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Sample records for alks mrna coding

  1. Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

    PubMed

    Marzec, M; Liu, X; Wong, W; Yang, Y; Pasha, T; Kantekure, K; Zhang, P; Woetmann, A; Cheng, M; Odum, N; Wasik, M A

    2011-03-17

    The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

  2. Pleiotrophin induces neurite outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the ALK/GSK3beta/beta-catenin signaling in developing mouse neurons.

    PubMed

    Yanagisawa, Hiroko; Komuta, Yukari; Kawano, Hitoshi; Toyoda, Masashi; Sango, Kazunori

    2010-01-01

    Pleiotrophin (PTN) is highly expressed in the nervous system during embryogenesis; however, little is known about its functional role in neural development. By using whole mount in situ hybridization, we observed that the expression pattern of PTN was similar to that of Wnt3a; PTN mRNA was abundant in the nervous tissue along the dorsal midline and in the forelimb and hindlimb buds of embryonic mice (E8.5-E12.5). Treatment with recombinant PTN (100ng/ml) induced phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear localization of beta-catenin and up-regulation of growth-associated protein (GAP)-43 mRNA in cultured embryonic mouse (E14.5) neurons. Furthermore, recombinant PTN enhanced neurite outgrowth from cortical explants embedded in Matrigel. These PTN-induced biochemical changes and neurite outgrowth were attenuated by the co-treatment with anti-anaplastic lymphoma kinase (ALK) antibodies, but not with anti-protein tyrosine phosphatase (PTP)zeta antibodies. These findings imply that ALK is involved in the PTN signaling on neural development.

  3. A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer

    PubMed Central

    Ma, Yibao; Yu, Chunrong; Mohamed, Esraa M.; Shao, Huanjie; Wang, Li; Sundaresan, Gobalakrishnan; Zweit, Jamal; Idowu, Michael; Fang, Xianjun

    2016-01-01

    A high rate of aerobic glycolysis is a hallmark of malignant transformation. Accumulating evidence suggests that diverse regulatory mechanisms mediate this cancer-associated metabolic change seen in a wide spectrum of cancer. The echinoderm microtubule associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein is found in approximately 3-7% of non-small cell lung carcinomas (NSCLC). Molecular evidence and therapeutic effectiveness of FDA-approved ALK inhibitors indicated that EML4-ALK is a driving factor of lung tumorigenesis. A recent clinical study showed that NSCLC harboring EML4-ALK rearrangements displayed higher glucose metabolism compared to EML4-ALK-negative NSCLC. In the current work, we presented evidence that EML4-ALK is coupled to overexpression of hexokinase II (HK2), one of the rate-limiting enzymes of the glycolytic pathway. The link from EML4-ALK to HK2 upregulation is essential for a high rate of glycolysis and proliferation of EML4-ALK-rearranged NSCLC cells. We identified hypoxia-inducible factor 1α (HIF1α) as a key transcription factor to drive HK2 gene expression in normoxia in these cells. EML4-ALK induced hypoxia-independent but glucose-dependent accumulation of HIF1α protein via both transcriptional activation of HIF1α mRNA and the PI3K-AKT pathway to enhance HIF1α protein synthesis. The EML4-ALK-mediated upregulation of HIF1α, HK2 and glycolytic metabolism was also highly active in vivo as demonstrated by FDG-PET imaging of xenografts grown from EML4-ALK-positive NSCLC cells. Our data reveal a novel EML4-ALK-HIF1α-HK2 cascade to enhance glucose metabolism in EML4-ALK-positive NSCLC. PMID:27132509

  4. Discovery of Proteomic Code with mRNA Assisted Protein Folding

    PubMed Central

    Biro, Jan C

    2008-01-01

    The 3x redundancy of the Genetic Code is usually explained as a necessity to increase the mutation-resistance of the genetic information. However recent bioinformatical observations indicate that the redundant Genetic Code contains more biological information than previously known and which is additional to the 64/20 definition of amino acids. It might define the physico-chemical and structural properties of amino acids, the codon boundaries, the amino acid co-locations (interactions) in the coded proteins and the free folding energy of mRNAs. This additional information, which seems to be necessary to determine the 3D structure of coding nucleic acids as well as the coded proteins, is known as the Proteomic Code and mRNA Assisted Protein Folding. PMID:19330085

  5. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    PubMed

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK.

  6. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK

    PubMed Central

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK. PMID:27471553

  7. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    PubMed

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK. PMID:27471553

  8. Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

    PubMed

    Spangler, Jacob B; Feltus, Frank Alex

    2013-01-01

    Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

  9. A "liaison dangereuse" between AUF1/hnRNPD and the oncogenic tyrosine kinase NPM-ALK.

    PubMed

    Fawal, Mohamad; Armstrong, Florence; Ollier, Severine; Dupont, Henri; Touriol, Christian; Monsarrat, Bernard; Delsol, Georges; Payrastre, Bernard; Morello, Dominique

    2006-10-15

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

  10. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  11. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

    PubMed

    van Beilen, J B; Panke, S; Lucchini, S; Franchini, A G; Röthlisberger, M; Witholt, B

    2001-06-01

    The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS: PMID:11390693

  12. The Signal Sequence Coding Region Promotes Nuclear Export of mRNA

    PubMed Central

    Palazzo, Alexander F; Springer, Michael; Shibata, Yoko; Lee, Chung-Sheng; Dias, Anusha P; Rapoport, Tom A

    2007-01-01

    In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs. PMID:18052610

  13. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

  14. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS.

    PubMed

    Zhang, Qian; Wang, Hongyi; Kantekure, Kanchan; Paterson, Jennifer C; Liu, Xiaobin; Schaffer, Andras; Paulos, Chrystal; Milone, Michael C; Odum, Niels; Turner, Suzanne; Marafioti, Teresa; Wasik, Mariusz A

    2011-09-15

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative enhancer region, of the ICOS gene, whereas cutaneous T-cell lymphoma cell lines, which strongly express ICOS, show no methylation of the island. Treatment of the ALK(+) TCL cell lines with DNA methyltransferase inhibitor reversed the CpG island methylation and augmented the expression of ICOS mRNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth promoting receptor. These data also show that the DNA methylation status of the intronic CpG island affects transcriptional activity of the ICOS gene and, consequently, modulates the concentration of the expressed ICOS protein.

  15. Sequence of the 3'-noncoding and adjacent coding regions of human gamma-globin mRNA.

    PubMed Central

    Poon, R; Kan, Y W; Boyer, H W

    1978-01-01

    In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present. Images PMID:318163

  16. On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23.

    PubMed

    Nicole, L M; Tanguay, R M

    1987-03-01

    The specificity of action of antisense RNA for one of Drosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translation in vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45 degrees C) and low stringency (37 degrees C) conditions. This suggests that the 5' leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

  17. Shear induced collateral artery growth modulated by endoglin but not by ALK1

    PubMed Central

    Seghers, Leonard; de Vries, Margreet R; Pardali, Evangelia; Hoefer, Imo E; Hierck, Beerend P; ten Dijke, Peter ten; Goumans, Marie Jose; Quax, Paul HA

    2012-01-01

    Transforming growth factor-beta (TGF-β) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-β receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis. PMID:22436015

  18. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

    PubMed

    Patrinou-Georgoulas, M; John, H A

    1977-10-01

    A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

  19. Precision medicine in NSCLC and pathology: how does ALK fit in the pathway?

    PubMed

    Kerr, K M; López-Ríos, F

    2016-09-01

    The evolution of personalised medicine in lung cancer has dramatically impacted diagnostic pathology. Current challenges centre on the growing demands placed on small tissue samples by molecular diagnostic techniques. In this review, expert recommendations are provided regarding successful identification of anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC). Steps to correctly process and conserve tumour tissue during diagnostic testing are essential to ensure tissue availability. For example, storing extra tissue sections ready for molecular diagnostic steps allows faster testing and preserves tissue. Fluorescence in situ hybridisation (FISH) is commonly used to detect ALK rearrangements, with most laboratories favouring screening by immunohistochemistry followed by a confirmatory FISH assay. Reverse transcription-polymerase chain reaction can also identify ALK fusion gene mRNA transcripts but can be limited by the quality of RNA and the risk that rare fusion variants may not be captured. Next-generation sequencing (NGS) technology has recently provided an alternative method for detecting ALK rearrangements. While current experience is limited, NGS is set to become the most efficient approach as an increasing number of genetic abnormalities is required to be tested. Upfront, reflex testing for ALK gene rearrangement should become routine as ALK tyrosine kinase inhibitor therapy moves into the first-line setting. Guidelines recommend that EGFR and ALK tests are carried out in parallel on all confirmed and potential adenocarcinomas, and this is more efficient in terms of tissue usage and testing turnaround time for both of these actionable gene alterations. The practice of sequential testing is not recommended. Identification of ALK rearrangements is now essential for the diagnosis of NSCLC, underpinned by the benefits of ALK inhibitors. As scientific understanding and diagnostic technology develops, ALK testing will continue to be an

  20. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1).

    PubMed

    Marzec, Michal; Zhang, Qian; Goradia, Ami; Raghunath, Puthiyaveettil N; Liu, Xiaobin; Paessler, Michele; Wang, Hong Yi; Wysocka, Maria; Cheng, Mangeng; Ruggeri, Bruce A; Wasik, Mariusz A

    2008-12-30

    The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells strongly express the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1), as determined on the mRNA and protein level. The CD274 expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as demonstrated by inhibition of the NPM/ALK function in ALK+TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting CD274 expression in IL-3-depleted BaF3 cells transfected with the wild-type NPM/ALK, but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. NPM/ALK induces CD274 expression by activating its key signal transmitter, transcription factor STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo, as shown in the gel electromobility shift and chromatin immunoprecipitation assays, and is required for the PD-L1 gene expression, as demonstrated by siRNA-mediated STAT3 depletion. These findings identify an additional cell-transforming property of NPM/ALK and describe a direct link between an oncoprotein and an immunosuppressive cell-surface protein. These results also provide an additional rationale to therapeutically target NPM/ALK and STAT3 in ALK+TCL. Finally, they suggest that future immunotherapeutic protocols for this type of lymphoma may need to include the inhibition of NPM/ALK and STAT3 to achieve optimal clinical efficacy.

  1. Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: Combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-05-26

    ABSTRACT-The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused whether on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRN abundance and non-random features in coding sequences (e.g. codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together...

  2. Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant.

    PubMed

    Bernstein, P L; Herrick, D J; Prokipcak, R D; Ross, J

    1992-04-01

    Polysome-associated c-myc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an approximately 75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the approximately 75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.

  3. IL-2R common gamma-chain is epigenetically silenced by nucleophosphin-anaplastic lymphoma kinase (NPM-ALK) and acts as a tumor suppressor by targeting NPM-ALK.

