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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers. Unexpectedly, the inducibility of the CD11c gene promoter showed distinct kinetics and magnitude on the PMA-, SB-, GM-CSF-triggered differentiation. Moreover, SB synergized with either PMA or GM-CSF in enhancing both the CD11c promoter activity and the cell surface expression of p150,95 on differentiating U937 cells. Furthermore, we showed the existence of a c-Myb-binding site at -85, the importance of the -99/-61 region in the CD11c promoter inducibility during PMA- or SB-triggered differentiation, and the dependency of the GM-CSF and PMA responsiveness of the CD11c promoter on an intact AP-1-binding site located at -60. These results, together with the lack of functional effect of mutations disrupting the Sp1-and Myb-binding sites within the proximal region of the CD11c promoter, indicate that the myeloid differentiation pathways indicated by SB and phorbol esters (or GM-CSF) activate a distinct set of transcription factors and show that the myeloid differentiation-inducibility of the CD11c gene maps to the -99/-53 proximal region of the promoter.
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PMID:Granulocyte-macrophage colony-stimulating factor, phorbol ester, and sodium butyrate induce the CD11c integrin gene promoter activity during myeloid cell differentiation. 757 38

Adenosine triphosphate (ATP) bioluminescence was used to determine whether there was a linear relationship between cultured cell number and measured luminescence using the luciferin-luciferase reaction. In all the cells tested including peripheral blood mononuclear cells (MNC), MOLT-4, HL-60, TF-1, NFS-60 and L-929 cell lines there was a significant correlation as determined by Spearman's rank correlation coefficient (p > 0.00001). These observations were then used to determine whether ATP bioluminescence could be used as a suitable substitute for tritiated thymidine uptake as a measure of cell proliferation. The cell lines MOLT-4, HL-60, TF-1 and NFS-60 showed a strong correlation between thymidine uptake and ATP bioluminescence (p > 0.00001 for all cell types). Additionally the ATP method could detect the cytokine dependent proliferation on TF-1 and NFS-60 cells by granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) respectively. The tumour necrosis factor alpha (TNF)-induced cytotoxic effect on L-929 cells could also be accurately detected using this method. It would therefore appear to be possible to use ATP bioluminescence in the detection of cytokine activity in a number of different bioassays.
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PMID:The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. 768 Jun 99

Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 10(11) dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 +/- 8 days, with a mean population doubling time of 3.87 +/- 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9-24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/10(6) cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-alpha (TNF-alpha) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Primary fibroblasts from human adults as target cells for ex vivo transfection and gene therapy. 784 93

We have reported modulation, by cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) and by hormonal cyclic-adenosine-monophosphate (cAMP) agonists, of hematopoietic growth factor production in the murine marrow adherent cell line +/+(-)1.LDA11. Previously, we reported that increased intracellular cAMP levels inhibited bioactive granulocyte-macrophage colony-stimulatory factor (GM-CSF) production stimulated by IL-1 or by the synergistic stimulus of IL-1 plus TNF-alpha. On the other hand, increased intracellular cAMP stimulated IL-6 synthesis in +/+(-)1.LDA11 cells. In addition, cAMP was additive with either IL-1 or IL-1 plus TNF-alpha in inducing production of soluble IL-6. In the present study, these observations were pursued mechanistically at the level of messenger RNA (mRNA) production. Northern blot analysis of steady-state mRNA for GM-CSF revealed induction by treatment of +/+(-)1.LDA11 cells with IL-1 or with TNF-alpha. The combined stimulation by IL-1 plus TNF-alpha resulted in supra-additive increases in GM-CSF expression by +/+(-)1.LDA11. Addition to stromal cells of the soluble cAMP agonist 8-bromo-cAMP (8BrcAMP) at 0.5 to 1 mM stimulated IL-6 mRNA expression acting alone, and it was additive with IL-1 or IL-1 plus TNF-alpha in stimulating IL-6 expression. On the other hand, 8BrcAMP inhibited GM-CSF mRNA expression stimulated by IL-1 or IL-1 plus TNF-alpha. Inhibition of GM-CSF mRNA by 8BrcAMP was time-dependent, starting 120 to 180 minutes posttreatment. In addition, inhibition of GM-CSF transcript expression in +/+(-)1.LDA11 by 8BrcAMP required the expression of a labile protein. Nuclear run-on assays revealed that GM-CSF and IL-6 genes were transcriptionally induced in +/+(-)1.LDA11 by incubation with IL-1 plus TNF-alpha. IL-6 transcription was further enhanced by 8BrcAMP co-incubation. More sensitive experiments using a luciferase reporter vector containing the GM-CSF promoter region were necessary to convincingly establish the role of TNF-alpha and 8BrcAMP on transcriptional induction of the GM-CSF gene in +/+(-)1.LDA11 stromal cells. Considering these results and an effect of 8BrcAMP on decreasing GM-CSF transcript stability in actinomycin-D (act-D) decay experiments, we conclude that the inhibitory effect of 8BrcAMP on GM-CSF expression is exerted at the posttranscriptional level. These data demonstrate that the intracellular level of cAMP has an important discriminatory role on expression of the cytokines GM-CSF and IL-6 in a model stromal cell line.
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PMID:Granulocyte-macrophage colony-stimulating factor expression is regulated at transcriptional and posttranscriptional levels in a murine bone marrow stromal cell line. 806 90

