generate macrophagic colonies in a murine bone marrow colony assay, but only stimulates small macrophagic colonies of 40-50 cells in a human bone marrow colony assay. We report here that recombinant human granulocytic/macrophage colony stimulating factor (rhGM-CSF) at concentrations in the range of picograms enhances the responsiveness of bone marrow progenitors to M-CSF activity, resulting in an increased number of macrophagic colonies of up to 300 cells. Polyclonal antiserum against M-CSF did not alter colony formation of bone marrow progenitors incubated with GM-CSF at optimal concentration (1-10 ng/ml) for these in vitro assays. Thus, GM-CSF at higher concentrations (nanogram range) can by itself, elicit macrophagic colonies, and at lower concentrations (picogram range) acts to enhance the responsiveness of these progenitors to M-CSF.

scholarly journals Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture

Blood ◽  
1990 ◽  
Vol 75 (11) ◽  
pp. 2118-2129 ◽  
Author(s):  
LH Coutinho ◽  
A Will ◽  
J Radford ◽  
R Schiro ◽  
NG Testa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Bone Marrow Culture ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Nonadherent Cells ◽  
Stimulating Factor

We have studied the effects of recombinant human granulocyte colony- stimulating factor (rhG-CSF), hG macrophage-CSF (hGM-CSF), and gibbon interleukin-3 (gIL-3) on cell proliferation and differentiation in human long-term bone marrow culture (LTBMC). hG-CSF induced a maximal increase of 2.3-fold in both total nonadherent cells and GM cluster- forming cells, but only an increase of 1.7-fold in GM-colony-forming cell (GM-CFC) numbers, influencing mainly neutrophil differentiation. Cultures treated with hGM-CSF demonstrated a peak of 12.8-, 21- and 3.2- fold elevations in total nonadherent cells, cluster, and GM-CFC, respectively, and influenced differentiation of neutrophils, monocytes, eosinophils, and lymphocytes. Cultures treated with gIL-3 demonstrated the largest expansion in the GM-CFC population, reaching a maximum of 5.3-fold in relation to that of unstimulated controls. IL-3 treatment also increased the numbers of GM clusters and mature cells (including all myeloid cells and lymphocytes) 7.8- and 4.8-fold, respectively. Similar quantitative and qualitative changes were induced by G-CSF, GM- CSF, and IL-3 in LTBMCs of patients in remission after treatment for acute lymphoblastic leukemia or Hodgkin's lymphoma. Overall, the expansion of GM progenitor cells in cultures treated with growth factors was larger in the adherent cell layer than in the nonadherent cell fraction. In addition, hGM-CSF, gIL-3, and hG-CSF to a less extent, increased the cycling rates of GM-CFC progenitors located in the adherent layer. These results indicate that hG-CSF is a much less potent stimulus of hematopoiesis in LTBMC than the other CSFs assayed, and that the increases in cell production after treatment with G-CSF, GM-CSF, or IL-3 may be achieved by primary expansion of different cell populations within the hierarchy of the hematopoietic system. The effects of the growth factors were transient and the longevity of hematopoiesis in the cultures was not altered, suggesting that treatment with IL-3, GM-CSF, or G-CSF had not compromised the ability of primitive cells to give rise to mature cells. This indicates that the stromal microenvironment in LTBMC can override potential differentiation-inducing activities of the CSFs.

scholarly journals Production of granulocyte-macrophage colony-stimulating factor by Abelson virus-induced tumorigenic mast cell lines

