Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The c-myb protooncogene plays a major role in regulating the process of in vitro and in vivo hematopoiesis via its activity as transcriptional regulator in hematopoietic progenitor cells. Since the bone marrow microenvironment appears to regulate in vivo hematopoiesis by maintaining the growth of multipotent progenitors via secretion of specific cytokines, we asked whether c-myb is also required for the proliferation of and/or cytokine production by stromal cells that generate fibroblast-like colonies (fibroblast colony-forming units [CFU-F]). Using the reverse transcriptase polymerase chain reaction technique, we detected low levels of c-myb mRNA transcripts in human normal bone marrow fibroblasts. Treatment of these cells with c-myb antisense oligodeoxynucleotides caused downregulation of c-myb expression, decreased in the number of marrow CFU-F colonies (approximately 54% inhibition) and in the cell number within residual colonies (approximately 80%), and downregulation of granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) mRNA expression. Transfection of T98G glioblastoma cells, in which expression of c-myb, GM-CSF, and SCF mRNAs is undetectable or barely detectable, with a plasmid containing a full-length c-myb cDNA under the control of the SV40 promoter induced the expression of biologically active SCF and GM-CSF in these cells. Regulation of GM-CSF expression by c-myb was due in part to transactivation of the GM-CSF promoter. These results indicate that, in addition to regulating hematopoietic cell proliferation, c-myb is also required for proliferation of and cytokines synthesis by bone marrow fibroblasts.
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PMID:Regulation of proliferation and cytokine expression of bone marrow fibroblasts: role of c-myb. 768 94

As an approach for characterizing the molecules involved in the proliferation and differentiation of hematopoietic stem cells we have compared the ability of four murine stromal cell lines, MS-5, MS-K, both derived from Dexter cultures, BMS1 and BMS2 both derived from Whitlock-Witte cultures, to sustain murine long term hematopoiesis and to express the major hematopoietic cytokine genes. As opposed to the three other cell lines, MS-5 supports the maintenance of stem cells for up to 4-5 weeks. However, reconstituting stem cell output was reduced while clonogenic cell (day 12 and day 8 spleen colony-forming units, granulo-macrophagic, and erythroid progenitor cells) output was markedly increased. This hematopoietic-promoting activity is at least in part mediated by soluble molecules since medium conditioned with MS-5 cells was able to partially complement the nonsupportive cell line BMS1. The comparative study of the cytokine gene expression in MS-5 and in the nonsupportive cell lines included Northern blot and reverse transcriptase-polymerase chain reaction analysis of messenger RNA for interleukin-1, -3, -6, granulo-macrophage-colony-stimulating factor (GM-CSF), granulocyte-CSF, macrophage-CSF, stem cell factor, transforming growth factor-beta, tumor necrosis factor-alpha, macrophage inflammatory protein-1 alpha, and leukemia inhibitory factor. None of these molecules or their association were found to clearly confer to the MS-5 cell line its hematopoietic-promoting activity raising the possibility that uncharacterized molecule(s) would be involved in the proliferation and differentiation of stem cells.
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PMID:Hematopoietic-promoting activity of the murine stromal cell line MS-5 is not related to the expression of the major hematopoietic cytokines. 770 74

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.
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PMID:Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes. 783 72

We noted previously that ischemic acute tubular necrosis (ATN) induces local expression of MHC products in renal epithelium. The present investigations were conducted to establish the role of IFN-gamma in the regulation of MHC antigen expression in ATN and to explore the changes in cytokine and growth factor expression induced by ischemic renal injury. We produced unilateral ischemic ATN in mice by clamping the left renal pedicle. MHC class I and II steady state mRNA induction was assessed by northern blot analysis, and MHC product was quantified by the extent of binding of radiolabeled monoclonals to tissue homogenates. The steady state mRNA levels for IFN-gamma, IL-2, IL-10, and granulocyte-macrophage CSF were assessed by reverse transcriptase polymerase chain reaction and the levels for transforming growth factor-beta 1 and prepro-epidermal growth factor (ppEGF) were assessed by Northern blot analysis. In the injured kidneys, steady state mRNA levels for IFN-gamma, IL-2, IL-10, granulocyte-macrophage CSF, and transforming growth factor beta-1 were increased, whereas ppEGF mRNA was markedly decreased. The MHC expression was inhibited by treatment of mice with an anti-IFN-gamma mAb (R4-6A2). Murine EGF, administered in an attempt to accelerate recovery, did not reduce the cytokine and MHC changes. These data indicate that ischemic injury, and possibly other forms of injury, triggers a complex circuit of proinflammatory cytokines. This "injury response" could be relevant to clinical renal transplants, where ATN is associated with poor graft outcome.
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PMID:Ischemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-gamma, transforming growth factor-beta 1, granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-10. 787 62