    PubMed

    Zhang, Qian; Wang, Hong Yi; Liu, Xiaobin; Bhutani, Gauri; Kantekure, Kanchan; Wasik, Mariusz

    2011-07-19

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by certain neural cells, becomes highly oncogenic, when aberrantly expressed in nonneural tissues as a fusion protein with nucleophosphin (NPM) and other partners. The reason why NPM-ALK succeeds in transforming specifically CD4(+) T lymphocytes remains unknown. The IL-2R common γ-chain (IL-2Rγ) is shared by receptors for several cytokines that play key roles in the maturation and growth of normal CD4(+) T lymphocytes and other immune cells. We show that IL-2Rγ expression is inhibited in T-cell lymphoma cells expressing NPM-ALK kinase as a result of DNA methylation of the IL-2Rγ gene promoter. IL-2Rγ promoter methylation is induced in malignant T cells by NPM-ALK. NPM-ALK acts through STAT3, a transcription factor that binds to the IL-2Rγ gene promoter and enhances binding of DNA methyltransferases (DNMTs) to the promoter. In addition, STAT3 suppresses expression of miR-21, which selectively inhibits DNMT1 mRNA expression. Reconstitution of IL-2Rγ expression leads to loss of the NPM-ALK protein and, consequently, apoptotic cell death of the lymphoma cells. These results demonstrate that the oncogenic tyrosine kinase NPM-ALK induces epigenetic silencing of the IL-2Rγ gene and that IL-2Rγ acts as a tumor suppressor by reciprocally inhibiting expression of NPM-ALK.

  4. Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

    PubMed Central

    Wroblewska, Zuzanna; Olejniczak, Mikolaj

    2016-01-01

    The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs. PMID:27154968

  5. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex

    SciTech Connect

    Zain, S.B.; Salim, M.; Chou, W.G.; Sajdel-Sulkowska, E.M.; Majocha, R.E.; Marotta, C.A.

    1988-02-01

    To gain insight into factors associated with the excessive accumulation of ..beta..-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, the authors cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)/sup +/ RNA from AD cortices was used for the preparation of lambdagt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the ..beta..-amyloid domain and corresponded to 75% of the coding region and approx. = 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, they conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

  6. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex.

    PubMed Central

    Zain, S B; Salim, M; Chou, W G; Sajdel-Sulkowska, E M; Majocha, R E; Marotta, C A

    1988-01-01

    To gain insight into factors associated with the excessive accumulation of beta-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, we cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)+ RNA from AD cortices was used for the preparation of lambda gt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the beta-amyloid domain and corresponded to 75% of the coding region and approximately equal to 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, we conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product. Images PMID:2893379

  7. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus

    PubMed Central

    1988-01-01

    Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains. PMID:2843548

  8. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

    NASA Technical Reports Server (NTRS)

    Shibata, K.; Abe, S.; Davies, E.

    2001-01-01

    Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

  9. The ALK gene, an attractive target for inhibitor development.

    PubMed

    Tartari, Carmen J; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2011-01-01

    Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase that belongs to the Insulin receptor subfamily involved as full length receptor in neural development. Even if the expression of ALK protein is down-regulated in the adults, the ALK full length is expressed in different types of tumors. Moreover, chromosomal rearrangements, involving the alk gene, can occur leading the formation of different ALK fusion proteins characterized by the kinase domain of ALK fused to several partners that determine cellular localization. Structural investigation and characterization of the ALK kinase domain in absence of its crystal structure constituted the basis of development of ALK small molecule inhibitors. Here, we described normal function of the ALK receptor and its role in tumors; formation of the constitutively activated ALK fusion proteins and we reported an update of developed small molecule inhibitors of the ALK kinase activity. PMID:21513493

  10. The role of the ALK receptor in cancer biology.

    PubMed

    Hallberg, B; Palmer, R H

    2016-09-01

    A vast array of oncogenic variants has been identified for anaplastic lymphoma kinase (ALK). Therefore, there is a need to better understand the role of ALK in cancer biology in order to optimise treatment strategies. This review summarises the latest research on the receptor tyrosine kinase ALK, and how this information can guide the management of patients with cancer that is ALK-positive. A variety of ALK gene alterations have been described across a range of tumour types, including point mutations, deletions and rearrangements. A wide variety of ALK fusions, in which the kinase domain of ALK and the amino-terminal portion of various protein partners are fused, occur in cancer, with echinoderm microtubule-associated protein-like 4 (EML4)-ALK being the most prevalent in non-small-cell lung cancer (NSCLC). Different ALK fusion proteins can mediate different signalling outputs, depending on properties such as subcellular localisation and protein stability. The ALK fusions found in tumours lack spatial and temporal regulation, which can also affect dimerisation and substrate specificity. Two ALK tyrosine kinase inhibitors (TKIs), crizotinib and ceritinib, are currently approved in Europe for use in ALK-positive NSCLC and several others are in development. These ALK TKIs bind slightly differently within the ATP-binding pocket of the ALK kinase domain and are associated with the emergence of different resistance mutation patterns during therapy. This emphasises the need to tailor the sequence of ALK TKIs according to the ALK signature of each patient. Research into the oncogenic functions of ALK, and fast paced development of ALK inhibitors, has substantially improved outcomes for patients with ALK-positive NSCLC. Limited data are available surrounding the physiological ligand-stimulated activation of ALK signalling and further research is needed. Understanding the role of ALK in tumour biology is key to further optimising therapeutic strategies for ALK

  11. Evidence Suggesting That Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors.

    PubMed

    Amin, Amit Dipak; Rajan, Soumya S; Liang, Winnie S; Pongtornpipat, Praechompoo; Groysman, Matthew J; Tapia, Edgar O; Peters, Tara L; Cuyugan, Lori; Adkins, Jonathan; Rimsza, Lisa M; Lussier, Yves A; Puvvada, Soham D; Schatz, Jonathan H

    2015-07-15

    The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of certain cancers, including 2% to 7% of non-small cell lung cancers (NSCLC) and ∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK(+) NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications. PMID:26018086

  12. Evidence Suggesting that Discontinuous Dosing of ALK Kinase Inhibitors May Prolong Control of ALK+ Tumors

    PubMed Central

    Amin, Amit Dipak; Rajan, Soumya S.; Liang, Winnie S.; Pongtornpipat, Praechompoo; Groysman, Matthew J.; Tapia, Edgar O.; Peters, Tara L.; Cuyugan, Lori; Adkins, Jonathan; Rimsza, Lisa M.; Lussier, Yves A.; Puvvada, Soham D.; Schatz, Jonathan H.

    2015-01-01

    The anaplastic lymphoma kinase ALK is chromosomally rearranged in a subset of certain cancers, including 2–7% non-small cell lung cancers (NSCLC) and ~70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors crizotinib and ceritinib are approved for relapsed ALK+ NSCLC, but acquired resistance to these drugs limits median progression-free survival on average to ~10 months. Kinase domain mutations are detectable in 25–37% of resistant NSCLC samples, with activation of bypass signaling pathways detected frequently with or without concurrent ALK mutations. Here we report that, in contrast to NSCLC cells, drug resistant ALCL cells show no evidence of bypassing ALK by activating alternate signaling pathways. Instead, drug resistance selected in this setting reflects upregulation of ALK itself. Notably, in the absence of crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested or killed cells, allowing a prolonged control of drug-resistant tumors in vivo with the administration of discontinuous rather than continuous regimens of drug dosing. Furthermore, even when drug resistance mutations were detected in the kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We confirmed these findings derived from human ALCL cells in murine pro-B cells that were transformed to cytokine independence by ectopic expression of an activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK activation functions as a double-edged sword for tumor cell viability, with potential therapeutic implications. PMID:26018086

  13. Rationale for co-targeting IGF-1R and ALK in ALK fusion positive lung cancer

    PubMed Central

    Lovly, Christine M.; McDonald, Nerina T.; Chen, Heidi; Ortiz-Cuaran, Sandra; Heukamp, Lukas C.; Yan, Yingjun; Florin, Alexandra; Ozretić, Luka; Lim, Diana; Wang, Lu; Chen, Zhao; Chen, Xi; Lu, Pengcheng; Paik, Paul K.; Shen, Ronglai; Jin, Hailing; Buettner, Reinhard; Ansén, Sascha; Perner, Sven; Brockmann, Michael; Bos, Marc; Wolf, Jürgen; Gardizi, Masyar; Wright, Gavin M.; Solomon, Benjamin; Russell, Prudence A.; Rogers, Toni-Maree; Suehara, Yoshiyuki; Red-Brewer, Monica; Tieu, Rudy; de Stanchina, Elisa; Wang, Qingguo; Zhao, Zhongming; Johnson, David H.; Horn, Leora; Wong, Kwok-Kin; Thomas, Roman K.; Ladanyi, Marc; Pao, William

    2014-01-01

    The ALK tyrosine kinase inhibitor (TKI), crizotinib, shows significant activity in patients whose lung cancers harbor ALK fusions but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the intriguing clinical observation of a patient with ALK fusion+ lung cancer who had an ‘exceptional response’ to an IGF-1R antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor, IRS-1, and IRS-1 knockdown enhances the anti-tumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK/IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, IGF-1R/IRS-1 levels are increased in biopsy samples from patients progressing on crizotinib therapy. Collectively, these data support a role for the IGF-1R/IRS-1 pathway in both ALK TKI-sensitive and TKI-resistant states and provide biological rationale for further clinical development of dual ALK/IGF-1R inhibitors. PMID:25173427

  14. Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia.

    PubMed

    Giuriato, Sylvie; Foisseau, Marianne; Dejean, Emilie; Felsher, Dean W; Al Saati, Talal; Demur, Cécile; Ragab, Ashraf; Kruczynski, Anna; Schiff, Claudine; Delsol, Georges; Meggetto, Fabienne

    2010-05-20

    NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) and TPM3-ALK (nonmuscular tropomyosin 3-anaplastic lymphoma kinase) are oncogenic tyrosine kinases implicated in the pathogenesis of human ALK-positive lymphoma. We report here the development of novel conditional mouse models for ALK-induced lymphomagenesis, with the use of the tetracycline regulatory system under the control of the EmuSRalpha enhancer/promoter. The expression of either oncogene resulted in the arrest of the differentiation of early B cells and lymphomagenesis. We also observed the development of skin keratoacanthoma lesions, probably because of aberrant ALK expression in keratinocytes. The inactivation of the ALK oncogene on doxycycline treatment was sufficient to induce sustained regression of both hematopoietic tumors and skin disease. Importantly, treatment with the specific ALK inhibitor (PF-2341066) also reversed the pathologic states, showing the value of these mouse models for the validation of ALK tyrosine kinase inhibitors. Thus, our results show (1) that NPM-ALK and TPM3-ALK oncogenes are sufficient for lymphoma/leukemia development and required for tumor maintenance, hence validating ALK as potentially effective therapeutic target; and (2) for the first time, in vivo, the equal tumorigenic potential of the NPM-ALK and TPM3-ALK oncogenic tyrosine kinases. Our models offer a new tool to investigate in vivo the molecular mechanisms associated with ALK-induced lymphoproliferative disorders.