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1074-1081
Author(s):  
SW Chung ◽  
PM Wong ◽  
G Shen-Ong ◽  
S Ruscetti ◽  
T Ishizaka ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Northern Analysis ◽  
Pokeweed Mitogen ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We have recently described a system that supports the development of continuously growing and tumorigenic cell lines after infection of individual multilineage hematopoietic colonies with Abelson murine leukemia virus (A-MuLV). We now provide definitive evidence that these transformed lines express features characteristic of mast cells. Although these lines have been maintained in some cases for more than a year in the absence of exogenous growth factors other than those present in fetal calf serum, colony formation could consistently after 2 months, and variably after 5 months, be shown to be increased several fold when pokeweed mitogen-stimulated spleen cell conditioned medium (CM) was added to the cultures. CM from the A-MuLV-transformed lines was then tested for its ability to stimulate hematopoietic colony formation by cells from both fetal and adult tissues. Four of four randomly selected cell lines produced factors that were active on erythropoietic, granulopoietic, and in some cases pluripotent progenitors. Removal of viral particles from the CM from one of the lines (27d1) by either heat inactivation or high-speed centrifugation did not alter the colony-stimulating activity detected. When CM from 27d1 cells was tested for its ability to stimulate the proliferation of interleukin 3 (IL3) granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent FDC-P1 cells, a positive result was obtained. This stimulatory activity was not reduced in the presence of neutralizing anti-IL 3 immunoglobulin (Ig), suggesting that the activity detected was GM-CSF and not IL 3. This was confirmed by the lack of expression of the IL 3 gene in 27d1 cells as determined by Northern analysis of 27d1 cell RNA. Furthermore, S1 analysis of mRNA from 27d1 cells as well as two other lines indicated that the GM-CSF gene in all three was transcriptionally active. Taken together, these data suggest that A- MuLV transformation of normal mast cells or their precursors under certain conditions commonly activates the production of GM-CSF.

scholarly journals A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM- CSF), G-CSF interleukin-3, and canine G-CSF

Blood ◽  
1990 ◽  
Vol 76 (3) ◽  
pp. 523-532 ◽  
Author(s):  
WP Hammond ◽  
TC Boone ◽  
RE Donahue ◽  
LM Souza ◽  
DC Dale
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Cell Counts ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Cyclic Hematopoiesis ◽  
Stimulating Factor

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG- CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.

scholarly journals Chemokines and Growth Factors Produced by Lymphocytes in the Incompetent Great Saphenous Vein

2019 ◽  
Vol 2019 ◽  
pp. 1-10
Author(s):  
Ewa Grudzińska ◽  
Sławomir Grzegorczyn ◽  
Zenon P. Czuba
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Saphenous Vein ◽  
Oscillatory Flow ◽  
Great Saphenous Vein ◽  
Upper Limbs ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The role of cytokines in the pathogenesis of chronic venous disease (CVD) remains obscure. It has been postulated that oscillatory flow present in incompetent veins causes proinflammatory changes. Our earlier study confirmed this hypothesis. This study is aimed at assessing chemokines and growth factors (GFs) released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting reflux in GSV, blood was derived from the cubital vein and from the incompetent saphenofemoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Eotaxin, interleukin 8 (IL-8), macrophage inflammatory protein 1 A and 1B (MIP-1A and MIP-1B), interferon gamma-induced protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 5 (IL-5), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF) were assessed in culture supernatants by a Bio-Plex assay. Higher concentrations of eotaxin and G-CSF were revealed in the incompetent GSV, compared with the concentrations in the patients’ upper limbs. The concentrations of MIP-1A and MIP-1B were higher in the CVD group while the concentration of VEGF was lower. In the stimulated cultures, the concentration of G-CSF proved higher in the incompetent GSV, as compared with the patients’ upper limbs. Between the groups, the concentration of eotaxin was higher in the CVD group, while the IL-5 and MCP-1 concentrations were lower. IL-8, IP-10, FGF, GM-CSF, and PDGF-BB did not reveal any significant differences in concentrations between the samples. These observations suggest that the concentrations of chemokines and GFs are different in the blood of CVD patients. The oscillatory flow present in incompetent veins may play a role in these changes. However, the role of cytokines in CVD requires further study.

scholarly journals Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture

Blood ◽  
1990 ◽  
Vol 75 (11) ◽  
pp. 2118-2129 ◽  
Author(s):  
LH Coutinho ◽  
A Will ◽  
J Radford ◽  
R Schiro ◽  
NG Testa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Bone Marrow Culture ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Nonadherent Cells ◽  
Stimulating Factor