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.
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PMID:Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. 813 80

Introduction of v-src or c-src527F, a transforming mutant of the c-src proto-oncogene, into the growth factor-dependent cell line FDCP-1 resulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth factor independence was oncogene-dependent; that is, the cells were growth factor-independent at the permissive temperature but became growth factor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The c-src2A,527F mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane localization sequence, induced growth factor independence. This suggests that the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated that conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF was confirmed by a quantitative enzyme-linked immunosorbent assay (ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infected FDCP-1 cells was demonstrated by PCR amplification of cDNAs with primers specific for GM-CSF. While GM-CSF mRNA was detected in FDCP/ts29 cells grown at 34 degrees C, it was not observed in cells infected with the tsLA29 mutant grown at the nonpermissive temperature of 39 degrees C. Transfection of v-src-infected FDCP-1 cells with a GM-CSF promoter reporter plasmid revealed src-dependent expression of luciferase; that is, while expression was observed at the permissive temperature, no expression was detected in FDCP/ts29 clone 6 cells grown at the nonpermissive temperature. No expression of the GM-CSF promoter reporter plasmid was observed in uninfected FDCP-1 cells.
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PMID:Induction of growth factor-independence and GM-CSF secretion by the v-src oncogene in murine myeloid cells does not require membrane association of pp60v-src. 864 39

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hemopoietic growth factor that is expressed in activated T cells, fibroblasts, macrophages, and endothelial cells. Although GM-CSF does not appear to be essential for normal hemopoiesis, overexpression of GM-CSF has been implicated in the pathogenesis of some diseases such as myeloid leukemia and chronic inflammation. An NF-kappaB/Rel binding site within the GM-CSF promoter, termed the kappaB element appears to be important for controlling expression in reporter gene assays in response to a number of stimuli in T cells. We investigated oligonucleotide-directed triple helix formation across this regulatory sequence as a potential tool to inhibit GM-CSF gene transcription. A 15-base oligonucleotide, GM3, was targeted to a purine-rich region in the GM-CSF proximal promoter, which overlaps the kappaB element. Gel mobility shift assays and DNase I footprinting demonstrated that GM3 formed a sequence-specific collinear triplex with its double-stranded DNA target. Triplex formation by GM3 blocked recombinant and nuclear NF-kappaB proteins binding to the GM-CSF element. GM3 also caused selective inhibition of the human T-cell lymphotrophic virus-1 Tax transactivator-induced luciferase activity from a reporter construct driven by the GM-CSF promoter in Jurkat T cells. Finally, GM3 greatly reduced the concentration of endogenous GM-CSF mRNA induced by different stimuli in Jurkat T cells but did not affect interleukin 3 mRNA levels in the same cells. We conclude that the kappaB element in the GM-CSF promoter plays a central role in the transcriptional activation of the endogenous GM-CSF gene. Colinear triplex formation acts as a selective transcriptional repressor of the GM-CSF gene and may have potential therapeutic application in cases of undesirable overexpression of this protein.
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PMID:DNA triplex formation selectively inhibits granulocyte-macrophage colony-stimulating factor gene expression in human T cells. 866 66

