UNIPROT:P04141 - FACTA Search

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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)

The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus-mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune-deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte-macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418-resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.
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PMID:Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays. 185 80

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.
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PMID:Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells. 208 Nov 93

We compared the efficiency of retroviral vector (N2)-mediated transfer of the bacterial neomycin resistance gene (NeoR) into adult and fetal hematopoietic progenitors of sheep and humans by assessing their ability to form colonies in the presence of lethal doses of the neomycin analogue G418 in vitro. Fetal cells from both sheep and humans exhibited a higher degree of NeoR transfer than adult cells. The overall level of NeoR expression was significantly higher for sheep than human cells. The transfer/expression of NeoR into adult human bone marrow hematopoietic progenitors was not affected by the presence or absence of T cells and monocyte/macrophages. The efficiency of NeoR transfer into both adult and fetal human cells, however, was improved when transduction was carried out in the presence of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor. These results demonstrate the greater efficiency of NeoR gene transfer into fetal hematopoietic progenitors, which may provide a basis for the relatively higher efficiency of the in utero approach to gene therapy.
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PMID:Retroviral vector-mediated transfer of the bacterial neomycin resistance gene into fetal and adult sheep and human hematopoietic progenitors in vitro. 240 26

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.
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PMID:Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src. 311 87

A full length clone of murine fms-like tyrosine kinase 3 [flt3, also known as fetal liver kinase 2 (flk2)] was constructed from sequences obtained from a brain complementary DNA (cDNA) library and from cDNA prepared from the cell line Tikaut. In the absence of a ligand to study the function of Flt3, a chimeric molecule was constructed comprising the extracellular domain of murine c-Fms and the transmembrane and cytoplasmic domains of Flt3. A plasmid encoding the chimeric receptor was cotransfected along with a plasmid conferring neomycin resistance into FDC-P1 cells that do not normally express c-fms or flt3 and require granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 for growth. Two types of clones were obtained following selection in GM-CSF and G418. Two of seven clones had the capacity for M-CSF-dependent colony formation in semisolid medium, indicating that the cytoplasmic domain of Flt3 can transduce a proliferative signal. From the remaining clones, M-CSF-dependent clonogenic cells could be selected by prior bulk liquid culture in M-CSF. It has been shown previously that the GM-CSF-dependent proliferative capacity is strongly inhibited by M-CSF in FDC-P1 cells engineered to express full length c-fms. This phenomenon was also observed with FD/fms-flt3 cells that were clonogenic in M-CSF. Stimulation of FD/fms or FD/fms-flt3 cells in liquid culture by M-CSF caused differentiation of a small proportion of cells along the myelomonocytic pathway which was enhanced by the combination of M-CSF and GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fms-like tyrosine kinase 3 catalytic domain can transduce a proliferative signal in FDC-P1 cells that is qualitatively similar to the signal delivered by c-Fms. 804 61

Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Number and location of AUUUA motifs: role in regulating transiently expressed RNAs. 819 53

The human pluripotent UT-7 cell line is growth factor-dependent for proliferation and differentiation. We have previously shown that (1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) induce a myeloid and erythroid pattern of differentiation, respectively; (2) GM-CSF acts predominantly over Epo for cell differentiation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo receptors (Epo-R) at the mRNA and binding site levels; and (4) in contrast, Epo has no effect on GM-CSF receptor (GM-CSF-R) expression. These results suggested that UT-7 cell commitment or differentiation may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors. To test this hypothesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constructed: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3' sequence placed under the transcriptional control of the viral long terminal repeat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R infected clones (12, 6, 10) and one UT-7/neomycin clone (Neo) were selected in medium containing G418. After growth factor deprivation (18 hours), Epo-Rs were expressed at the same level (approximately 6,000 receptors per cell) in all four clones 12, 6, 10, Neo, and in parental UT-7 cells, and exhibited similar affinity (0.1 to 0.2 nmol/L). Cross-linking experiments showed that Epo is associated with three proteins of about 66, 85, and 100 kD in cells of parental UT-7, as well as in cells of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the cross-linking on parental UT-7 cells, but not on cells of clone 12, demonstrating that more than 90% cell surface Epo-Rs were of murine origin. The presence of GM-CSF significantly reduced the number of Epo-Rs expressed on parental UT-7 cells, but not on cells of clones 12, 10, and 6. HuEpo-R Ab inhibited Epo-induced parental UT-7 cell growth, but not that of cells of clone 12, suggesting that the muEpo-R is able to induce human UT-7 cell proliferation. When cells of clone 12 were switched from a medium containing GM-CSF to one with Epo, cell surface glycophorin A (GPA) was induced, as in parental UT-7 cells without inhibition by the huEpo-R Ab, demonstrating that the muEpo-R is also able to transduce a differentiation signal in human cells. However, in cells of clones 12, 6, 10 and Neo, as well as in parental UT-7 cells, the induction of GPA by Epo was inhibited by GM-CSF. This finding demonstrates that, although GM-CSF does not downregulate muEpo-R binding sites on UT-7/muEpo-R infected clones, it still inhibits the effects of Epo on cell differentation. Therefore, hierarchical regulation induced by growth factors for cell commitment or differntiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels.
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PMID:Inhibition of the erythropoietin-induced erythroid differentiation by granulocyte-macrophage colony-stimulating factor in the human UT-7 cell line is not due to a negative regulation of the erythropoietin receptor. 863 20

The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.
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PMID:Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus. 1067 Jun 49

We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosabetageo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5' rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF-treated and -infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACA was recently defined as a coactivator of c-jun-mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34(+) cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis. (Blood. 2000;95:3750-3757)
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PMID:Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach. 1084 6