  15. The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling.

    PubMed

    Staber, Philipp B; Vesely, Paul; Haq, Naznin; Ott, Rene G; Funato, Kotaro; Bambach, Isabella; Fuchs, Claudia; Schauer, Silvia; Linkesch, Werner; Hrzenjak, Andelko; Dirks, Wilhelm G; Sexl, Veronika; Bergler, Helmut; Kadin, Marshall E; Sternberg, David W; Kenner, Lukas; Hoefler, Gerald

    2007-11-01

    Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK-positive than in NPM-ALK-negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK-expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK-positive and -negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK-negative ALCL, the mTOR pathway is active in NPM-ALK-positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK-positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK-positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target.

  16. Identification of protein-coding sequences using the hybridization of 18S rRNA and mRNA during translation.

    PubMed

    Xing, Chuanhua; Bitzer, Donald L; Alexander, Winser E; Vouk, Mladen A; Stomp, Anne-Marie

    2009-02-01

    We introduce a new approach in this article to distinguish protein-coding sequences from non-coding sequences utilizing a period-3, free energy signal that arises from the interactions of the 3'-terminal nucleotides of the 18S rRNA with mRNA. We extracted the special features of the amplitude and the phase of the period-3 signal in protein-coding regions, which is not found in non-coding regions, and used them to distinguish protein-coding sequences from non-coding sequences. We tested on all the experimental genes from Saccharomyces cerevisiae and Schizosaccharomyces pombe. The identification was consistent with the corresponding information from GenBank, and produced better performance compared to existing methods that use a period-3 signal. The primary tests on some fly, mouse and human genes suggests that our method is applicable to higher eukaryotic genes. The tests on pseudogenes indicated that most pseudogenes have no period-3 signal. Some exploration of the 3'-tail of 18S rRNA and pattern analysis of protein-coding sequences supported further our assumption that the 3'-tail of 18S rRNA has a role of synchronization throughout translation elongation process. This, in turn, can be utilized for the identification of protein-coding sequences.

  17. Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions.

    PubMed

    Chen, Jin; Coakley, Arthur; O'Connor, Michelle; Petrov, Alexey; O'Leary, Seán E; Atkins, John F; Puglisi, Joseph D

    2015-11-19

    Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements. PMID:26590426

  18. CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

    PubMed Central

    Voeller, Donna; Gower, Arjan; Kim, In-Kyu; Zhang, Yu-Wen; Giaccone, Giuseppe

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients. PMID:27078848

  19. The absence of the long 3'-non-translated region in mRNA coding for eye lens alpha A2-crystallin of the frog (Rana temporaria).

    PubMed

    Tomarev, S I; Zinovieva, R D; Dolgilevich, S M; Krayev, A S; Skryabin, K G; Gause, G G

    1983-10-01

    The nucleotide sequence of a cloned cDNA (clone pRt(1)297; GENE (1982) 17, 131) coding for a 18 kDa polypeptide of the frog eye lens has been determined. The sequence, 791 nucleotide in length has only one long open reading frame (447 nucleotides). The derived amino acid sequence in this frame has greater than 90% homology with the region 25-173 of alpha A2-crystallin amino acid sequence from a related frog species Rana pipiens. The 5'-terminal part of mRNA corresponding to the first 24 amino acids of alpha A2-crystallin has been lost in cloning and substituted by an artefactual sequence. The 3'-terminal part appears to be intact as follows from the presence of the universal poly(A) addition site and poly(A) tract. The 3'-nontranslated region present in frog alpha A2-crystallin mRNA (130 nucleotides) is about 4-times shorter than in mammalian alpha A2-crystallin mRNA. Intact alpha A2-crystallin mRNA with a size of about 700 nucleotides as determined by Northern blot hybridization is about twice smaller than corresponding mammalian mRNAs.

  20. [Fragment of mRNA coding part that is complementary to region 1638-1650 of wheat 18S rRNA functions as a translational enhancer].

    PubMed

    Zhigaĭlov, A V; Babaĭlova, E S; Polimbetova, N S; Graĭfer, D M; Karpova, G G; Iskakov, B K

    2012-01-01

    Possible involvement of 18S rRNA fragment 1638-1650 including basements of the helices h44 and h28 and nucleotides of the ribosomal decoding site in the cap-independent translation initiation on plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions within the 40S ribosomal subunit. It is found that the sequence complementary to the 18S rRNA fragment 1638-1650 is able to enhance efficiency of a reporter mRNA translation when placed just after the initiation codon. The results obtained indicate that in the course of the cap-independent translation initiation, complementary interactions can occur between mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site.

  1. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors.

    PubMed

    Ceccon, Monica; Mologni, Luca; Bisson, William; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2013-02-01

    The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

  2. A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1

    PubMed Central

    Wright, Jane E; Gaidatzis, Dimos; Senften, Mathias; Farley, Brian M; Westhof, Eric; Ryder, Sean P; Ciosk, Rafal

    2011-01-01

    RNA-binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP-mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD-1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD-1 interactions with the transcriptome, we identified GLD-1-associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD-1 association with mRNA is determined by the strength and number of 7-mer GLD-1-binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD-1/GBM-binding experiments to determine relative affinity, and in vivo, by ‘transplantation' experiments, where ‘weak' and ‘strong' GBMs imposed translational repression of increasing strength on a non-target mRNA. This study demonstrates that transcriptome-wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post-transcriptional regulatory networks. PMID:21169991

  3. Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance.

    PubMed Central

    Kaasen, I; Evensen, G; Seeberg, E

    1986-01-01

    We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair. Images PMID:3536857

  4. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein.

    PubMed

    Vishwamitra, Deeksha; Curry, Choladda V; Shi, Ping; Alkan, Serhan; Amin, Hesham M

    2015-09-01

    Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  5. Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

    PubMed

    Schedl, P; Artavanis-Tsakonas, S; Steward, R; Gehring, W J; Mirault, M E; Goldschmidt-Clermont, M; Moran, L; Tissières, A

    1978-08-01

    The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes. PMID:99246

  6. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

    PubMed

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-03-01

    Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

  7. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

    PubMed Central

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-01-01

    Summary Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  8. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

    PubMed

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-03-01

    Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  9. Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.

    PubMed

    Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

    2015-07-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases.

  10. Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice

    PubMed Central

    Lopez-Delisle, Lucille; Pierre-Eugène, Cécile; Bloch-Gallego, Evelyne; Birling, Marie-Christine; Duband, Jean-Loup; Durand, Estelle; Bourgeois, Thomas; Matrot, Boris; Sorg, Tania; Huerre, Michel; Meziane, Hamid; Roux, Michel J.; Champy, Marie-France; Gallego, Jorge; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2014-01-01

    The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations. PMID:24811761

  11. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

    PubMed

    Guan, J; Tucker, E R; Wan, H; Chand, D; Danielson, L S; Ruuth, K; El Wakil, A; Witek, B; Jamin, Y; Umapathy, G; Robinson, S P; Johnson, T W; Smeal, T; Martinsson, T; Chesler, L; Palmer, R H; Hallberg, B

    2016-09-01

    The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. PMID:27483357

  12. Silibinin suppresses NPM-ALK, potently induces apoptosis and enhances chemosensitivity in ALK-positive anaplastic large cell lymphoma.

    PubMed

    Molavi, Ommoleila; Samadi, Nasser; Wu, Chengsheng; Lavasanifar, Afsaneh; Lai, Raymond

    2016-05-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion protein carrying constitutively active tyrosine kinase, is known to be central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK+ALCL). Here, it is reported that silibinin, a non-toxic naturally-occurring compound, potently suppressed NPM-ALK and effectively inhibited the growth and soft agar colony formation of ALK+ALCL cells. By western blots, it was found that silibinin efficiently suppressed the phosphorylation/activation of NPM-ALK and its key substrates/downstream mediators (including STAT3, MEK/ERK and Akt) in a time- and dose-dependent manner. Correlating with these observations, silibinin suppressed the expression of Bcl-2, survivin and JunB, all of which are found to be upregulated by NPM-ALK and pathogenetically important in ALK+ALCL. Lastly, silibinin augmented the chemosensitivity of ALK+ALCL cells to doxorubicin, particularly the small cell sub-set expressing the transcriptional activity of Sox2, an embryonic stem cell marker. To conclude, the findings suggest that silibinin might be useful in treating ALK+ALCL.

  13. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

    PubMed Central

    Guan, J.; Tucker, E. R.; Wan, H.; Chand, D.; Danielson, L. S.; Ruuth, K.; El Wakil, A.; Witek, B.; Jamin, Y.; Umapathy, G.; Robinson, S. P.; Johnson, T. W.; Smeal, T.; Martinsson, T.; Chesler, L.; Palmer, R. H.

    2016-01-01

    ABSTRACT The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. PMID:27483357

  14. Analyses of Long Non-Coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium.

    PubMed

    Wang, Yueying; Xue, Songyi; Liu, Xiaoran; Liu, Huan; Hu, Tao; Qiu, Xiaotian; Zhang, Jinlong; Lei, Minggang

    2016-01-01

    Establishment of implantation in pig is accompanied by a coordinated interaction between the maternal uterine endometrium and conceptus development. We investigated the expression profiles of endometrial tissue on Days 9, 12 and 15 of pregnancy and on Day 12 of non-pregnancy in Yorkshire, and performed a comprehensive analysis of long non-coding RNAs (lncRNAs) in endometrial tissue samples by using RNA sequencing. As a result, 2805 novel lncRNAs, 2,376 (301 lncRNA and 2075 mRNA) differentially expressed genes (DEGs) and 2149 novel transcripts were obtained by pairwise comparison. In agreement with previous reports, lncRNAs shared similar characteristics, such as shorter in length, lower in exon number, lower at expression level and less conserved than protein coding transcripts. Bioinformatics analysis showed that DEGs were involved in protein binding, cellular process, immune system process and enriched in focal adhesion, Jak-STAT, FoxO and MAPK signaling pathway. We also found that lncRNAs TCONS_01729386 and TCONS_01325501 may play a vital role in embryo pre-implantation. Furthermore, the expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy). Our results first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases. PMID:26822553

  15. How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes.

    PubMed

    Doamekpor, Selom K; Sanchez, Ana M; Schwer, Beate; Shuman, Stewart; Lima, Christopher D

    2014-06-15

    Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II (Pol2) and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase•Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37°C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). We propose that the state of Thr1 phosphorylation comprises a binary "Spt5 CTD code" that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD. PMID:24939935

  16. A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase

    PubMed Central

    1992-01-01

    Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid- specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms. PMID:1577871

  17. A novel acquired ALK F1245C mutation confers resistance to crizotinib in ALK-positive NSCLC but is sensitive to ceritinib.