Platelets and megakaryocytes express Fc receptors for IgG which are encoded by the Fc gamma RIIA gene. In an effort to establish a cellular model for induction of Fc gamma RIIA expression during megakaryocyte development by hematopoietic growth factors, steady-state Fc gamma RIIA mRNA levels were monitored in c-kit receptor-positive megakaryocytic cells (M07e, HEL, and Dami) in response to c-kit ligand (KL; also known as stem cell factor, mast cell growth factor, or Steel factor). Northern blot analysis showed that exposure of cells to KL led to significant increases in Fc gamma RIIA levels in M07e (15 x at 24 hours), with smaller increases in HEL (1.9 x at 2 hours) and Dami (1.6 x at 24 hours) cells. K562 cells, which lack c-kit receptor, showed no effect of KL on modulating Fc gamma RIIA mRNA levels. The effects of KL were specific for Fc gamma RIIA, as there were no effects on platelet factor 4 (PF4), gamma-globin, or GATA-1 mRNA levels. Effects of KL, alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) and gamma-interferon (IFN-gamma), on surface Fc gamma RIIA expression were assessed by flow cytometry using anti-Fc gamma RII monoclonal antibody IV.3. In M07e cells, KL alone and in combination led to significant increases in the percentage of cells positive for surface Fc gamma RIIA and the mean cell fluorescence intensity. Transient transfection studies of an Fc gamma RIIA promoter-luciferase reporter gene in the presence or absence of KL showed increased reporter gene expression in KL-treated cells, with the largest increase (3.7-fold) in the M07e cells. In HEL and Dami cells, other cytokines active in megakaryocytopoiesis when used alone (interleukin-3 [IL-3], IL-6, IL-11, GM-CSF) had negligible activity in increasing reporter gene activity. These results suggest that increased levels of Fc gamma RIIA mRNA after KL treatment of M07e cells are a result, in part, of increased Fc gamma RIIA gene transcription. Our results indicate that M07e cells represent a cellular model for KL-induced Fc gamma RIIA expression in early megakaryocyte development.
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PMID:Human c-kit ligand (stem cell factor) induces platelet Fc receptor expression in megakaryoblastic cells. 876 99

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.
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PMID:Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine. 886 54

Macrophages are putative target cells for expressing an exogenous gene with therapeutical effects. Knowing that macrophages express membrane lectins mediating endocytosis of their ligands, DNA/glycosylated polylysine complexes were used to transfect human blood monocyte-derived macrophages. Monocytes from human peripheral blood were matured in culture for 7 days to differentiate into macrophage-like cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adherent cells, which displayed characteristic macrophage markers, CD 14, CD 11b, HLA-DR, and HLA-ABC antigens and mannose receptor, were transfected by DNA/glycosylated polylysine complexes in the presence of chloroquine. The luciferase reporter gene expression was maximal 24 hr after transfection with a DNA/mannosylated polylysine complex and by using plasmids in which the promoters (either the long terminal repeat of the human immunodeficiency virus or the human cytomegalovirus) drove the luciferase gene expression. Luciferase gene expression was lower when the promoter was the early region of the large T antigen of SV40 virus. Transfection mediated by DNA/mannosylated polylysine complexes was much more efficient than with DEAE-dextran or lipofectin. The possibility of transferring and expressing an exogenous gene into macrophage-like cells by using a nonimmunogenic synthetic vector as a DNA carrier opens new ways to develop nonviral gene therapy strategies.
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PMID:Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages. 891 94


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