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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

More than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-gamma as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK-expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK-transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK-transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL. (Blood. 2000;96:4319-4327)
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PMID:Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway. 1111 Jul 8

An inverse correlation between p27(Kip1) expression and proliferation has been recently established in tissues derived from human lymphomas. The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)/phospholipase C-gamma (PLCgamma) complex also appears to play an important role in cell proliferation and malignant transformation of anaplastic large cell lymphoma (ALCL). In this study, we report that SUDHL-1 and KARPAS 299 ALCL-derived cell lines present different sensitivity to the antiproliferative effect of recombinant adenovirus-mediated p27(Kip1) expression or to serum-starvation in culture media. The results indicate that exogenous p27(Kip1) may interact with the NPM-ALK/PLCgamma pathway in SUDHL-1 but not in KARPAS 299 cells. This interaction correlates with changes in cell cycle and cell morphology observed mainly in SUDHL-1 cells. The percentage of SUDHL-1 cells in S phase declines, whereas it is almost unchanged in KARPAS 299 cells as compared to the controls after 96 h of infection with the recombinant adenovirus. Furthermore KARPAS 299 cells are resistant to serum-starvation due to deficient p27(Kip1)-upregulation and G1 arrest, whereas SUDHL-1 cells respond with increased G1 phase and p27(Kip1)-upregulation after 48 h of serum-starvation. Both cell lines express appropriate variation of levels of cyclins E and A, and Rb-phosphorylation as expected by growing them in culture media with different FBS content. Although both cell lines express cyclin D2, SUDHL-1 cells only present high level of cyclin D3. Moreover SUDHL-1 cells express high level of PTEN and the PKB/Akt pathway is constitutively activated in both cell lines. Lastly SUDHL-1 cells show higher levels of phosphotyrosine-containing proteins that is correlated with a higher NPM-ALK-associated autophosphorylation activity compared to KARPAS 299 cells. Our study clearly identifies some of the biochemical differences that may explain the difference in sensitivity to antiproliferative stimuli shown by two cell lines derived from the same type of lymphoma.
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PMID:Biochemical differences between SUDHL-1 and KARPAS 299 cells derived from t(2;5)-positive anaplastic large cell lymphoma are responsible for the different sensitivity to the antiproliferative effect of p27(Kip1). 1149 42

Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.
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PMID:Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis. 1170 68

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.
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PMID:Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia. 1664 13

Erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), induces differentiation, cell-cycle arrest, and apoptosis of EGFR-negative myeloblasts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as in EGFR-negative cell lines representing these diseases (P39, KG-1, and HL 60). This off-target effect can be explained by inhibitory effects on JAK2. Apoptosis induction coupled to mitochondrial membrane permeabilization occurred independently from phenotypic differentiation. In apoptosis-sensitive AML cells, erlotinib caused a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14(ARF). Apoptosis-insensitive myeloblasts failed to manifest this translocation yet became sensitive to apoptosis induction by erlotinib when NPM-1 was depleted by RNA interference. Moreover, erlotinib reduced the growth of xenografted human AML cells in vivo. Erlotinib also killed CD34(+) bone marrow blasts from MDS and AML patients while sparing normal CD34(+) progenitors. This ex vivo therapeutic effect was once more associated with the nucleocytoplasmic translocation of NPM-1 and p14(ARF). One patient afflicted with both MDS and non-small cell lung cancer manifested hematologic improvement in response to erlotinib. In summary, we here provide novel evidence in vitro, ex vivo, and in vivo for the potential therapeutic efficacy of erlotinib in the treatment of high-risk MDS and AML.
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PMID:Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study. 1792 89

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.
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PMID:Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation. 1848 64

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.
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PMID:IL-21 contributes to JAK3/STAT3 activation and promotes cell growth in ALK-positive anaplastic large cell lymphoma. 1960 66

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) both represent highly heterogeneous entities on the basis of diverse cyto- and molecular genetic alterations with considerable influence on prognosis and therapeutic decisions. In recent years, insights into the complex network of molecular markers underlying this diversity have shown marked progress due to the detection of novel mutations, such as nucleophosmin gene (NPM1) in AML, and due to the description of cooperation pathways in leukemogenesis. Also, targeted therapeutic strategies are continuously expanding as illustrated by the tyrosine kinase inhibitor (TKI) imatinib for BCR-ABL positive ALL. Thus, molecular analysis based on various techniques, such as polymerase chain reaction (PCR) has become an essential part of the diagnostic panel for acute leukemia. In addition, cytomorphology, cytogenetics, fluorescence in situ hybridization (FISH), and immunophenotyping with multiparameter flow cytometry (MFC) need to be applied for diagnosis. During the course of disease, the residual leukemic cell load can be monitored by highly sensitive quantitative PCR techniques ("real-time PCR"). At present, new techniques, such as high throughput sequencing (next generation sequencing, NGS) or gene expression profiling with microarrays are being explored for use in hematological malignancies, and are being evaluated in preclinical studies. This demonstrates that molecular diagnostics for acute leukemias are in continuous development. This review summarizes the most important recurrent molecular markers seen in acute leukemias, their role in prognosis and therapy and provides an overview on the relevant PCR techniques.
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PMID:Molecular diagnostics in acute leukemias. 1981 44

The signaling network of protein kinase B(PKB)/Akt has been implicated in survival of lung cancer cells. However, understanding the relative contribution of the different isoform of Akt network is nontrival. Here, we report that Akt2 is highly expressed in human lung adenocarcinoma cell line A549 cells. Suppression of Akt2 expression in A549 cells results in notable inhibition of cell poliferation, soft agar growth, and invasion, accompanying by a decrease of nucleophosmin/B23 protein. Overexpression of Akt1 restores cancerous growth of A549 cells in B23-knockdown (KD) cells while Akt2 overexpression did not restore proliferating potential in cells with downregulated B23, thus suggesting Akt2 requires B23 to drive proliferation of lung cancer cell. Loss of functional Akt2 and B23 has similar defects on cell proliferation, apoptotic resistance and cell cycle regulation, while loss of Akt1 has less defects on cell proliferation, survival and cell cycle progression in A549 cells. Moreover, overexpression of B23 rescues the proliferative block induced as a consequence of loss of Akt2. Thus our data suggest that Akt2/B23 functions as an oncogenic unit to drive tumorigenesis of A549 lung cancer cells.
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PMID:Akt2 and nucleophosmin/B23 function as an oncogenic unit in human lung cancer cells. 2118 34

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.
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PMID:A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease. 2168 58


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