    PubMed

    Kodityal, Sandeep; Elvin, Julia A; Squillace, Rachel; Agarwal, Nikita; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J; Ou, Sai-Hong Ignatius

    2016-02-01

    The emergence of acquired anaplastic lymphoma kinase (ALK) resistant mutations is a common molecular mechanism underpinning disease progression during crizotinib treatment of ALK-positive (ALK+) non-small cell lung cancer (NSCLC) patients. Identifying acquired resistance mutations in ALK is paramount for tailoring future therapy with second generation ALK inhibitors and beyond. Comprehensive genomic profiling using hybrid-capture next generation sequencing has been successful in identifying acquired ALK resistance mutations. Here we described the emergence of an ALK F1245C mutation in an advanced ALK+ NSCLC patient (EML4-ALK variant 3a/b) who developed slow disease progression after a durable response to crizotinib. The patient was eventually switched to ceritinib with on-going clinical response. This is the first patient report that ALK F1245C is an acquired resistance mutation to crizotinib that can be overcome by ceritinib. PMID:26775591

  18. Prognostic significance of NPM-ALK fusion transcript overexpression in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Li, Chunmei; Takino, Hisashi; Eimoto, Tadaaki; Ishida, Takashi; Inagaki, Atsushi; Ueda, Ryuzo; Suzuki, Ritsuro; Yoshino, Tadashi; Nakagawa, Atsuko; Nakamura, Shigeo; Inagaki, Hiroshi

    2007-06-01

    In anaplastic large-cell lymphomas positive for anaplastic lymphoma kinase (ALK) protein, the ALK gene is most commonly fused to the NPM gene, and less commonly to TPM3, TFG, ATIC, and other rare genes. Although this lymphoma is generally associated with a favorable clinical outcome, 25% of the patients die of the disease within 5 years. In this study, we developed three assays, all of which can be used with archival formalin-fixed, paraffin-embedded tissues: (1) a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay for various X-ALK fusion genes, (2) a 5' rapid amplification of cDNA ends (RACE) assay to identify unknown fusion partners, and (3) a real-time RT-PCR assay to quantify the amount of the NPM-ALK fusion transcript. In 26 cases of ALK(+) anaplastic large-cell lymphoma, the RT-PCR assay showed that the ALK was fused to NPM in 21 cases, to TPM3 in three, and to TFG in one. The 5' RACE assay detected ATIC-ALK fusion in the remaining case. The real-time quantitative RT-PCR assay showed that the NPM-ALK transcript was over expressed in four of 20 quantifiable cases. Patients with NPM-ALK overexpression showed a significantly unfavorable overall survival compared with those with a low expression of this transcript. The RT-PCR and 5' RACE assays developed here may be useful for identification of known and unknown gene partners fused to the ALK gene. Overexpression of the NPM-ALK fusion transcript may be associated with a poor prognosis of the patients with ALK(+) anaplastic large-cell lymphomas.

  19. Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins

    SciTech Connect

    Kang, Chung Hyo; Yun, Jeong In; Lee, Kwangho; Lee, Chong Ock; Lee, Heung Kyoung; Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho; Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un; Jeong, Hye Gwang; Kim, Hyoung Rae; Park, Chi Hoon

    2015-08-28

    Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151-L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15–20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. - Highlights: • We synthesized KRCA-0008 derivatives having trifluoromethyl instead of chloride. • KRCA-0080 shows superior activity against several ALK mutants to KRCA-0008. • Cellular assays show our ALK inhibitors suppress only EML4-ALK positive cells. • Our ALK inhibitors induce G1/S arrest to lead apoptosis in H3122 cells. • KRCA-0080 has superior in vivo efficacy to crizotinib and KRCA-0008 by 15–20%.

  20. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. PMID:26464158

  1. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations.

  2. Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection

    SciTech Connect

    Dirks, R.W.; Raap, A.K.; Van Minnen, J.; Vreugdenhil, E.; Smit, A.B.; Van der Ploeg, M.

    1989-01-01

    To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a patch-like pattern. Nuclear and axoplasmic staining for mRNA was also observed.

  3. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

    PubMed Central

    Fennewald, M; Benson, S; Oppici, M; Shapiro, J

    1979-01-01

    We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes. Images PMID:479111

  4. Targeting ALK in neuroblastoma--preclinical and clinical advancements.

    PubMed

    Carpenter, Erica L; Mossé, Yael P

    2012-07-01

    Despite improvements in cancer therapies in the past 50 years, neuroblastoma remains a devastating clinical problem and a leading cause of childhood cancer deaths. Advances in treatments for children with high-risk neuroblastoma have, until recently, involved addition of cytotoxic therapy to dose-intensive regimens. In this era of targeted therapies, substantial efforts have been made to identify optimal targets for different types of cancer. The discovery of hereditary and somatic activating mutations in the oncogene ALK has now placed neuroblastoma among other cancers, such as melanoma and non-small-cell lung cancer (NSCLC), which benefit from therapies with oncogene-specific small-molecule tyrosine kinase inhibitors. Crizotinib, a small-molecule inhibitor of ALK, has transformed the landscape for the treatment of NSCLC harbouring ALK translocations and has demonstrated activity in preclinical models of ALK-driven neuroblastomas. However, inhibition of mutated ALK is complex when compared with translocated ALK and remains a therapeutic challenge. This Review discusses the biology of ALK in the development of neuroblastoma, preclinical and clinical progress with the use of ALK inhibitors and immunotherapy, challenges associated with resistance to such therapies and the steps being taken to overcome some of these hurdles.

  5. NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26a-dependent mechanism.

    PubMed

    Zhu, Haifeng; Vishwamitra, Deeksha; Curry, Choladda V; Manshouri, Roxsan; Diao, Lixia; Khan, Aarish; Amin, Hesham M

    2013-05-01

    NPM-ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T-cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM-ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM-ALK(+) T-cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM-ALK(+) T-cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM-ALK(+) T-cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM-ALK(+) T-cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post-transcriptional regulation. Consistently, we found that miR-26a contains potential binding sites and interacts with the 3'-UTR of iNOS. In addition, miR-26a was significantly decreased in NPM-ALK(+) T-cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR-26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR-26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR-26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM-ALK tyrosine kinase activity, suppresses MIR26A1 gene expression.

  6. Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition.

    PubMed

    Reiff, Tobias; Huber, Leslie; Kramer, Marco; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Rohrer, Hermann

    2011-11-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression. PMID:21989914

  7. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  8. Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity.

    PubMed

    Donella-Deana, Arianna; Marin, Oriano; Cesaro, Luca; Gunby, Rosalind H; Ferrarese, Anna; Coluccia, Addolorata M L; Tartari, Carmen J; Mologni, Luca; Scapozza, Leonardo; Gambacorti-Passerini, Carlo; Pinna, Lorenzo A

    2005-06-14

    The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases. PMID:15938644

  9. Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma.

    PubMed

    Armanious, Hanan; Gelebart, Pascal; Anand, Mona; Lai, Raymond

    2013-02-01

    It was previously reported that β-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), and the oncogenic effects of β-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK(+)ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of β-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK(+)ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK(+)ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the β-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.

  10. PF-06463922, an ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models

    PubMed Central

    Zou, Helen Y.; Friboulet, Luc; Kodack, David P.; Engstrom, Lars D.; Li, Qiuhua; West, Melissa; Tang, Ruth W.; Wang, Hui; Tsaparikos, Konstantinos; Wang, Jinwei; Timofeevski, Sergei; Katayama, Ryohei; Dinh, Dac M.; Lam, Hieu; Lam, Justine L.; Yamazaki, Shinji; Hu, Wenyue; Patel, Bhushankumar; Bezwada, Divya; Frias, Rosa L.; Lifshits, Eugene; Mahmood, Sidra; Gainor, Justin F.; Affolter, Timothy; Lappin, Patrick B.; Gukasyan, Hovhannes; Lee, Nathan; Deng, Shibing; Jain, Rakesh K; Johnson, Ted W.; Shaw, Alice T.; Fantin, Valeria R.; Smeal, Tod

    2015-01-01

    SUMMARY We report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor. Compared to other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to secondary ALK kinase domain mutations and/or due to the failed control of brain metastases. PMID:26144315

  11. The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways.

    PubMed

    Lim, Megan S; Carlson, Mary L; Crockett, David K; Fillmore, G Chris; Abbott, David R; Elenitoba-Johnson, Olaotan F; Tripp, Sheryl R; Rassidakis, George Z; Medeiros, L Jeffrey; Szankasi, Philippe; Elenitoba-Johnson, Kojo S J

    2009-08-20

    Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

  12. Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.

    PubMed

    Culbert, Andria L; Chakkalakal, Salin A; Theosmy, Edwin G; Brennan, Tracy A; Kaplan, Frederick S; Shore, Eileen M

    2014-05-01

    Bone morphogenetic protein (BMP) signaling is a critical regulator of cartilage differentiation and endochondral ossification. Gain-of-function mutations in ALK2, a type I BMP receptor, cause the debilitating disorder fibrodysplasia ossificans progressiva (FOP) and result in progressive heterotopic (extraskeletal) endochondral ossification within soft connective tissues. Here, we used murine mesenchymal progenitor cells to investigate the contribution of Alk2 during chondrogenic differentiation and heterotopic endochondral ossification (HEO). Alk2(R206H/+) (gain-of-function), Alk2(CKO) (loss-of-function), and wild-type mouse embryonic fibroblasts were evaluated for chondrogenic potential. Chondrogenic differentiation was accelerated in Alk2(R206H/+) cells, due in part to enhanced sensitivity to BMP ligand. In vivo, Alk2(R206H/+) cells initiated robust HEO and recruited wild-type cell contribution. Despite expression of other type I BMP receptors (Alk3 and Alk6), chondrogenesis of Alk2(CKO) cells was severely impaired by absence of Alk2 during early differentiation. Alk2 is therefore a direct regulator of cartilage formation and mediates chondrogenic commitment of progenitor cells. These data establish that at least one effect of ALK2 gain-of-function mutations in FOP patients is enhanced chondrogenic differentiation which supports formation of heterotopic endochondral bone. This establishes ALK2 as a plausible therapeutic target during early chondrogenic stages of lesion formation for preventing heterotopic bone formation in FOP and other conditions.

  13. New treatment options for ALK+ advanced non-small-cell lung cancer: critical appraisal of ceritinib

    PubMed Central

    Rothschild, Sacha I

    2016-01-01

    Rearrangements in ALK gene and EML4 gene were first described in 2007. This genomic aberration is found in about 2%–8% of non-small-cell lung cancer (NSCLC) patients. Crizotinib was the first ALK tyrosine kinase inhibitor licensed for the treatment of metastatic ALK-positive NSCLC based on a randomized Phase III trial. Despite the initial treatment response of crizotinib, disease progression inevitably develops after approximately 10 months of therapy. Different resistance mechanisms have recently been described. One relevant mechanism of resistance is the development of mutations in ALK. Novel ALK tyrosine kinase inhibitors have been developed to overcome these mutations. Ceritinib is an oral second-generation ALK inhibitor showing clinical activity not only in crizotinib-resistant ALK-positive NSCLC but also in treatment-naïve ALK-positive disease. In this paper, preclinical and clinical data of ceritinib are reviewed, and its role in the clinical setting is put into perspective. PMID:27217763

  14. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth.

    PubMed

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-09-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(-) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation-mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.

