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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)

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

Stem Cell Factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to synergistically interact with other cytokines, enhancing the responsiveness of haemopoietic precursors. In this study we have examined the effects of SCF, in combination with interleukin-3 (IL-3), on FDCP-Mix A4 cells, a murine, multipotent, haemopoietic progenitor cell line. Low concentration of IL-3 act to enhance cell survival but do not stimulate proliferation in A4 cells. Similarly, SCF when added alone, acts as a good survival stimulus, but is a poor proliferative stimulus. However, in combination with low concentrations of IL-3, SCF stimulates a synergistic enhancement of proliferation leading to a large increase in cell number after seven days. The synergy observed was not due to SCF stimulated alterations in the mRNA, protein levels or affinity of the IL-3 receptors. Therefore, interactions between cytokines at the level of cytoplasmic signalling pathways were investigated. IL-3 stimulated the rapid and transient tyrosine phosphorylation of several proteins (including those of molecular weights 130, 110, 100, 95, 80, 65, 50 and 45 kDa). Some of these proteins were identified as the Src Homology Collagen (SHC) protein, Janus kinase (JAK-2) and the Mitogen Activated Protein Kinase isoforms ERK 1 and ERK 2. IL-3 also stimulated a transient increase in the activity of both ERK 1 and 2. SCF stimulated a rapid and transient increase in the tyrosine phosphorylation of ERK 1 and ERK 2 although, coaddition of SCF with IL-3, caused no gross differences in the phosphorylation of SHC, JAK-2 or ERKs compared to those observed with IL-3 alone. Coaddition of SCF and low concentration of IL-3 stimulated a reproducible synergistic increase in the activity of ERK 2, whereas only an additive increase in the activity of ERK 1 was observed. These results suggest that ERK 2 activation represents a point at which the two pathways, stimulated by IL-3 and SCF, interact synergistically.
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PMID:Investigation of the molecular mechanisms underlying growth factor synergy: the role of ERK 2 activation in synergy. 971 13

The change of phenotypic traits in domestic animals and crops as a response to selective breeding mimics the much slower evolutionary change in natural populations. Here, we describe that the dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene encoding the mast/stem cell growth factor receptor (MGF), one gene duplication associated with a partially dominant phenotype and a splice mutation in one of the copies leading to the fully dominant allele. The splice mutation is a G to A substitution in the first nucleotide of intron 17 and leads to skipping of exon 17. The duplication is most likely a regulatory mutation affecting KIT expression, whereas the splice mutation is expected to cause a receptor with impaired or absent tyrosine kinase activity. Immunocytochemistry showed that this variant form is expressed in 17- to 19-day-old pig embryos. Hundreds of millions of white pigs around the world are assumed to be heterozygous or homozygous for the two mutations. [The EMBL accession numbers for porcine KIT1*0101, KIT1*0202, KIT2*0202, and KIT2*0101 are AJ223228-AJ223231, respectively.]
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PMID:Molecular basis for the dominant white phenotype in the domestic pig. 972 28

It is now accepted from studies in animal models that hematopoietic stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of CD34(+)CD45(+)Lin- high proliferative potential hematopoietic cells clustered on the ventral endothelium of the aorta. To characterize molecules involved in initial stem cell emergence, we first investigated the expression in that territory of known early hematopoietic regulators. We herein show that aorta-associated CD34(+) cells coexpress the tal-1/SCL, c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic and fetal hematopoietic progenitors later present in the liver and bone marrow. Next, CD34(+)CD45(+) aorta-associated cells were sorted by flow cytometry from a 5-week embryo and a cDNA library was constructed therefrom. Differential screening of that library with total cDNA probes obtained from CD34(+) embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in all developing endothelial cells in the yolk sac and embryo, which suggests its involvement in the genesis of both hematopoietic and vascular cell lineages in humans.
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PMID:Molecular identity of hematopoietic precursor cells emerging in the human embryo. 980 56

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk-1/KDR and Flt-1 respectively. Flk-1/KDR-deficient murine embryos manifest failure of blood-island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt-1 and Flk-1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt-1 and Flk-1/KDR receptors on stem cell rich CD34+ c-kit-R+ Rh123(low) cells. Further studies revealed that Fit-1 R mRNA, but not Flk-1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co-stimulated HEL cell proliferation as well as CFU-GM colony formation from approximately 15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.
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PMID:Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth. 988 8

