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)

It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reverse-transcription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and self-renewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1beta, IL-3, leukemia inhibitory factor, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor-enriched fetal liver hematopoietic cells resulted in a 40-fold to 80-fold expansion of total hematopoietic cells and in a 2.5-fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic-derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self-renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.
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PMID:Hematopoietic support and cytokine expression of murine-stable hepatocyte cell lines (MMH). 982 30

FLT3-ligand (FL) is a recently described cytokine that stimulates the proliferation and differentiation of hematopoietic progenitors both in vivo and in vitro and, when administered to mice, induces an accumulation of dendritic cells (DC) in different lymphoid and nonlymphoid organs and tissues, including the liver. We have studied the antitumor effect of FL administered alone or in combination with IL-12 in a day 3 murine liver metastasis model. FL significantly reduced the number of hepatic metastases (36.00 +/- 11.00 vs 92.00 +/- 10.19 in control group, p < 0.05). Histologic evaluation of the livers revealed that FL induced a significant infiltration of the tumor border by lymphocytes and DC associated with increased number of apoptotic figures. Immunohistochemical analysis demonstrated that FL significantly enhanced the number of DC in the liver parenchyma and within the liver metastases, as well as the number of CD4+ and CD8+ T lymphocytes. These data support the suggestion that DC may be directly involved in the antitumor effect of FL. Interestingly, the antitumor effect of FL was greatly reduced by the NK depletion. Combination of FL and IL-12 resulted in greater antitumor efficacy than these cytokines alone. In summary, we have shown that FL has significant antitumor effect on preexisting murine C3 liver tumors that is mediated by NK cells. We have also demonstrated that the FL/IL-12 combination has an enhanced antitumor activity in the same murine tumor model.
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PMID:FLT3-ligand administration inhibits liver metastases: role of NK cells. 983 2

The identification and study of genes expressed in hematopoietic stem/progenitor cells should further our understanding of hematopoiesis. Transcription factors in particular are likely to play important roles in maintaining the set of genes that define the stem/progenitor cell. We report here the identification of a putative KRAB-zinc finger gene (SZF1) from a cDNA library prepared from human bone marrow CD34+ cells. Characterization of SZF1 implicates its role in hematopoiesis. The predicted protein contains a highly conserved KRAB domain at the NH2 terminus and four zinc fingers of the C2H2 type at the COOH terminus. Two alternatively spliced products of SZF1 were isolated, which predict proteins of 421 (SZF1-1) and 361 (SZF1-2) amino acids, differing from each other only at the carboxy terminus. The two transcripts of SZF1 have different expression patterns. SZF1-2 is ubiquitously expressed, as indicated by Northern blot, RNase protection, and reverse transcriptase polymerase chain reaction. SZF1-1 expression, in contrast, was detected only in CD34+ cells. We recently isolated the promoter region for the stem/progenitor cell expressed FLT3/FLK-2/STK-1 gene and used this region to generate a reporter construct to test the effect of SZF1 expression. Cotransfection of the reporter construct with SZF1 constructs showed that SZF1-2 repressed transcription three- to fourfold, whereas SZF1-1 showed a lower level of repression. The expression pattern of SZF1 transcripts and the transcriptional repression of a CD34+-specific promoter demonstrate a possible role for SZF1 in hematopoietic stem/progenitor cell differentiation.
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PMID:SZF1: a novel KRAB-zinc finger gene expressed in CD34+ stem/progenitor cells. 1002 71

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

We analyzed tandem duplication in the juxtamembrane (JM) domain of the FLT3 (FMS-like tyrosine kinase 3/FLK2, CD135) gene in 94 children with acute myeloid leukemia (AML) and evaluated its correlation with clinical features. Longer polymerase chain reaction (PCR) products were observed in five patients; 1/3 of M0, 119 of M1, 1/39 of M2, 1/9 of M3 and 1/12 of M5. The sequence analyses of abnormal PCR products showed that all the abnormal products were derived from tandem duplications involving the JM domain and that all the lengthened sequences were in-frame as we previously reported. Statistical analyses revealed a significantly lower incidence of the tandem duplication in childhood AML patients than in adult patients (P < 0.05), and significantly shorter disease-free survival in patients with mutant FLT3 than in patients with wild-type FLT3 (P < 0.05). Our results suggest that the tandem duplication in the JM domain of the FLT3 gene is not a frequent phenomenon but might be a factor of poor prognosis in childhood patients with AML.
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PMID:Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia. The Children's Cancer and Leukemia Study Group, Japan. 1004 58