  15. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth

    PubMed Central

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-01-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies. PMID:26258416

  16. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    SciTech Connect

    Xu, Fei; Li, Hongling; Sun, Yong

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  17. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

    NASA Astrophysics Data System (ADS)

    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  18. Uterine ALK3 is essential during the window of implantation.

    PubMed

    Monsivais, Diana; Clementi, Caterina; Peng, Jia; Titus, Mary M; Barrish, James P; Creighton, Chad J; Lydon, John P; DeMayo, Francesco J; Matzuk, Martin M

    2016-01-19

    The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-β family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3(flox) (/flox)-Pgr-cre-positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor β (TGF-β) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity. PMID:26721398

  19. Timely topic: anaplastic lymphoma kinase (ALK) spreads its influence.

    PubMed

    Cheuk, W; Chan, J K

    2001-02-01

    Anaplastic lymphoma kinase (ALK) is normally not expressed in human tissues except selected sites in the nervous system. Its expression and constitutive activation as a result of a chromosomal translocation involving 2p23 plays a pivotal role in the genesis of anaplastic large cell lymphoma. ALK expression has been instrumental in defining a homogeneous subset from the category of anaplastic large cell lymphoma, characterised by occurrence in young patients, primary systemic presentation, favorable prognosis, a broad morphological spectrum, nuclear and/or cytoplasmic immunostaining for ALK protein, and a number of possible fusion partner genes such as NPM, ATIC, TFG, TPM3 and CLTCL. Recently ALK has been implicated in the genesis of another tumour type, the inflammatory myofibroblastic tumours. The ALK-positive examples occur in children and young adults, involving a variety of sites, such as the abdomen, mesentery, liver, bladder, mediastinum, lung and bone. The partner genes identified in some cases are TPM3 (tropomyosin 3) and TPM4 (tropomyosin 4). These molecular findings also further support the neoplastic nature of at least a subset of inflammatory myofibroblastic tumours.

  20. NPM-ALK and the JunB transcription factor regulate the expression of cytotoxic molecules in ALK-positive, anaplastic large cell lymphoma.

    PubMed

    Pearson, Joel D; Lee, Jason K H; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2011-01-30

    Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null immunophenotype that is most prevalent in children and young adults. The normal cellular counterpart of this malignancy is presumed to be the cytotoxic T lymphocyte (CTL), and this presumption is partly based on the observation that these tumour cells often express cytotoxic granules containing Granzyme B (GzB) and Perforin. Chromosomal translocations involving the gene encoding for the ALK tyrosine kinase are also characteristic of ALK+ ALCL, and the resulting fusion proteins (e.g. NPM-ALK) initiate signalling events important in ALK+ ALCL pathogenesis. These events include the elevated expression of JunB; an AP-1 family transcription factor that promotes ALK+ ALCL proliferation. In this report we demonstrate that JunB is a direct transcriptional activator of GzB and that GzB transcription is also promoted by NPM-ALK. We found that Perforin expression was not regulated by JunB, but was promoted by NPM-ALK in some cell lines and inhibited by it in others. In conclusion, our study makes the novel observation that signalling through NPM-ALK and JunB affect the expression of cytotoxic molecules in ALK+ ALCL. Moreover, these findings demonstrate the expression of GzB and Perforin in this lymphoma is not solely due its presumed CTL origin, but that oncogenic signalling is actively influencing the expression of these proteins.

  1. STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Wu, Chengsheng; Molavi, Ommoleila; Zhang, Haifeng; Gupta, Nidhi; Alshareef, Abdulraheem; Bone, Kathleen M; Gopal, Keshav; Wu, Fang; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Lai, Raymond

    2015-07-16

    The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

  2. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    PubMed

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  3. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alkylphenylpolyether-alk-a-nol-a-mines... Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical... as alkylphenylpolyether-alk-a-nol-a-mines (PMNs P-97-880/881/882) are subject to reporting under...

  4. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alkylphenylpolyether-alk-a-nol-a-mines... Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical... as alkylphenylpolyether-alk-a-nol-a-mines (PMNs P-97-880/881/882) are subject to reporting under...

  5. Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

    PubMed

    K Singh, V; Werner, S; Hackstein, H; Lennerz, V; Reiter, A; Wölfel, T; Damm-Welk, C; Woessmann, W

    2016-10-01

    Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

  6. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein1

    PubMed Central

    Vishwamitra, Deeksha; Curry, Choladda V.; Shi, Ping; Alkan, Serhan; Amin, Hesham M.

    2015-01-01

    Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm. PMID:26476082

  7. BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

    2013-12-01

    In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

  8. A patient previously treated with ALK inhibitors for central nervous system lesions from ALK rearranged lung cancer: a case report

    PubMed Central

    Kashima, Jumpei; Okuma, Yusuke; Hishima, Tsunekazu

    2016-01-01

    Background Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) are now preferentially treated with tyrosine kinase inhibitors (TKIs). However, patients treated with ALK inhibitors end up with acquired resistance. Case presentation We present a patient with recurrent ALK-rearranged NSCLC that developed multiple brain metastases and meningitis carcinomatosa after sequential treatment with several lines of cytotoxic chemotherapy, crizotinib, and alectinib. After the patient underwent retreatment with crizotinib as salvage therapy because of poor performance status, the intracranial metastatic foci and meningeal thickening were shrank within 1 week. Conclusion Our experience with this case suggests that alectinib may restore sensitivity to crizotinib or amplified pathway such as MET which bestowed alectinib resistance was inhibited with crizotinib. PMID:27785052

  9. Novel ALK inhibitor AZD3463 inhibits neuroblastoma growth by overcoming crizotinib resistance and inducing apoptosis

    PubMed Central

    Wang, Yongfeng; Wang, Long; Guan, Shan; Cao, Wenming; Wang, Hao; Chen, Zhenghu; Zhao, Yanling; Yu, Yang; Zhang, Huiyuan; Pang, Jonathan C.; Huang, Sophia L.; Akiyama, Yo; Yang, Yifan; Sun, Wenjing; Xu, Xin; Shi, Yan; Zhang, Hong; Kim, Eugene S.; Muscal, Jodi A.; Lu, Fengmin; Yang, Jianhua

    2016-01-01

    ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB. PMID:26786851

  10. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    PubMed Central

    Balkow, Aileen; Jagow, Johanna; Haas, Bodo; Siegel, Franziska; Kilić, Ana; Pfeifer, Alexander

    2015-01-01

    Objective Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor β (TGFβ) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. Methods Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. Results Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. Conclusions We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes. PMID:26266090

  11. Canadian consensus: inhibition of ALK-positive tumours in advanced non-small-cell lung cancer

    PubMed Central

    Melosky, B.; Agulnik, J.; Albadine, R.; Banerji, S.; Bebb, D.G.; Bethune, D.; Blais, N.; Butts, C.; Cheema, P.; Cheung, P.; Cohen, V.; Deschenes, J.; Ionescu, D.N.; Juergens, R.; Kamel-Reid, S.; Laurie, S.A.; Liu, G.; Morzycki, W.; Tsao, M.S.; Xu, Z.; Hirsh, V.

    2016-01-01

    Anaplastic lymphoma kinase (alk) is an oncogenic driver in non-small-cell lung cancer (nsclc). Chromosomal rearrangements involving the ALK gene occur in up to 4% of nonsquamous nsclc patients and lead to constitutive activation of the alk signalling pathway. ALK-positive nsclc is found in relatively young patients, with a median age of 50 years. Patients frequently have brain metastasis. Targeted inhibition of the alk pathway prolongs progression-free survival in patients with ALK-positive advanced nsclc. The results of several recent clinical trials confirm the efficacy and safety benefit of crizotinib and ceritinib in this population. Canadian oncologists support the following consensus statement: All patients with advanced nonsquamous nsclc (excluding pure neuroendocrine carcinoma) should be tested for the presence of an ALK rearrangement. If an ALK rearrangement is present, treatment with a targeted alk inhibitor in the first-line setting is recommended. As patients become resistant to first-generation alk inhibitors, other treatments, including second-generation alk inhibitors can be considered. PMID:27330348

  12. ALK-rearrangements and testing methods in non-small cell lung cancer: a review

    PubMed Central

    Shackelford, Rodney E.; Vora, Moiz; Mayhall, Kim; Cotelingam, James

    2014-01-01

    The anaplastic lymphoma tyrosine kinase (ALK) gene was first described as a driver mutation in anaplastic non-Hodgkin's lymphoma. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies, including 4-9% of non-small cell lung cancers (NSCLC). The tyrosine kinase inhibitor crizotinib is more effective than standard chemotherapeutic agents in treating ALK positive NSCLC, making molecular diagnostic testing for dysregulated ALK expression a necessary step in identifying optimal treatment modalities. Here we review ALKmediated signal transduction pathways and compare the molecular protocols used to identify dysregulated ALK expression in NSCLC. We also discuss the use of crizotinib and second generation ALK tyrosine kinase inhibitors in the treatment of ALK positive NSCLC, and the known mechanisms of crizotinib resistance in NSCLC. PMID:24955213

  13. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells.

    PubMed

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S; Young, Leah C; Kwak, Larry W; Ford, Richard J; Amin, Hesham M

    2009-07-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

  14. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells

    PubMed Central

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S.; Young, Leah C.; Kwak, Larry W.; Ford, Richard J.

    2009-01-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK+ ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK+ ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK+ ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK+ ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK+ ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK+ ALCL and possibly other types of malignant lymphoma. PMID:19423729

  15. Identification of a novel HIP1-ALK fusion variant in Non-Small-Cell Lung Cancer (NSCLC) and discovery of ALK I1171 (I1171N/S) mutations in two ALK-rearranged NSCLC patients with resistance to Alectinib.

    PubMed

    Ou, Sai-Hong Ignatius; Klempner, Samuel J; Greenbowe, Joel R; Azada, Michele; Schrock, Alexa B; Ali, Siraj M; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A

    2014-12-01

    Huntingtin-interacting protein 1 (HIP1) has recently been identified as a new fusion partner fused to anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC). To date, two variants of HIP1-ALK (H21; A20) and (H28; A20) have been identified in NSCLC. However, the response of patients with NSCLC harboring HIP1-ALK to ALK inhibitors and potential resistance mechanisms to such remain unknown. Here, we report a patient with NSCLC harboring a novel HIP1-ALK fusion variant (H30; A20). This patient and another patient with EML4-ALK variant 3a/b initially responded sequentially to crizotinib and then alectinib, a next-generation ALK inhibitor, but developed acquired resistance to alectinib with the presence of a mutation in amino acid residue 1171 (I1171N and I1171S respectively) located in the hydrophobic regulatory spine (R-spine) of the ALK kinase in both the cases as identified by a comprehensive next-generation sequencing-based assay performed on biopsies of new liver metastases that developed during alectinib treatment.

  16. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.

    PubMed

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  17. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers

    PubMed Central

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  18. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    PubMed

    Lovisa, Federica; Cozza, Giorgio; Cristiani, Andrea; Cuzzolin, Alberto; Albiero, Alessandro; Mussolin, Lara; Pillon, Marta; Moro, Stefano; Basso, Giuseppe; Rosolen, Angelo; Bonvini, Paolo

    2015-01-01

    ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  19. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.