Growth factors (cytokines) are considered to be key regulators of hematopoiesis, in particular by stimulating growth or maintaining viability mainly of progenitor cells, but also of more mature cells. We examined cytokine-stimulated survival of constitutively growth factor-dependent acute myeloid leukemia (AML)-derived cell lines. The cells from the four cell lines MUTZ-2 (AML M2-derived), OCI/AML5 (AML M4), TF-1 (AML M6) and UT-7 (AML M7) undergo apoptosis quickly in the absence of cytokines in serum-free medium: half-lives of serum- and factor-deprived cells ranged from 14 to 64 h. Here, we analyzed the survival-promoting and apoptosis-inhibiting properties of FLT3 ligand (FL) using the viable cell count as an indicator of programmed cell death. The receptor for FL belongs to the class III family of receptor tyrosine kinases which also includes c-kit, the receptor for stem cell factor (SCF). FL extended the survival of cell lines MUTZ-2 and OCI/AML5, but was not effective for cell lines TF-1 and UT-7. In OCI/AML5, the action of FL was evident both in first promoting survival and then stimulating proliferation slightly. In MUTZ-2, depending on the concentration used, FL extended survival by 64-135% compared with control cells. SCF alone prolonged cell survival of MUTZ-2 as well, however, FL and the combination of FL+SCF was significantly more active. Thus, FL alone, and in combination with SCF, was active in promoting survival and proliferation of human AML cells in vitro.
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PMID:FLT3 ligand inhibits apoptosis and promotes survival of myeloid leukemia cell lines. 1004 31

The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor alpha (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci-KIT, PDGFRA, albumin (ALB), and MC1R-in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals.
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PMID:Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping. 1005 24

Megakaryocytopoiesis is the process by which bone marrow progenitor cells develop into mature megakaryocytes, which in turn produce platelets required for normal hemostasis. The development of this hematopoietic lineage depends on a variety of growth factors and cytokines. Growth factor-dependent tyrosine kinase receptors important in megakaryocytopoiesis include c-Kit, fibroblast growth factor receptor, the RON receptor, and the macrophage colony-stimulating factor receptor. Binding of growth factors to their respective receptors results in receptor dimerization and subsequent autophosphorylation on tyrosine residues. Tyrosine autophosphorylations become sites of association for cytoplasmic signaling molecules via their SH2 domains. Some of these molecules are themselves cytoplasmic tyrosine kinases such as the Src kinases, TEC, and CHK. Others are molecules such as phospholipase C-gamma, phosphoinositol 3-kinase, Shc, GTPase-activating protein, and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. These molecules generate second messengers, regulate the phosphorylation of other downstream molecules, and also regulate the phosphorylation of the receptor itself. The different cytoplasmic components activate pathways involved in either changes in cell growth or changes in the cytoskeleton that affect maturation of the cell. Cytokine receptors also generate signals involved in growth and differentiation. Some of these second messengers overlap with those of the receptor tyrosine kinases. Others, such as the JAKs/STATs, are involved in transcriptional control and are unique to the signaling mediated by cytokine receptors. We describe the contribution of these different signals to the growth/differentiation processes of megakaryocytes. We also describe the contribution of receptor and nonreceptor tyrosine phosphatases to these processes. Lastly, we have compiled selected methods related to the study of protein phosphorylation in megakaryocytes.
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PMID:Regulation of megakaryocytopoiesis and platelet production by tyrosine kinases and tyrosine phosphatases. 1008 Sep 10

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.
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PMID:Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells. 1034 Apr 8

The biological effects of flt3-L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG-CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low-level expression, flt3-L exerted synergistic effects with a combination of c-kit ligand, IL-3, IL-6, GM-CSF and G-CSF, mainly to induce proliferation of CD34+/CD38- cells. In addition, flt3-L increased the detection of HPP-CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3-L is active on circulating early mobilized haemopoietic progenitors, despite the low- level expression of its receptor.
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PMID:Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3-L. 1046 May 91


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