Abstract We have studied the effects of recombinant human granulocyte colony- stimulating factor (rhG-CSF), hG macrophage-CSF (hGM-CSF), and gibbon interleukin-3 (gIL-3) on cell proliferation and differentiation in human long-term bone marrow culture (LTBMC). hG-CSF induced a maximal increase of 2.3-fold in both total nonadherent cells and GM cluster- forming cells, but only an increase of 1.7-fold in GM-colony-forming cell (GM-CFC) numbers, influencing mainly neutrophil differentiation. Cultures treated with hGM-CSF demonstrated a peak of 12.8-, 21- and 3.2- fold elevations in total nonadherent cells, cluster, and GM-CFC, respectively, and influenced differentiation of neutrophils, monocytes, eosinophils, and lymphocytes. Cultures treated with gIL-3 demonstrated the largest expansion in the GM-CFC population, reaching a maximum of 5.3-fold in relation to that of unstimulated controls. IL-3 treatment also increased the numbers of GM clusters and mature cells (including all myeloid cells and lymphocytes) 7.8- and 4.8-fold, respectively. Similar quantitative and qualitative changes were induced by G-CSF, GM- CSF, and IL-3 in LTBMCs of patients in remission after treatment for acute lymphoblastic leukemia or Hodgkin's lymphoma. Overall, the expansion of GM progenitor cells in cultures treated with growth factors was larger in the adherent cell layer than in the nonadherent cell fraction. In addition, hGM-CSF, gIL-3, and hG-CSF to a less extent, increased the cycling rates of GM-CFC progenitors located in the adherent layer. These results indicate that hG-CSF is a much less potent stimulus of hematopoiesis in LTBMC than the other CSFs assayed, and that the increases in cell production after treatment with G-CSF, GM-CSF, or IL-3 may be achieved by primary expansion of different cell populations within the hierarchy of the hematopoietic system. The effects of the growth factors were transient and the longevity of hematopoiesis in the cultures was not altered, suggesting that treatment with IL-3, GM-CSF, or G-CSF had not compromised the ability of primitive cells to give rise to mature cells. This indicates that the stromal microenvironment in LTBMC can override potential differentiation-inducing activities of the CSFs.

scholarly journals Production of granulocyte-macrophage colony-stimulating factor by Abelson virus-induced tumorigenic mast cell lines

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1074-1081 ◽  
Author(s):  
SW Chung ◽  
PM Wong ◽  
G Shen-Ong ◽  
S Ruscetti ◽  
T Ishizaka ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Northern Analysis ◽  
Pokeweed Mitogen ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract We have recently described a system that supports the development of continuously growing and tumorigenic cell lines after infection of individual multilineage hematopoietic colonies with Abelson murine leukemia virus (A-MuLV). We now provide definitive evidence that these transformed lines express features characteristic of mast cells. Although these lines have been maintained in some cases for more than a year in the absence of exogenous growth factors other than those present in fetal calf serum, colony formation could consistently after 2 months, and variably after 5 months, be shown to be increased several fold when pokeweed mitogen-stimulated spleen cell conditioned medium (CM) was added to the cultures. CM from the A-MuLV-transformed lines was then tested for its ability to stimulate hematopoietic colony formation by cells from both fetal and adult tissues. Four of four randomly selected cell lines produced factors that were active on erythropoietic, granulopoietic, and in some cases pluripotent progenitors. Removal of viral particles from the CM from one of the lines (27d1) by either heat inactivation or high-speed centrifugation did not alter the colony-stimulating activity detected. When CM from 27d1 cells was tested for its ability to stimulate the proliferation of interleukin 3 (IL3) granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent FDC-P1 cells, a positive result was obtained. This stimulatory activity was not reduced in the presence of neutralizing anti-IL 3 immunoglobulin (Ig), suggesting that the activity detected was GM-CSF and not IL 3. This was confirmed by the lack of expression of the IL 3 gene in 27d1 cells as determined by Northern analysis of 27d1 cell RNA. Furthermore, S1 analysis of mRNA from 27d1 cells as well as two other lines indicated that the GM-CSF gene in all three was transcriptionally active. Taken together, these data suggest that A- MuLV transformation of normal mast cells or their precursors under certain conditions commonly activates the production of GM-CSF.

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