    PubMed

    Zhu, Dongxing; Mackenzie, Neil Charles Wallace; Shanahan, Catherine M; Shroff, Rukshana C; Farquharson, Colin; MacRae, Vicky Elizabeth

    2015-01-01

    The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.

  20. Alk7 Depleted Mice Exhibit Prolonged Cardiac Repolarization and Are Predisposed to Ventricular Arrhythmia

    PubMed Central

    Ying, Shaozhen; Cao, Hong; Hu, He; Wang, Xin; Tang, Yanhong; Huang, Congxin

    2016-01-01

    We aimed to investigate the role of activin receptor-like kinase (ALK7) in regulating cardiac electrophysiology. Here, we showed that Alk7-/- mice exhibited prolonged QT intervals in telemetry ECG recordings. Furthermore, Langendorff-perfused Alk7-/- hearts had significantly longer action potential duration (APD) and greater incidence of ventricular arrhythmia (AV) induced by burst pacing. Using whole-cell patch clamp, we found that the densities of repolarizing K+ currents Ito and IK1 were profoundly reduced in Alk7-/- ventricular cardiomyocytes. Mechanistically, the expression of Kv4.2 (a major subunit of Ito carrying channel) and KCHIP2 (a key accessory subunit of Ito carrying channel), was markedly decreased in Alk7-/- hearts. These findings suggest that endogenous expression of ALK7 is necessary to maintain repolarizing K+ currents in ventricular cardiomyocytes, and finally prevent action potential prolongation and ventricular arrhythmia. PMID:26882027

  1. Targeting brain metastases in ALK-rearranged non-small-cell lung cancer.

    PubMed

    Zhang, Isabella; Zaorsky, Nicholas G; Palmer, Joshua D; Mehra, Ranee; Lu, Bo

    2015-10-01

    The incidence of brain metastases has increased as a result of improved systemic control and advances in imaging. However, development of novel therapeutics with CNS activity has not advanced at the same rate. Research on molecular markers has revealed many potential targets for antineoplastic agents, and a particularly important aberration is translocation in the ALK gene, identified in non-small-cell lung cancer (NSCLC). ALK inhibitors have shown systemic efficacy against ALK-rearranged NSCLC in many clinical trials, but the effectiveness of crizotinib in CNS disease is limited by poor blood-brain barrier penetration and acquired drug resistance. In this Review, we discuss potential pathways to target ALK-rearranged brain metastases, including next generation ALK inhibitors with greater CNS penetration and mechanisms to overcome resistance. Other important mechanisms to control CNS disease include targeting pathways downstream of ALK phosphorylation, increasing the permeability of the blood-brain barrier, modifying the tumour microenvironment, and adding concurrent radiotherapy.

  2. MiR-193a-5p Targets the Coding Region of AP-2α mRNA and Induces Cisplatin Resistance in Bladder Cancers

    PubMed Central

    Zhou, Ji; Duan, Huaxin; Xie, Yu; Ning, Yichong; Zhang, Xing; Hui, Na; Wang, Chunqing; Zhang, Jian; Zhou, Jianlin

    2016-01-01

    Transcription factor AP-2 alpha (AP-2α or TFAP2A) is a newly identified prognostic marker of chemotherapy; its expression is positively correlated with chemosensitivity and survival of cancer patients. Using computational programs, we predicted that the coding region of AP-2α gene contains a potential miRNA response element (MRE) of miR-193a-5p, and the single nucleotide polymorphism (SNP) site (c.497A>G, rs111681798) resides within the predicted MRE. The results of luciferase assays and Western blot analysis demonstrated that miR-193a-5p negatively regulated the expression of AP-2α proteins, but have no influence on the mutant AP-2α (c.497A>G). Infection with lentiviral AP-2α gene or miR-193a-5p inhibitor in the bladder cancer cells decreased migration and cisplatin resistance, while knockdown of AP-2α gene or overexpression of miR-193a-5p in the urothelial cell line SV-HUC-1 increased migration and cisplatin resistances. We concluded that miR-193a-5p induced cisplatin resistance by repressing AP-2α expression in bladder cancer cells. PMID:27698912

  3. MiR-193a-5p Targets the Coding Region of AP-2α mRNA and Induces Cisplatin Resistance in Bladder Cancers

    PubMed Central

    Zhou, Ji; Duan, Huaxin; Xie, Yu; Ning, Yichong; Zhang, Xing; Hui, Na; Wang, Chunqing; Zhang, Jian; Zhou, Jianlin

    2016-01-01

    Transcription factor AP-2 alpha (AP-2α or TFAP2A) is a newly identified prognostic marker of chemotherapy; its expression is positively correlated with chemosensitivity and survival of cancer patients. Using computational programs, we predicted that the coding region of AP-2α gene contains a potential miRNA response element (MRE) of miR-193a-5p, and the single nucleotide polymorphism (SNP) site (c.497A>G, rs111681798) resides within the predicted MRE. The results of luciferase assays and Western blot analysis demonstrated that miR-193a-5p negatively regulated the expression of AP-2α proteins, but have no influence on the mutant AP-2α (c.497A>G). Infection with lentiviral AP-2α gene or miR-193a-5p inhibitor in the bladder cancer cells decreased migration and cisplatin resistance, while knockdown of AP-2α gene or overexpression of miR-193a-5p in the urothelial cell line SV-HUC-1 increased migration and cisplatin resistances. We concluded that miR-193a-5p induced cisplatin resistance by repressing AP-2α expression in bladder cancer cells.

  4. Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

    PubMed

    Ceccon, M; Merlo, M E Boggio; Mologni, L; Poggio, T; Varesio, L M; Menotti, M; Bombelli, S; Rigolio, R; Manazza, A D; Di Giacomo, F; Ambrogio, C; Giudici, G; Casati, C; Mastini, C; Compagno, M; Turner, S D; Gambacorti-Passerini, C; Chiarle, R; Voena, C

    2016-07-21

    Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

  5. Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary?

    PubMed

    Toyokawa, Gouji; Seto, Takashi; Takenoyama, Mitsuhiro; Ichinose, Yukito

    2015-12-01

    Anaplastic lymphoma kinase (ALK) has been identified to exert a potent transforming activity through its rearrangement in non-small cell lung cancer (NSCLC), and patients (pts) with ALK rearrangement can be treated more successfully with ALK inhibitors, such as crizotinib, alectinib, and ceritinib, than with chemotherapy. Despite the excellent efficacy of ALK inhibitors, resistance to these drugs is inevitably encountered in most ALK-rearranged pts. Cases of resistance are subtyped into three groups, i.e., systemic, oligo, and central nervous system (CNS) types, with the CNS being used to be considered a sanctuary. With regard to the management of CNS lesions in pts with ALK+ NSCLC, a growing body of evidence has gradually demonstrated the intracranial (IC) efficacy of ALK inhibitor (ALKi) in ALK+ NSCLC pts with brain metastases (BMs). Although the efficacy of crizotinib for the CNS lesions remains controversial, a recent retrospective investigation of ALK+ pts with BM enrolled in PROFILE 1005 and PROFILE 1007 demonstrated that crizotinib is associated with a high disease control rate for BM. However, BM comprises the most common site of progressive disease in pts with or without baseline BMs, which is a serious problem for crizotinib. Furthermore, alectinib can be used to achieve strong and long-lasting inhibitory effects on BM. In addition to alectinib, the IC efficacy of other next-generation ALK inhibitors, such as ceritinib, AP26113 and PF-06463922, has been demonstrated. In this article, we review the latest evidence regarding the BM and IC efficacy of ALK inhibitors in pts with ALK+ NSCLC.

  6. Molecular characterization of coding sequences and analysis of Toll-like receptor 3 mRNA expression in water buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus).

    PubMed

    Dhara, Animesh; Saini, Mohini; Das, Dhanjit K; Swarup, Devendra; Sharma, Bhaskar; Kumar, Satish; Gupta, Praveen K

    2007-01-01

    Toll-like receptor 3 (TLR3), an antiviral innate immunity receptor recognizes double-stranded RNA, preferably of viral origin and induces type I interferon production, which causes maturation of phagocytes and subsequent release of chemical mediators from phagocytes against some viral infections. The present study has characterized TLR3 complementary DNA (cDNA) in buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus). TLR3 coding sequences of both buffalo and nilgai were amplified from cultured dendritic cell cDNA and cloned in pGEMT-easy vector for characterization by restriction endonucleases and nucleotide sequencing. Sequence analysis reveals that 2,715-bp-long TLR3 open reading frame encoding 904 amino acids in buffalo as well as nilgai is similar to that of cattle. Buffalo TLR3 has 98.6 and 97.9% identity at nucleotide level with nilgai and cattle, respectively. Likewise, buffalo TLR3 amino acids share 96.7% identity with cattle and 97.8% with nilgai. Non-synonymous substitutions exceeding synonymous substitutions indicate evolution of this receptor through positive selection among these three ruminant species. Buffalo and nilgai appear to have diverged from a common ancestor in phylogenetic analysis. Predicted protein structures of buffalo and nilgai TLR3 from deduced amino acid sequences indicate that the buffalo and nilgai TLR3 ectodomain may be more efficient in ligand binding than that of cattle. Furthermore, TLR3 messenger RNA expression in tissues as quantified by real-time PCR was found higher in nilgai than buffalo.

  7. NPM-ALK signals through glycogen synthase kinase 3β to promote oncogenesis.

    PubMed

    McDonnell, S R P; Hwang, S R; Basrur, V; Conlon, K P; Fermin, D; Wey, E; Murga-Zamalloa, C; Zeng, Z; Zu, Y; Elenitoba-Johnson, K S J; Lim, M S

    2012-08-01

    Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3β. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3β (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3β levels. Phosphorylation of pS(9)-GSK3β by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3β substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3β. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3β inhibition. Furthermore, stable knockdown of GSK3β conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3β and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3β and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3β by PI3K/AKT. The subsequent inhibition of

  8. Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer

    PubMed Central

    Amatu, Alessio; Somaschini, Alessio; Cerea, Giulio; Bosotti, Roberta; Valtorta, Emanuele; Buonandi, Pasquale; Marrapese, Giovanna; Veronese, Silvio; Luo, David; Hornby, Zachary; Multani, Pratik; Murphy, Danielle; Shoemaker, Robert; Lauricella, Calogero; Giannetta, Laura; Maiolani, Martina; Vanzulli, Angelo; Ardini, Elena; Galvani, Arturo; Isacchi, Antonella; Sartore-Bianchi, Andrea; Siena, Salvatore

    2015-01-01

    Background: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. Methods: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. Results: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. Conclusions: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC. PMID:26633560

  9. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

    PubMed

    Witek, Barbara; El Wakil, Abeer; Nord, Christoffer; Ahlgren, Ulf; Eriksson, Maria; Vernersson-Lindahl, Emma; Helland, Åslaug; Alexeyev, Oleg A; Hallberg, Bengt; Palmer, Ruth H

    2015-01-01

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

  10. New strategies in neuroblastoma: Therapeutic targeting of MYCN and ALK.

    PubMed

    Barone, Giuseppe; Anderson, John; Pearson, Andrew D J; Petrie, Kevin; Chesler, Louis

    2013-11-01

    Clinical outcome remains poor in patients with high-risk neuroblastoma, in which chemoresistant relapse is common following high-intensity conventional multimodal therapy. Novel treatment approaches are required. Although recent genomic profiling initiatives have not revealed a high frequency of mutations in any significant number of therapeutically targeted genes, two exceptions, amplification of the MYCN oncogene and somatically acquired tyrosine kinase domain point mutations in anaplastic lymphoma kinase (ALK), present exciting possibilities for targeted therapy. In contrast with the situation with ALK, in which a robust pipeline of pharmacologic agents is available from early clinical use in adult malignancy, therapeutic targeting of MYCN (and MYC oncoproteins in general) represents a significant medicinal chemistry challenge that has remained unsolved for two decades. We review the latest approaches envisioned for blockade of ALK activity in neuroblastoma, present a classification of potential approaches for therapeutic targeting of MYCN, and discuss how recent developments in targeting of MYC proteins seem to make therapeutic inhibition of MYCN a reality in the clinic.