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34(+) cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34(+) cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34(-)CD1a- CD14(+) (p14(+)) and CD34(-)CD1a-CD14(-) (p14(-)) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14(-), CD83(-)) macropinocytic DC. Mature (CD1a+, CD14(-), CD83(+)) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-alpha (TNF). In addition, p14(-) cells generated CD14(+) cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34(+) cells may improve future prospects for immunotherapy.
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PMID:Long-term culture of human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem cell factor induces extensive amplification of a CD34(-)CD14(-) and a CD34(-)CD14(+) dendritic cell precursor. 1009 Sep 33

Relapse is a major cause of treatment failure in acute myeloid leukaemia (AML), and is usually accompanied by resistance to chemotherapy. To study whether relapse is accompanied by genetic alterations, we compared N-ras, p53 and FLT3 gene mutations in paired samples obtained at initial diagnosis and first relapse. 28 patients with relapsed AML were studied, and their duration of complete remission ranged from 133 to 989 d (mean 318 d). Karyotype changes were observed at relapse in 11 patients. Point mutations of the N-ras gene were positive at both stages (+/+) in three patients, positive at initial diagnosis and negative at relapse (+/-) in three patients, and negative at initial diagnosis and positive at relapse (-/+) in two patients. Internal tandem duplications of the FLT3 gene (FLT3/ITD) were +/+ in five patients, +/- in one patient, and -/+ in six patients. The p53 gene mutations were +/+ in two patients, +/- in one patient, and -/- in 25 patients. FLT3/ITD and mutant p53 at relapse were associated with short survival after relapse. These results indicate that relapse is frequently accompanied by molecular alterations that include the loss and/or acquisition of mutations. Thus relapse can be understood as clonal shift or collateral succession rather than clonal progression.
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PMID:Molecular evolution of acute myeloid leukaemia in relapse: unstable N-ras and FLT3 genes compared with p53 gene. 1019 23

FLT3 ligand (FL) acting through its tyrosine kinase receptor FLT3 has pleiotropic and potent effects on hematopoietic cells. The well-described involvement of this ligand-receptor pair in physiological hematopoiesis raised the question whether FL and FLT3 also play a role in the pathobiology of leukemia. Following the early discovery of high receptor expression by myeloid leukemia cells, several investigators have focused their attention on these cells, both primary acute myeloid leukemia (AML) cells and continuous human myeloid leukemia cell lines. Regardless of the morphological FAB subtype, the vast majority of AML cases were FLT3-positive both at the mRNA and protein level; among the myeloid cell lines, predominantly the monocytic and myelocytic cell lines were FLT3-positive whereas the erythrocytic and megakaryocytic cell lines were FLT3-negative. Virtually all cell lines studied expressed FL transcripts; the finding that some cell lines displayed both ligand and receptor indicates the possibility of autocrine, intracrine or paracrine stimulatory loops. In vitro growth assays showed that FL caused a proliferative response in a high percentage of AML cases. Only constitutively growth factor-dependent myelocytic cell lines increased their proliferation upon incubation with FL whereas all growth factor-independent cell lines were refractory to FL stimulation. Combinations of FL with various cytokines (e.g. G-CSF, GM-CSF, IL-3, M-CSF, PIXY-321, SCF) had synergistic or additive mitogenic effects. Finally, FL had significant anti-apoptotic, survival-promoting effects on primary AML cells and myeloid cell lines under serum-free culture conditions. On the strength of the above findings, it can be concluded that the FL-FLT3 signaling system may play a certain, albeit probably not causal role in the development of human leukemias. Dissection of the exact molecular pathways that lead to proliferation and/or anti-apoptosis of myeloid leukemia cells as well as the detailed elucidation of the possible contribution of the FL-FLT3 genes to leukemogenesis remain future challenges.
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PMID:Effects of FLT3 ligand on proliferation and survival of myeloid leukemia cells. 1019 24

Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients' sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system.
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PMID:Endogenous FLT-3 ligand serum levels are associated with disease stage in patients with myelodysplastic syndromes. 1021 61

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.
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PMID:Efficient detection and selection of immature rhesus monkey and human CD34+ hematopoietic cells expressing the enhanced green fluorescent protein (EGFP). 1021 69


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