Gene/Protein Disease Symptom Drug Enzyme Compound
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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 aim of this study was to develop non-radioactive cell line proliferation assays. The human leukemic cell line TF1 (Kitamura et al., 1989) was used for the determination of the specific biological activity of recombinant human (rhu) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhu Interleukin 3 (IL-3) by a simple and economical fluorometric assay with a sensitivity similar to the measurement of 3H-thymidine uptake. The TF1 cell line responds to rhu IL-3, rhu GM-CSF and to a lesser extent to rhu Erythropoietin (EPO) and mast cell growth factor (MGF), but not to rhu G-CSF. It is dependent upon rhu GM-CSF for survival in culture. For the proliferation assay 1 x 10(4) TF1 cells were incubated with 20 ng - 0.256 pg rhu GM-CSF or rhu IL-3 at 37 degrees C and 5% CO2 in humidified atmosphere. After 48 h the cells were washed twice with PBS and were incubated with 4-Methylumbelliferyl-heptanoate for 60 min. Fluorescence was determined on a Titertek Fluoroskan II (Flow Lab.), and results were given as fluorescence units using a 355 nm excitation filter and a 480 nm emission filter. The developed assay showed an interassay variability lower than 15%. The sensitivity of the proliferation assays in the same range as the thymidine incorporation assays.
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PMID:Development of a rapid, highly sensitive, non-radioactive assay system for hematopoietic growth factors. 180 97

Apoptosis (programmed cell death) regulates cell population size. To determine the mechanisms whereby hematopoietic growth factors (HGFs) modulate apoptosis in human myeloid leukemic cells, we evaluated the roles of protein and mRNA synthesis for altering apoptosis in growth factor-stimulated vs. quiescent leukemic TF1 cells. Lysates of cells from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemic cell line TF1 were separated into high molecular weight (HMW) pellets of intact DNA and supernatants of fragmented low MW (LMW) DNA, and the DNA purified from these fractions was quantified. In the absence of both GM-CSF and fetal bovine serum (FBS), 70% of the DNA was fragmented after 3 days in culture, with a characteristic apoptotic ladder-like pattern on agarose gel electrophoresis, whereas this proportion had initially been < 5%. In contrast, less than 5% of the DNA was fragmented in cells incubated with GM-CSF plus FBS or GM-CSF alone. Delayed addition of GM-CSF, but not FBS, permitted partial rescue of the cells, inhibiting increasing rates of accumulation of fragmented DNA. When the macro-molecular synthesis inhibitor cycloheximide (CHX) or actinomycin D (Act D) was present for 26 hours in the absence of GM-CSF and FBS, apoptosis was inhibited. In contrast, in the presence of GM-CSF or FBS, apoptosis was enhanced upon addition of CHX or Act D. The latter effect persisted even with the late addition of CHX. These findings indicate that disparate mechanisms of enhancing or inhibiting apoptosis exist in myeloid leukemic cells related to environmental conditions, including HGF-regulated cellular synthesis of distinct proteins and mRNA.
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PMID:Modulation of apoptosis in human myeloid leukemic cells by GM-CSF. 787 43

gP130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XGI hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells.
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PMID:gp130 transducing receptor cross-linking is sufficient to induce interleukin-6 type responses. 866 9

Two major isoforms of PML-RARalpha are associated with (15;17)-positive acute promyelocytic leukemia (APL); however, functional differences between these isoforms have been difficult to define, and the molecular mechanism by which each isoform contributes to the pathogenesis of APL is not fully understood. To address these issues, the 'short' (S) and 'long' (L) isoforms of PML-RARalpha were constitutively expressed in the factor-dependent human erythroleukemia cell line, TF1. Expression of the L, but not the S, isoform inhibited growth of these cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the absence of GM-CSF, the S isoform partially protected against apoptosis, while the L isoform accelerated cell death. Treatment with all-trans retinoic acid (ATRA) inhibited cell growth and caused apoptosis only in PML-RARalpha-expressing cells, and these effects of ATRA were more marked in cells expressing the L isoform. ATRA treatment also led to downregulation of bcl-2 and endogenous RARalpha in PML-RARalpha-expressing cells, but had little effect on the level of exogenously expressed PML-RARalpha. We conclude that (1) subtle differences exist in the biologic activities of the L and S isoforms of PML-RARalpha, and (2) both isoforms are capable of transducing an ATRA-mediated signal that leads to downregulation of bcl-2 and induction of programmed cell death.
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PMID:Constitutive expression of the promyelocytic leukemia-associated oncogene PML-RARalpha in TF1 cells: isoform-specific and retinoic acid-dependent effects on growth, bcl-2 expression, and apoptosis. 955 92