  11. Lumican binds ALK5 to promote epithelium wound healing.

    PubMed

    Yamanaka, Osamu; Yuan, Yong; Coulson-Thomas, Vivien Jane; Gesteira, Tarsis Ferreira; Call, Mindy K; Zhang, Yujin; Zhang, Jianhua; Chang, Shao-Hsuan; Xie, Changchun; Liu, Chia-Yang; Saika, Shizuya; Jester, James V; Kao, Winston W-Y

    2013-01-01

    Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

  12. Detection of novel and potentially actionable anaplastic lymphoma kinase (ALK) rearrangement in colorectal adenocarcinoma by immunohistochemistry screening

    PubMed Central

    Wang, Kai; Kim, Sun Young; Jang, Jiryeon; Kim, Seung Tae; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Park, Young Suk; Lee, Jiyun; Lee, Woo Yong; Park, Yoon Ah; Huh, Jung Wook; Yun, Seong Hyeon; Do, In-Gu; Kim, Seok Hyung; Balasubramanian, Sohail; Stephens, Philip J.; Ross, Jeffrey S.; Li, Gang Gary; Hornby, Zachary; Ali, Siraj M.; Miller, Vincent A.; Kim, Kyoung-Mee; Ou, Sai-Hong Ignatius

    2015-01-01

    Purpose Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies. Experimental designs Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial. Results Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22–21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib. Conclusions ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors. PMID:26172300

  13. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice

    PubMed Central

    Pfeifer, Kathrin; Rivera, Victor M.; Guan, Jikui; Palmer, Ruth H.; Hallberg, Bengt

    2016-01-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments. PMID:27049722

  14. NPM/ALK mutants resistant to ASP3026 display variable sensitivity to alternative ALK inhibitors but succumb to the novel compound PF-06463922.

    PubMed

    Mologni, Luca; Ceccon, Monica; Pirola, Alessandra; Chiriano, Gianpaolo; Piazza, Rocco; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2015-03-20

    ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.

  15. Total Body Metabolic Tumor Response in ALK Positive Non-Small Cell Lung Cancer Patients Treated with ALK Inhibition

    PubMed Central

    Koole, Michel J. B.; Bongaerts, Alphons H. H.; Pruim, Jan; Groen, Harry J. M.

    2016-01-01

    Background In ALK-positive advanced NSCLC, crizotinib has a high response rate and effectively increases quality of life and survival. CT measurement of the tumor may insufficiently reflect the actual tumor load changes during targeted therapy with crizotinib. We explored whether 18F-FDG PET measured metabolic changes are different from CT based changes and studied the impact of these changes on disease progression. Methods 18F-FDG PET/CT was performed prior to and after 6 weeks of crizotinib treatment. Tumor response on CT was classified with RECIST 1.1, while 18F-FDG PET response was assessed according to the 1999 EORTC recommendations and PERCIST criteria. Agreement was assessed using McNemars test. During follow-up, patients received additional PET/CT during crizotinib treatment and second generation ALK inhibition. We assessed whether PET was able to detect progression earlier then CT. Results In this exploratory study 15 patients were analyzed who were treated with crizotinib. There was a good agreement in the applicability of CT and 18F-FDG PET/CT using the EORTC recommendations. During first line crizotinib and subsequent second line ALK inhibitors, PET was able to detect progression earlier then CT in 10/22 (45%) events of progression and in the others disease progression was detected simultaneously. Conclusion In advanced ALK positive NSCLC PET was able to detect progressive disease earlier than with CT in nearly half of the assessments while both imaging tests performed similar in the others. PMID:27137772

  16. Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

    PubMed

    Gonzales, Cara B; De La Chapa, Jorge J; Saikumar, Pothana; Singha, Prajjal K; Dybdal-Hargreaves, Nicholas F; Chavez, Jeffery; Horning, Aaron M; Parra, Jamie; Kirma, Nameer B

    2016-08-01

    Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation. PMID:27424178

  17. Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

    PubMed Central

    Facchinetti, Francesco; Di Maio, Massimo; Graziano, Paolo; Bria, Emilio; Rossi, Giulio; Novello, Silvia

    2016-01-01

    Crizotinib is an oral inhibitor of anaplastic lymphoma kinase (ALK) with remarkable clinical activity in patients suffering from ALK-rearranged non-small cell lung cancer (NSCLC), accounting to its superiority compared to chemotherapy. Unfortunately, virtually all ALK-rearranged tumors acquire resistance to crizotinib, frequently within one year since the treatment initiation. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and structurally different compounds. Second-generation ALK inhibitors such as ceritinib (LDK378), alectinib (CH5424802/RO5424802) and brigatinib (AP26113) have shown relevant clinical activity, consequently fostering their rapid clinical development and their approval by health agencies. The third-generation inhibitor lorlatinib (PF-06463922), selectively active against ALK and ROS1, harbors impressive biological potency; its efficacy in reversing resistance to crizotinib and to other ALK inhibitors is being proven by early clinical trials. The NTRK1-3 and ROS1 inhibitor entrectinib (RXDX-101) has been reported to act against NSCLC harboring ALK fusion proteins too. Despite the quick development of these novel agents, several issues remain to be discussed in the treatment of patients suffering from ALK-rearranged NSCLC. This position paper will discuss the development, the current evidence and approvals, as long as the future perspectives of new ALK inhibitors beyond crizotinib. Clinical behaviors of ALK-rearranged NSCLC vary significantly among patients and differential molecular events responsible of crizotinib resistance account for the most important quote of this heterogeneity. The precious availability of a wide range of active anti-ALK compounds should be approached in a critical and careful perspective, in order to develop treatment strategies tailored on the disease evolution of every single patient. PMID:27413712

  18. Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

    PubMed Central

    Wiesner, Thomas; Lee, William; Obenauf, Anna C.; Ran, Leili; Murali, Rajmohan; Zhang, Qi Fan; Wong, Elissa W. P.; Hu, Wenhuo; Scott, Sasinya N.; Shah, Ronak H.; Landa, Iñigo; Button, Julia; Lailler, Nathalie; Sboner, Andrea; Gao, Dong; Murphy, Devan A.; Cao, Zhen; Shukla, Shipra; Hollmann, Travis J.; Wang, Lu; Borsu, Laetitia; Merghoub, Taha; Schwartz, Gary K.; Postow, Michael A.; Ariyan, Charlotte E.; Fagin, James A.; Zheng, Deyou; Ladanyi, Marc; Busam, Klaus J.; Berger, Michael F.; Chen, Yu; Chi, Ping

    2016-01-01

    Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ~ 11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites1. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation. PMID:26444240

  19. RANBP2-ALK fusion combined with monosomy 7 in acute myelomonocytic leukemia.

    PubMed

    Lim, Ji-Hun; Jang, Seongsoo; Park, Chan-Jeoung; Cho, Young-Uk; Lee, Je-Hwan; Lee, Kyoo-Hyung; Lee, Jin-Ok; Shin, Jong-Yeon; Kim, Jong-Il; Huh, Jooryung; Seo, Eul-Ju

    2014-01-01

    Anaplastic lymphoma receptor tyrosine kinase (ALK) is located on chromosome 2p23; the chromosomal rearrangements of this gene are common genetic alterations, resulting in the creation of multiple fusion genes involved in tumorigenesis. However, the presence of an ALK fusion in myeloid malignancies is extremely rare. We report a case of acute myelomonocytic leukemia in a 31-year-old woman with an unusual rearrangement between RAN-binding protein 2 (RANBP2) and ALK and a karyotype of 45,XX,inv(2)(p23q21),-7[20]. We detected an ALK rearrangement using fluorescence in situ hybridization, identified the ALK fusion partner by using RNA transcriptome sequencing, and demonstrated the RANBP2-ALK fusion transcript by reverse transcriptase--PCR and Sanger sequencing. Immunohistochemistry for ALK showed strong staining of the nuclear membrane in leukemic cells. The patient had an unfavorable clinical course. Our results, together with a literature review, suggest the RANBP2-ALK fusion combined with monosomy 7 may be related to a unique clonal hematologic disorder of childhood and adolescence, characterized by myelomonocytic leukemia and a poor prognosis.

  20. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  1. Inhibition of Rac controls NPM-ALK-dependent lymphoma development and dissemination.

    PubMed

    Colomba, A; Giuriato, S; Dejean, E; Thornber, K; Delsol, G; Tronchère, H; Meggetto, F; Payrastre, B; Gaits-Iacovoni, F

    2011-06-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM-ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM-ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM-ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas.

  2. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    PubMed Central

    Herhaus, Lina; Al-Salihi, Mazin A.; Dingwell, Kevin S.; Cummins, Timothy D.; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C.; Sapkota, Gopal P.

    2014-01-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  3. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling.

    PubMed

    Herhaus, Lina; Al-Salihi, Mazin A; Dingwell, Kevin S; Cummins, Timothy D; Wasmus, Lize; Vogt, Janis; Ewan, Richard; Bruce, David; Macartney, Thomas; Weidlich, Simone; Smith, James C; Sapkota, Gopal P

    2014-05-01

    Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis. PMID:24850914

  4. A Functional Landscape of Resistance to ALK Inhibition in Lung Cancer

    PubMed Central

    Wilson, Frederick H.; Johannessen, Cory M.; Piccioni, Federica; Tamayo, Pablo; Kim, Jong Wook; Van Allen, Eliezer M.; Corsello, Steven M.; Capelletti, Marzia; Calles, Antonio; Butaney, Mohit; Sharifnia, Tanaz; Gabriel, Stacey B.; Mesirov, Jill P.; Hahn, William C.; Engelman, Jeffrey A.; Meyerson, Matthew; Root, David E.; Jänne, Pasi A.; Garraway, Levi A.