Although it is well established that homeobox (HOX) genes play a key role in normal human embryogenesis, the expression and function of HOX genes in normal hematopoiesis is largely unknown. We have investigated by reverse transcriptase-polymerase chain reaction the mRNA expression of HOXB cluster genes (3' to 5' position in the cluster: from HOXB2 through B9) in 72% to 88% purified hematopoietic progenitor cells (HPCs) from adult peripheral blood induced in liquid suspension culture to gradual erythroid or granulopoietic (largely eosinophilic) differentiation and maturation by differential growth factor (GF) stimulus (ie, low-dose interleukin-3 [IL-3] and granulocyte-macrophage colony-stimulating factor [GM-CSF] and high-dose erythropoietin, or saturating amounts of IL-3/GM-CSF, respectively). Only B3 is expressed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 hours and then through differentiation and maturation in erythroid and granulopoietic cultures. HOXB4 and B5 are induced at slightly later times and expressed through maturation in both lineages, whereas B6 is selectively induced in granulocytic differentiation. B2 is transiently expressed at low level in the granulopoietic pathway, whereas it is detected only in advanced stages of erythropoiesis: B7, B8, and B9 are essentially not detected. Functional studies were performed with antisense phosphorothioate oligomers to HOX mRNAs and included control analysis of the targeted mRNA. The results are strictly coherent with the HOX mRNA expression pattern: (1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of both erythroid and granulomonocytic colony formation (similarly, alpha-B3 treatment of K562 cell line causes a significant dose-related inhibition of cell proliferation); (2) alpha-B6 selectively and markedly inhibits granulomonocytic colony formation; (3) alpha-B4 and alpha-B5 cause a significant, less pronounced decrease of both colony types; (4) finally, alpha-B2 and alpha-B7, -B9 exert little and no effect, respectively. These studies provide novel evidence on the coordinate expression of selected HOXB cluster genes in erythropoiesis and granulopoiesis, particularly in the early stages of differentiation: B3 apparently functions as a master gene in early hematopoiesis, whereas B6 exerts a key selective function in the granulopoietic pathway.
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PMID:Key functional role and lineage-specific expression of selected HOXB genes in purified hematopoietic progenitor differentiation. 794 19

OL-2, a highly branched (1-->3)-beta-D-glucan, is an antitumor glucan showing strong hematopoietic activity with weaker adjuvant activity than schizophyllan (SPG), another antitumor glucan and one which is used clinically. This paper deals with the gene expression of cytokines in mice by OL-2 and SPG in order to characterize their immunopharmacological activity. Gene expression was examined by a reverse transcriptase-polymerase chain reaction method after intraperitoneal administration of OL-2 or SPG (250 micrograms/mouse). The OL-2 administered mice strongly expressed the interleukin 1 receptor antagonist (IL-1ra) gene but SPG administered mice did not. The difference would be strongly related to the antigen-specific response between OL-2 and SPG. In the genes related to haematopoiesis, OL-2 induced G-CSF and GM-CSF, but SPG induced IL-3. These differences would relate to the pattern of haematopoietic response. Comparing the cytokine gene expression in ICR and AKR mice by OL-2 administration, the changes in cytokine gene expression were less in AKR mice administered OL-2. These findings suggest that the immunopharmacological characteristics of OL-2 are closely related, at least in part, to the activation of the complement system. The data shown in this paper also suggest that cytokine gene expression by beta-glucan would be significantly affected by the structure of these glucans.
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PMID:Analysis of cytokine mRNAs induced by the administration of a highly branched (1-->3)-beta-D-glucan, OL-2. 800 Mar 84

The in vitro sensitivity of human chronic myeloid leukemia-blast crisis and chronic phase (CML-BC and CML-CP, respectively) cells as well as adherent cell-depleted, T lymphocyte-depleted normal bone marrow cells (A-T-NBMC) to various concentrations of mafosfamide (ASTA Z7654), was examined by colony formation assay in the presence of IL-3 and GM-CSF, to test the possibility of purging of BMC from CML cells. Colony formation by CML cells was inhibited more efficiently than by NBMC. After the incubation with 50 micrograms/ml or 100 micrograms/ml of mafosfamide, the growth of leukemic CFU-GM was totally abrogated in 2/11 or 9/11 cases of CML-BC and in 1/7 or 6/7 cases of CML-CP, respectively. At the same time the CFU-GM arising from normal BMC were not inhibited totally with 50 or 100 micrograms/ml of the drug in any of five experiments. CML cells were still unable to form secondary colonies, while normal BMC were capable of regrowth. The CD34+ cells isolated form CML-BC and CML-CP patients were also more susceptible to mafosfamide cytotoxicity in comparison to CD34+ cells derived from NBMC. To confirm the possibility of purging, CML-BC cells were mixed with NBMC (1:1) and incubated with mafosfamide. Finally, the growing colonies were examined for the presence of bcr/abl hybrid gene by reverse transcriptase-Taq polymerase chain reaction (RT-PCR) and specific hybridization. The bcr/abl gene was not detected in the colonies growing after 100 micrograms/ml, and the signal was diminished after incubation with 50 micrograms/ml of mafosfamide, as compared to control. These results strongly suggest that high concentrations of mafosfamide may be useful for the purging of autologous BMC from CML cells.
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PMID:Successful mafosfamide purging of bone marrow from chronic myelogenous leukemia (CML) cells. 813 96