Sphingosine, a sphingolipid breakdown product, has been proposed as an apoptosis-inducing agent. In this study, we examined the effect of sphingosine in bcl-2-overexpressing cells compared with cells that do not express the bcl-2 gene. The human erythroleukemic cell line TF1, which lacks bcl-2 expression, was easily induced to undergo apoptotic cell death by a variety of stimuli, including depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) or exposure to methylmethane sulfonate (MMS) (100 microg/mL), ultraviolet light (15 J/m2), X-ray irradiation (20 Gy), or sphingosine, a sphingolipid breakdown product (5 microM). In contrast, bcl-2 transfectants of TF1 (TF1-bcl2), which we established, were resistant to most of these treatments but remained sensitive to sphingosine. Neither C2- nor C6-ceramide (short-chain ceramide) induced apoptosis in TF1-mock and TF1-bcl2 cells. Sphingosine-induced apoptosis could not be inhibited by fumonisin B1, which can prevent conversion of sphingosine to ceramide, suggesting that sphingosine itself, not ceramide, possesses apoptosis-inducing capability. Western blotting, which revealed a 21-kDa bax protein in untreated cells, revealed the presence of an additional 18-kDa protein in GM-CSF-depleted and MMS- or sphingosine-treated TF1-mock cells. In TF1-bcl2 cells, this protein was not detected after GM-CSF depletion or MMS treatment, but was observed after sphingosine treatment. Immunoprecipitation with anti-bcl2 antibody, followed by immunoblotting with anti-bax antibody, showed that both the 21-kDa bax protein and the 18-kDa protein heterodimerized with bcl-2 protein. These results suggest that sphingosine is a unique reagent for apoptosis and that it can overcome bcl-2 gene expression. Furthermore, induction of 18-kDa bax-related protein may play an important role in apoptosis. Sphingosine, but not ceramide, may prove applicable as a reagent for future cytotoxic drugs used to treat intractable tumors overexpressing bcl-2.
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PMID:Analysis of bax protein in sphingosine-induced apoptosis in the human leukemic cell line TF1 and its bcl-2 transfectants. 980 50

Survival and proliferation of cells of a human myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence of granulocyte-macrophage colony-stimulating factor or interleukin-3. Upon hormone withdrawal these cells stop proliferating and undergo apoptotic process. In this report we demonstrate that a controlled increase in [Ca2+]i induces hormone-independent survival and proliferation of TF-1 cells. We found that moderate elevation of [Ca2+]i by the addition of cyclopiasonic-acid protected TF1 cells from apoptosis. Furthermore, a higher, but transient elevation of [Ca2+]i by ionomycin treatment induced cell proliferation. In both cases caspase-3 activity was reduced, and Bcl-2 was up-regulated. Higher elevation of [Ca2+]i by ionomycin induced MEK-dependent biphasic ERK1/2 activation, sufficient to move the cells from G0/G1 to S/M phases. Meanwhile, activation of ERK1/2, phosphorylation of the Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate elevation of [Ca2+]i, on the other hand, caused a delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all the effects of increasing [Ca2+]i, indicating that the MAPK/ERK pathway activation is essential for TF-1 cell survival and proliferation. Based on these results we suggest that the elevation of the [Ca2+]i may influence the cytokine dependence of hemopoietic progenitors and may contribute to pathological hematopoiesis.
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PMID:Calcium induces cell survival and proliferation through the activation of the MAPK pathway in a human hormone-dependent leukemia cell line, TF-1. 1264 64

Novel agents to treat acute myeloid leukemia (AML) are needed with increased efficacy and specificity. We have synthesized a dual-specificity fusion toxin DTU2GMCSF composed of the catalytic and translocation domains of diphtheria toxin (DT) fused to the granulocyte-macrophage colony-stimulating factor (GM-CSF) in which the DT furin cleavage site 163RVRRSV170 is modified to a urokinase plasminogen activator (uPA) cleavage site 163GSGRSA170, termed U2. DTU2GMCSF was highly toxic to the TF1-vRaf AML cell line (proliferation inhibition assay; IC50 = 3.14 pM), and this toxicity was greatly inhibited following pretreatment with anti-uPA and anti-GM-CSF antibodies. The activity of this toxin was then tested on a larger group of 13 human AML cell lines; 5 of the 13 cell lines were sensitive to DTU2GMCSF. An additional 5 of the 13 cell lines became sensitive when exogenous pro-uPA was added. Sensitivity to DTU2GMCSF strongly correlated with the expression levels of uPA receptors (uPARs) and GM-CSF receptors (GM-CSFRs) as well as with total uPA levels. DTU2GMCSF was less toxic to normal cells expressing uPAR or GMCSFR alone, that is, human umbilical vein endothelial cells and peripheral macrophages, respectively. These results indicate that DTU2GMCSF may be a selective and potent agent for the treatment of patients with AML.
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PMID:A urokinase-activated recombinant diphtheria toxin targeting the granulocyte-macrophage colony-stimulating factor receptor is selectively cytotoxic to human acute myeloid leukemia blasts. 1516 68

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.
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PMID:Production and characterization of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica. 2262 58