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)

The interaction between p145(c-KIT) and p210(bcr-abl) in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34(+) cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34(+) cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34(+) cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) from CML CD34(+)CD38(+) cells and the more primitive CML CD34(+)CD38(-) cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34(+) cells showed exclusive growth of Ph+ cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of beta1-integrin-mediated adhesion of CML CD34(+) cells to fibronectin was not increased when compared with the effect on normal CD34(+) cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.
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PMID:Stem cell factor as a single agent induces selective proliferation of the Philadelphia chromosome positive fraction of chronic myeloid leukemia CD34(+) cells. 974 86

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

The escape of malignant cells from the immune response against the tumor may result from a defective differentiation or function of professional antigen-presenting cells (APC), ie, dendritic cells (DC). To test this hypothesis, the effect of human renal cell carcinoma cell lines (RCC) on the development of DC from CD34(+) progenitors was investigated in vitro. RCC cell lines were found to release soluble factors that inhibit the differentiation of CD34(+) cells into DC and trigger their commitment towards monocytic cells (CD14(+)CD64(+)CD1a-CD86(-)CD80(-)HLA-D Rlow) with a potent phagocytic capacity but lacking APC function. RCC CM were found to act on the two distinct subpopulations emerging in the culture at day 6 ([CD14(+)CD1a-] and [CD14(-)CD1a+]) by inhibiting the differentiation into DC of [CD14(+)CD1a-] precursors and blocking the acquisition of APC function of the [CD14(-)CD1a+] derived DC. Interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) were found to be responsible for this phenomenon: antibodies against IL-6 and M-CSF abrogated the inhibitory effects of RCC CM; and recombinant IL-6 and/or M-CSF inhibited the differentiation of DC similarly to RCC CM. The inhibition of DC differentiation by RCC CM was preceeded by an induction of M-CSF receptor (M-CSFR; CD115) and a loss of granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR; CD116) expression at the surface of CD34(+) cells, two phenomenon reversed by anti-IL-6/IL-6R and anti-M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, the results suggest that the inhibition of DC development could represent a frequent mechanism by which tumor cells will escape immune recognition.
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PMID:Inhibition of the differentiation of dendritic cells from CD34(+) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. 984 45

Myosin heavy chain (MHC) isoform expression was evaluated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) to clarify a possible link between gastrointestinal stromal tumor (GIST) and interstitial cells of Cajal (ICCs) in the gastrointestinal (GI) tract. Using monoclonal antibodies against MHC isoforms, 18 of 27 GISTs (67%) showed immunoreactivity for non-smooth-muscle myosin or the embryonic form of MHC (SMemb), but only one tumor showed immunoreactivity for smooth muscle cell (SMC)-specific isoforms (SM1 and SM2). Co-expression of KIT or CD34, which is also expressed in GIST and ICCs, was demonstrated in 18 (100%) and 16 SMemb-positive tumors (89%), respectively. Otherwise, the expression of SMemb in GIST was not correlated with the patient's age or sex, tumor size, histological grade of GIST, or expression of mesenchymal cell markers, such as alpha-smooth muscle actin (alpha-SMA) or S100 protein. By double-fluorescence immunostaining of the tunica muscularis of the GI tract wall, co-expression of KIT, CD34, and SMemb was demonstrated in ICCs, which were negative for SM1 and SM2. RT-PCR analysis confirmed that GIST expressed SMemb-mRNA, which lacked neuronal cell-specific inserts of 30 bp. These facts further strengthen the current hypothesis that GIST is a tumor of ICCs.
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PMID:Embryonic form of smooth muscle myosin heavy chain (SMemb/MHC-B) in gastrointestinal stromal tumor and interstitial cells of Cajal. 991 14

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+ cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34(+) and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1(+) endothelial cells on the OP9 stromal layer, while TEK- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.
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PMID:In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region. 1002 83

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

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

The RET gene product represents the signal-transducing molecule of a surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF), which includes GDNFR-alpha as a ligand-binding component. By a semi-quantitative competitive RT-PCR approach, we have analysed the relative abundances of RET transcripts in blasts purified from 40 acute myeloid leukaemia (AML) cases, revealing significant amounts of RET transcripts in 60% of AML cases (24/40). RT-PCR data was confirmed by immunocytochemical detection of RET protein in leukaemic blasts. The highest RET mRNA levels, almost exclusively confined to FAB M4/M5 AMLs, directly correlated with the presence on leukaemic cells of adhesion molecules and surface structures typically expressed by blasts of monocytic lineage and were inversely associated with the expression of the stem cell antigen CD34. Consistently, differentiation of the monoblastic cell line U937 resulted in an up-regulated expression of RET proto-oncogene, which was maximal upon exposure to agents inducing a more complete monocytic differentiation. Finally, while transcripts specific for GDNF and GDNFR-alpha were never found in leukaemic blasts, stromal cells of the haemopoietic microenvironment expressed, in the absence of RET, significant amounts of both GDNF and GDNFR-alpha. Our results suggest a role for RET in the functional regulation of AMLs through interactions with GDNF- and GDNFR-alpha-producing stromal cells.
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PMID:The RET receptor tyrosine kinase, but not its specific ligand, GDNF, is preferentially expressed by acute leukaemias of monocytic phenotype and is up-regulated upon differentiation. 1023 87

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.
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PMID:Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. 1034 58

The purpose of this report is to demonstrate the expression of very recently identified surface antigens on CD34+ and AC133+ bone marrow (BM) cells. Coexpression analysis of AC133 and defined antigens on CD34+ BM cells revealed that the majority of the CD164+, CD135+, CD117+, CD38low, CD33+, and CD71low cells resides in the AC133+ population. In contrast, most of the CD10+ and CD19+ B cell progenitors and a fraction of the CD71high population are AC133-, indicating that CD34+AC133+ cells are enriched in primitive and myeloid progenitor cells, whereas CD34+AC133- cells mainly consist of B cell and late erythroid progenitors. This corresponds to the highly reduced percentage of CD10+ B cells and the absence of CD71high erythroid progenitors on AC133+ selected BM cells. A portion of 0.2-0.7% of the AC133+ selected cells do not coexpress CD34. These cells are very small and define a uniform CD71-, CD117-, CD10-, CD38low, CD135+, HLA-DRhigh, CD45+ population with unknown delineation. Four color analysis on CD34+CD38- BM cells revealed that virtually all of these primitive cells express AC133. Using an improved liposome-enhanced labeling technique for the staining of weakly expressed antigens, subsets of this population could be identified which express the angiopoietin receptors TIE (67.6%) and TEK (36.8%), the vascular endothelial growth factor receptors FLT1 (7%), FLT4 (3.2%), and KDR (10.4%), or the receptor tyrosine kinases HER-2 (15.4%) and FLT3 (CD135; 77.6%). Our results suggest that the CD34+CD38- population is heterogeneous with respect to the expression of the analyzed receptor tyrosine kinases.
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PMID:Expression of novel surface antigens on early hematopoietic cells. 1037 8


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