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)

Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 microM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.
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PMID:In vivo treatment of mutant FLT3-transformed murine leukemia with a tyrosine kinase inhibitor. 1072 Jan 29

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (</= 100 pg/ml).
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PMID:FLT3 ligand gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. 1073 51

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.
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PMID:Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation. 1084 96

The corticosteroid-treated animal is well established as an experimental model for the study of Pneumocystis carinii pneumonitis (PCP). Latent or acquired infection with P. carinii in the murine lung progresses to fatal pneumonitis when the host is profoundly immunocompromized. In this study the effects of five immunomodulators; recombinant CD40 ligand (CD40L), bryostatin 1, recombinant FLT3 ligand (FLT3L), recombinant granulocyte colony-stimulating factor (G-CSF) and recombinant interleukin-15 (IL-15) were investigated against PCP in a dexamethasone immunosuppressed Sprague-Dawley rat model. The majority of rats (70%) treated with CD40L at the onset of dexamethasone immunosuppression were protected against PCP. When CD40L was given after 10 days of immunosuppression, only 40% of the rats resolved the infection. However, 95% of the control animals developed PCP. Immunosuppressed rats treated with bryostatin 1, an immune activator had a partial (50%) protection against P. carinii infection. In contrast, daily administration of FLT3L, IL-15 or G-CSF provided no protection against P. carinii infection.
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PMID:Effect of CD40 ligand and other immunomodulators on Pneumocystis carinii infection in rat model. 1096 50

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In 20% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred IL-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of IL-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation.
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PMID:Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation. 1102 52

About 30%-40% of hematopoietic stem cells in human fetal liver of 3-5 months are in S phase of cell cycle, much higher than the ratio of 10% of that in adult bone marrow. The existance of highly active hematopoietic stem cell proliferation stimulators is probably its molecular basis. CFU-S "suicide rate" in rats was adopted to detect the effective substance. Through several steps of separation, we obtained a relatively purified substance of 35 kDa, termed it as FLS-4. CD 34 positive cord blood cells were sorted and assayed for their response to FLS-4 in 3H-TdR incorporation assay. The response to FLS-4 alone was approximately 1 times the background response seen with no factor added. In combination with IL-6 and IL-3 produced response that was 2.9 and 6.5 fold respectively greater than that observed with no factor added, but was weakly in comparison with the effects of SCF. In combination with GM-CSF or IL-3, FLS-4 can stimulate the formation of blast-colonies. The results indicate that the FLS-4 is very likely to be a novel hematopoietic stem cell proliferation stimulator. In physical or biological characteristics, it exhibited a unique character different from IL-3, IL-6, GM-CSF, SCF or FLT3 ligand those are known to have hematopoietic stem cell proliferation stimulating activity. During the period of active hematopoiesis in fetal liver, FLS-4 might be the candidate in triggering hematopoietic stem cells from resting G0 to S phase.
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PMID:[Studies of a 35 KDa substance from human fetal liver on the regulation of hematopoiesis]. 1103 18

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.
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PMID:FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group. 1109 Dec

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34+ cells. The effect of cytokine prestimulation on transduction efficiency and the population of uncommitted CD34+CD38- cells was determined. CD34+ cells harvested from umbilical cord blood were kept in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL-3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04). However, prestimulation with SCF in combination with IL-3 and IL-6 also reduced the percentage of CD34+ cells (p = 0.02). Then, prestimulation with SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34+CD38- cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytokine combinations increased the transduction efficiency, but reduced the fraction of CD34+ cells. Importantly, the use of FLT3 ligand seemed to preserve the population of uncommitted cells.
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PMID:FLT3 ligand preserves the uncommitted CD34+CD38- progenitor cells during cytokine prestimulation for retroviral transduction. 1109 93

Cord blood (CB) stem cell transplantations have been associated with delayed hematopoietic engraftment. This has most likely been due to the limited numbers of hematopoietic short-term repopulating cells in CB. Ex vivo expansion of CB has been attempted, and expansion of CD34-enriched CB has been successful; however, CD34 enrichment procedures are in general associated with substantial cell loss. Thus, we have studied culture conditions for expansion of nonenriched CB. Nonenriched CB cells were cultured for 21 days in the presence of conditioned medium from the HS-5 stromal cell line and FLT3-L or alternatively in the presence of FLT3-L, stem cell factor (SCF), megakaryocite growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF) (FSMG), either on fibronectin fragment CH-296-coated dishes or on uncoated dishes. With all four culture conditions, the number of mononuclear cells initially decreased until day 7 and then increased until the end of the expansion cultures. Overall expansion using HS-5 and FLT3-L resulted in superior expansion of MNC and CFU-C (44-/34-fold) for both cultures with and without CH-296 compared to FSMG (18-/17-fold). Expansion on CH-296 was less efficient than expansion on tissue culture-treated wells without CH-296 for both conditions. We then studied the best time for transduction on nonenriched CB. In contrast to enriched CD34 cells, we found for both conditions, HS-5/FLT3-L and growth factor cocktail, higher transduction efficiencies when cells were transduced on day 7 as compared to day 2. Gene transfer rates up to 45% were achieved with both conditions, which corresponded with the increased number of cells in S phase on day 7 compared to day 2. We conclude that HS-5 and FLT-3L allow efficient expansion and transduction of nonenriched CB.
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PMID:Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L. 1109

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.
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PMID:Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells. 1112 Jul 82


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