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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-beta1 (TGF-beta1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-beta1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-beta1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-beta1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-beta1 but rapidly up-modulated by anti-TGF-beta1. (3) Anti-TGF-beta1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3(low) and IL6-Rlow). According to these results, we propose the working model of 'High Proliferative Potential-Quiescent cells' to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-beta1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.
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PMID:High proliferative potential-quiescent cells: a working model to study primitive quiescent hematopoietic cells. 962 49

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.
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PMID:Structure-function analysis of FLT3 ligand-FLT3 receptor interactions using a rapid functional screen. 965 58

The effects of thrombopoietin (TPO; c-mpl ligand), FLT3/FLK-2 ligand (FL), and interleukin-6 (IL-6) on the survival of murine hematopoietic long-term reconstituting cells (LTRC) were studied by using lineage-negative, Sca-1-positive, c-kit-positive (Lin-Sca-1(+)c-kit+) marrow cells from 5-fluorouracil-treated mice. We tested the ability of these cytokines to maintain the viability of LTRC by transplanting the cultured cells to lethally irradiated Ly-5 congenic mice together with compromised marrow cells. As a single agent, only TPO could maintain the LTRC. Neither IL-6 nor FL was effective by itself, but they acted synergistically to maintain the LTRC. We examined whether the maintenance of LTRC by these cytokines was due to the survival of stem cells or was the result of active cell divisions and self-renewal. To monitor cell division, we used membrane dye PKH26. Enriched cells were stained with PKH26 on day 0 and incubated in suspension culture with TPO or with IL-6 and FL for 7 days. On day 7, PKH26(low) and PKH26(high) cells were prepared by sorting and their in vivo reconstituting abilities were tested by transplantation into lethally irradiated Ly-5 congenic mice together with compromised marrow cells. PKH26(high) populations cultured with both TPO alone and the combination of IL-6 and FL showed greater reconstitution activity than that of PKH26(low) populations. These data indicate that TPO alone and the combination of IL-6 and FL can support the survival of stem cells without stimulating their active cell proliferation.
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PMID:Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. 965 44

Development of the hematopoietic lineages is partially under the control of hematopoietic receptors with tyrosine kinase activity (RTK). To compare the cellular functions of two of the class III RTK, FLT3 and KIT, a murine chimeric FMS/FLT3 (FF3) receptor was expressed ectopically using retroviral infection, in normal IL3-derived cultured mast cells. Stimulation of the chimeric receptor produced a full mitogenic signal and led to mast cell maturation, as occurs upon activation of the endogenous KIT receptor. When introduced into mast cells derived from KIT-deficient White spotting (W) mutant mice, the FF3 receptor bypassed their mitogenic defect. KIT activation induced a synergistic mitogenic activity in mast cells upon IL3 stimulation, whereas FF3 appeared to down-modulate the IL3 response. Adhesion to fibronectin was specifically associated with KIT signaling.
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PMID:Specific and common activities of the FLT3 and KIT tyrosine kinase receptors revealed by the use of cultured mast cells. 966 95

In this report we evaluated the exact expression pattern of c-Kit on mobilized peripheral blood (PB) CD34+ cells. Using a monoclonal antibody against CD117 antigen (95C3), flow cytometric analysis revealed that approximately 25% of the mobilized PB CD34+ cells coexpress c-Kit. This cell fraction showed a considerable heterogeneity with respect to c-Kit expression, consisting of a small fraction with high levels of c-Kit (4.2%) (CD34+/CD117high fraction) and a larger proportion of cells expressing low levels of this antigen (21.0%) (CD34+/CD117low fraction). Clonogenic assays showed that CD34+/CD117high cell fraction consisted almost exclusively of erythroid progenitors, in contrast to CD34+/CD117low cell subset which gave rise mostly to granulocyte-monocyte colonies. The majority of CFU-GEMM and the most primitive week 6 cobblestone area forming cells (CAFCs) segregated in the CD34+/CD117low cell subset, suggesting the highest content of multipotential progenitors within this cell fraction. None of the sorted cell subsets was able to produce reactive oxygen intermediates (ROI). However, ex vivo expansion of the sorted subsets with interleukin 3, stem cell factor and FLT3 ligand for 2 weeks resulted in a significant production of O2- and H2O2/HOCl by CD34+/CD117low cell fraction, compared to the same sorted but not expanded counterparts. According to the major content of multipotential hematopoietic progenitors and highest capacity to generate sufficient amounts of ROI after ex vivo expansion, we suggest that CD34+/CD117low cell subset would be one of the most potential candidates for transplantation in patients with acute lymphoblastic leukemia, which lack c-Kit antigen expression.
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PMID:Phenotypic and functional characterization of mobilized peripheral blood CD34+ cells coexpressing different levels of c-Kit. 966 40

Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with psi-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38- subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG-rhMGDF increased the transduction efficiency into CD34+CD38(-)-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers.
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PMID:Retrovirus-mediated gene transfer into human CD34+38low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo. 968 21

The fate of hematopoietic progenitor cells (HPCs) in the bone marrow (BM) microenvironment is determined by two different interactions: 1) they adhere (via integrins) to both extracellular matrix molecules and BM stromal cells; and 2) stromal cells produce cytokines that influence their survival, proliferation, differentiation, and mobilization. The ligands for the protein tyrosine kinase receptors c-KIT and FLT3/FLK2, stem cell factor (SCF), and FL are produced by BM stromal cells and are known to affect several facets of hematopoiesis. We studied another protein tyrosine kinase receptor, c-MET, and its ligand hepatocyte growth factor (HGF), also known as scatter factor (SF), which play a similar role in hematopoiesis. c-MET mRNA is expressed in immature human BM HPCs (CD34+CD33- or CD34+CD38-), but not in more mature HPCs (CD34+CD33+ or CD34+CD38+). The ligand HGF/SF is predominantly produced by BM stromal cells at both the mRNA and protein levels. We confirmed functionally that HGF/SF alone has no effect on proliferation of HPCs, but that when combined with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 it acts as a synergistic proliferative factor, although not as potently as kit-ligand or FLT-3/FLK-2 ligand. Furthermore, HGF/SF promotes adhesion of HPCs to immobilized fibronectin. HGF/SF-induced adhesion to fibronectin is probably caused by activation of the integrins alpha4beta1 and alpha5beta1, insofar as we were able to block this interaction by using monoclonal blocking antibodies directed against these integrin subunits. Addition of the tyrosine-phosphorylation inhibitor genistein inhibited HGF/SF-induced adhesion, supporting the idea that HGF/SF-induced effects are the result of signaling via the receptor c-MET after ligand binding. The enhanced adhesion of HGF/SF to fibronectin proved to be beneficial for the maintenance of the colony-forming potential of HPCs. HGF/SF alone and especially in combination with fibronectin prolongs survival of GM colony-forming cells in liquid culture. Our data indicate that HGF/SF is a polyfunctional cytokine in the BM microenvironment. It is produced by human BM stromal cells and directly or indirectly promotes proliferation, adhesion, and survival of human HPCs.
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PMID:Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+). 969 10

Cord blood (CB) provides an alternative source of stem cells for transplantation, although in a considerable number of cases CB transplantation is followed by long periods of aplasia. Ex vivo expansion has the capacity to generate large amounts of progenitors, and it has been proposed that expanded cells might be beneficial in overcoming these long periods of aplasia. We describe the biological characteristics of cord blood compared to other sources of stem cells (BM and PB), and report the effects of FLT3-L and MIP-1alpha when added to a combination of SCF, IL-3 and IL-6 in pre-clinical short-term, serum-free expansion cultures of CB-derived CD34+ cells. After 6 days, this culture system was able to generate considerable expansion rates in the committed compartment (between 8.16- and 17.26-fold for CFU-GM, and 21.58- and 36.53-fold for the BFU-E/CFU-Mix), and the CD34+ population (between 11.25- and 25.42-fold). Moreover, this culture system was also able to maintain the week 5 CAFC population, particularly when both FLT3-L and MIP-1alpha were present (91% of the input level). Thus, we have described a pre-clinical protocol for ex vivo expansion of CB CD34+ cells in a short-term, static, serum-free system, where a high generation of committed progenitor cells is achieved together with CAFC maintenance.
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PMID:Expansion of cord blood progenitor cells. 971 2

An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To elucidate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gain-of-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.
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PMID:Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. 973 79

The aim of this study was to evaluate the ex vivo expansion of normal CD34+ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF + MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10(3) to 1 x 10(5)CD34+ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39+/-0.5% in expanded cultures derived from fractions initiated at 5 x 10(3), 10(4) or 10(5) cells/ml and 45+/-6% in cultured cells obtained from starting fractions containing 5 x 10(4) cells/ml, as compared to 58+/-4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H2O2, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45+/-4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.
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PMID:Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for a large-scale clinical applications. 981 1


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