Neutrophils and Langerhans cells participate in inflammatory reactions within the human cornea. Because granulocyte-macrophage (GM)-CSF is a chemotactic and activating factor for these two cell types, we investigated whether this cytokine is produced by human corneal epithelial cells and corneal fibroblasts. Cultures of each cell type were exposed to increasing concentrations of IL-1 alpha or TNF-alpha. Culture supernatants were assayed for GM-CSF by using ELISA and cytokine mRNA levels were monitored by using reverse transcriptase-PCR. IL-1 alpha treatment of both cell types resulted in the appearance of GM-CSF mRNA and the production of > 480 pg protein/10(6) cells. However, TNF-alpha treatment yielded divergent results. Stimulation of epithelial cells with TNF-alpha resulted in the appearance of > 560 GM-CSF mRNA molecules per cell and production of > 1300 pg GM-CSF/10(6) cells. In contrast, stimulation of corneal fibroblasts resulted in < 16 GM-CSF mRNA molecules/cell and < 60 pg GM-CSF/10(6) cells. Binding studies with 125I-labeled TNF-alpha revealed that corneal fibroblasts had as many receptor sites as did corneal epithelial cells. Furthermore, corneal fibroblasts could respond to TNF-alpha-receptor-mediated signal transduction because they produced nanogram amounts of IL-6 after being treated with this cytokine. The results suggest that both cell types synthesize GM-CSF in response to IL-1 alpha, but that only corneal epithelial cells produce significant amounts of GM-CSF after TNF-alpha exposure. Differences in the responses of the two cell types to TNF-alpha may reflect a means of limiting accumulation of neutrophils and Langerhans cells and, thus, minimize corneal damage.
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PMID:Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines. 820 39

Peritoneal injection of thioglycollate medium (TM) to mice results in a dramatic increase in total number of peritoneal macrophages within 48 to 72 hours. Unlike resident macrophages, a fraction (10 to 20%) of these newly arrived young macrophages, designated as macrophage colony-forming cells (M-CFC), are highly proliferative and formed macrophage colonies in vitro in the presence of either macrophage or granulocyte-macrophage colony-stimulating factor (M-CSF or GM-CSF). Using a reverse transcriptase polymerase chain reaction (RT-PCR) technique, peritoneal exudate macrophages (PEM) obtained 2 to 5 days after a single TM injection actively expressed mRNA for recombinant murine macrophage inflammatory protein-1 alpha (rmMIP-1 alpha). Yet none or only a trace amount of mRNA for MIP-1 alpha was detected in normal resident macrophages or PEM obtained 7 days after TM treatment. The effect of rmMIP-1 alpha on the induction of exudate M-CFC was investigated. Multiple intraperitoneal (IP) administration of rmMIP-1 alpha caused a marked increase in the total number of peritoneal M-CFC and macrophages similar to but weaker than the increase in TM-injected mice. The total number of neutrophils, mast cells, and eosinophils also increased, but with different kinetics, following multiple injections of rmMIP-1 alpha. rmMIP-1 alpha alone did not stimulate the proliferation of M-CFC, nor did it potentiate their responsiveness to either rmGM-CSF or recombinant human (rh) M-CSF in vitro. Taken together, our results suggest that MIP-1 alpha released by exudate macrophages is a major chemoattractant responsible for the migration of M-CFC from the circulation to the peritoneal cavity during a TM-induced inflammatory response.
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PMID:Induction of murine peritoneal macrophage colony-forming cells by peritoneal administration of macrophage inflammatory protein-1 alpha. 840 40

Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross-reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down-modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the "distal" GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR ("pure" progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase-polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the "proximal" HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL-6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain-potentiation mechanism.
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PMID:Cascade transactivation of growth factor receptors in early human hematopoiesis. 845 93


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