    2015-01-01

    Summary We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein kinase C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naïve controls, supporting a role for identified resistance mechanisms in clinical resistance. PMID:25759024

  5. VCL-ALK Renal Cell Carcinoma in Children With Sickle-cell Trait

    PubMed Central

    Smith, Nathaniel E.; Deyrup, Andrea T.; Marinño-Enriquez, Adrian; Fletcher, Jonathan A.; Bridge, Julia A.; Illei, Peter B.; Netto, George J.; Argani, Pedram

    2015-01-01

    We report the third case of a renal cell carcinoma bearing a fusion of the vinculin (VCL) and anaplastic lymphoma kinase (ALK) genes. Like the 2 other reported cases, this neoplasm occurred in a young patient (6 y old) with sickle-cell trait and demonstrated distinctive morphologic features including medullary epicenter, discohesive polygonal or spindle-shaped cells with prominent cytoplasmic vacuoles, and prominent lymphocytic infiltrate. The neoplastic cells demonstrated focal membranous labeling for ALK protein by immunohistochemistry, ALK gene rearrangement by fluorescence in situ hybridization, and a specific VCL-ALK gene fusion by reverse transcriptase polymerase chain reaction. VCL-ALK renal cell carcinoma may represent the eighth sickle-cell nephropathy. PMID:24698962

  6. Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

    PubMed

    Wen, Y; Bekhor, I

    1993-10-01

    A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

  7. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    PubMed

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

  8. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma

    PubMed Central

    Gunby, Rosalind H.; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W.; Tartari, Carmen J.; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G.; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-01-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK+ ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK+ cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells. PMID:17537995

  9. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    PubMed

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells. PMID:17537995

  10. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  11. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL

    PubMed Central

    Boccalatte, Francesco E.; Voena, Claudia; Riganti, Chiara; Bosia, Amalia; D'Amico, Lucia; Riera, Ludovica; Cheng, Mangeng; Ruggeri, Bruce; Jensen, Ole N.; Goss, Valerie L.; Lee, Kimberly; Nardone, Julie; Rush, John; Polakiewicz, Roberto D.; Comb, Michael J.; Chiarle, Roberto

    2009-01-01

    Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy. PMID:18845790

  12. Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.

    PubMed

    Conde, Esther; Hernandez, Susana; Prieto, Mario; Martinez, Rebeca; Lopez-Rios, Fernando

    2016-06-01

    The development of several ALK inhibitors means that the importance of accurately identifying ALK-positive lung cancer has never been greater. Therefore, it is crucial that ALK testing assays become more standardized. The aim of this review is to comment on the recently FDA-approved VENTANA ALK (D5F3) Companion Diagnostic (CDx) Assay. This kit provides high sensitivity and specificity for the detection of ALK rearrangements and seamless integration into the laboratory workflow, with a fully automated analytical phase and fast interpretation. The use of controls increases the sensitivity and specificity and a dichotomous scoring approach enhances reproducibility.

  13. Analysis of gene expression profile of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma.

    PubMed

    Bohling, Sandra D; Jenson, Stephen D; Crockett, David K; Schumacher, Jonathan A; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2008-03-01

    Anaplastic large cell lymphoma (ALCL) comprises a group of non-Hodgkin lymphomas characterized by the expression of the CD30/Ki-1 antigen. A subset of ALCL is characterized by chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2. While the most common translocation is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5, up to 12 other translocations partners of the ALK gene have been identified. One of these is the t(1;2)(q25;p23) which results in the formation of the chimeric protein TPM3-ALK. While several of the signaling pathways induced by NPM-ALK have been elucidated, those involved in ALCLs harboring TPM3-ALK are largely unknown. In order to investigate the expression profiles of ALCLs carrying the NPM-ALK and TPM3-ALK fusions, we carried out cDNA microarray analysis of two ALCL tissue samples, one expressing the NPM-ALK fusion protein and the other the TPM3-ALK fusion protein. RNA was extracted from snap-frozen tissues, labeled with fluorescent dyes and analyzed using cDNAs microarray containing approximately 9,200 genes and expressed sequence tags (ESTs). Quantitative fluorescence RT-PCR was performed to validate the cDNA microarray data on nine selected gene targets. Our results show a significant overlap of genes deregulated in the NPM-ALK and TPM-ALK positive lymphomas. These deregulated genes are involved in diverse cellular functions, such as cell cycle regulation, apoptosis, proliferation, and adhesion. Interestingly, a subset of the genes was distinct in their expression pattern in the two types of lymphomas. More importantly, many genes that were not previously associated with ALK positive lymphomas were identified. Our results demonstrate the overlapping and unique transcriptional patterns associated with the NPM-ALK and TPM3-ALK fusions in ALCL.

  14. Personalized treatment in advanced ALK-positive non-small cell lung cancer: from bench to clinical practice

    PubMed Central

    Passaro, Antonio; Lazzari, Chiara; Karachaliou, Niki; Spitaleri, Gianluca; Pochesci, Alessia; Catania, Chiara; Rosell, Rafael; de Marinis, Filippo

    2016-01-01

    The discovery of anaplastic lymphoma kinase (ALK) gene rearrangements and the development of tyrosine kinase inhibitors (TKI) that target them have achieved unprecedented success in the management of patients with ALK-positive non-small cell lung cancer (NSCLC). Despite the high efficacy of crizotinib, the first oral ALK TKI approved for the treatment of ALK-positive NSCLC, almost all patients inevitably develop acquired resistance, showing disease progression in the brain or in other parenchymal sites. Second- or third-generation ALK TKIs have shown to be active in crizotinib-pretreated or crizotinib-naïve ALK-positive patients, even in those with brain metastases. In this review, the current knowledge regarding ALK-positive NSCLC, focusing on the biology of the disease and the available therapeutic options are discussed. PMID:27799783

  15. A novel Patient Derived Tumorgraft model with TRAF1-ALK Anaplastic Large Cell Lymphoma translocation

    PubMed Central

    Abate, Francesco; Todaro, Maria; van der Krogt, Jo-Anne; Boi, Michela; Landra, Indira; Machiorlatti, Rodolfo; Tabbo’, Fabrizio; Messana, Katia; Barreca, Antonella; Novero, Domenico; Gaudiano, Marcello; Aliberti, Sabrina; Di Giacomo, Filomena; Tousseyn, Thomas; Lasorsa, Elena; Crescenzo, Ramona; Bessone, Luca; Ficarra, Elisa; Acquaviva, Andrea; Rinaldi, Andrea; Ponzoni, Maurilio; Longo, Dario Livio; Aime, Silvio; Cheng, Mangeng; Ruggeri, Bruce; Piccaluga, Pier Paolo; Pileri, Stefano; Tiacci, Enrico; Falini, Brunangelo; Pera-Gresely, Benet; Cerchietti, Leandro; Iqbal, Javeed; Chan, Wing C; Shultz, Leonard D.; Kwee, Ivo; Piva, Roberto; Wlodarska, Iwona; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio

    2016-01-01

    Although Anaplastic Large Cell Lymphomas (ALCL) carrying Anaplastic Lymphoma Kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human Patient Derived Tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and of NFkB pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells lacking PRDM1/Blimp-1 and with c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to down-regulation of p50/p52 and lymphoma growth inhibition. Moreover a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Moreover, a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, but the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable to validate the role of druggable molecules, predict therapeutic responses and are helpful tools for the implementation of patient specific therapies. PMID:25533804

  16. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    PubMed

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  17. De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation

    PubMed Central

    Chen, Wanqiu; Sun, Zhengda; Han, Zhenying; Jun, Kristine; Camus, Marine; Wankhede, Mamta; Mao, Lei; Arnold, Tom; Young, William L.; Su, Hua

    2014-01-01

    Background and Purpose In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor (VEGF) stimulation induces brain AVMs (bAVMs) in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal VEGF stimulation is sufficient to induce bAVM in the adult mouse. Methods Focal angiogenesis was induced in the brain of eight-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing VEGF (AAV-VEGF). Two weeks later, EC-Alk1 deletion was induced by tamoxifen (TM) treatment. Vascular morphology was analyzed, and EC proliferation and Dysplasia Index (number of vessels with diameter >15μm per 200 vessels) were quantified10 days after TM administration. Results Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of TM-treated Pdgfb-iCreER;Alk12f/2f mice. Induced bAVMs were marked by increased Dysplasia Index (P<0.001), and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. Conclusions Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo bAVM. PMID:24457293

  18. PDGFR blockade is a rational and effective therapy for NPM-ALK-driven lymphomas.

    PubMed

    Laimer, Daniela; Dolznig, Helmut; Kollmann, Karoline; Vesely, Paul W; Schlederer, Michaela; Merkel, Olaf; Schiefer, Ana-Iris; Hassler, Melanie R; Heider, Susi; Amenitsch, Lena; Thallinger, Christiane; Staber, Philipp B; Simonitsch-Klupp, Ingrid; Artaker, Matthias; Lagger, Sabine; Turner, Suzanne D; Pileri, Stefano; Piccaluga, Pier Paolo; Valent, Peter; Messana, Katia; Landra, Indira; Weichhart, Thomas; Knapp, Sylvia; Shehata, Medhat; Todaro, Maria; Sexl, Veronika; Höfler, Gerald; Piva, Roberto; Medico, Enzo; Ruggeri, Bruce A; Cheng, Mangeng; Eferl, Robert; Egger, Gerda; Penninger, Josef M; Jaeger, Ulrich; Moriggl, Richard; Inghirami, Giorgio; Kenner, Lukas

    2012-11-01

    Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin's lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK-triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK-triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK(+) ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.

  19. The pathobiology of the oncogenic tyrosine kinase NPM-ALK: a brief update.

    PubMed

    Lai, Raymond; Ingham, Robert J

    2013-04-01

    Extensive research has been carried out in the past two decades to study the pathobiology of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which is an oncogenic fusion protein found exclusively in a specific type of T-cell lymphoid malignancy, namely ALK-positive anaplastic large cell lymphoma. Results from these studies have provided highly useful insights into the mechanisms by which a constitutively tyrosine kinase, such as NPM-ALK, promotes tumorigenesis. Several previous publications have comprehensively summarized the advances in this field. In this review, we provide readers with a brief update on specific areas of NPM-ALK pathobiology. In the first part, the NPM-ALK/signal transducer and activator of transcription 3 (STAT3) signaling axis is discussed, with an emphasis on the existence of multiple biochemical defects that have been shown to amplify the oncogenic effects of this signaling axis. Specifically, findings regarding JAK3, SHP1 and the stimulatory effects of several cytokines including interleukin (IL)-9, IL-21 and IL-22 are summarized. New concepts stemming from recent observations regarding the functional interactions among the NPM-ALK/STAT3 axis, β catenin and glycogen synthase kinase 3β will be postulated. Lastly, new mechanisms by which the NPM-ALK/STAT3 axis promotes tumorigenesis, such as its modulations of Twist1, hypoxia-induced factor 1α, CD274, will be described. In the second part, we summarize recent data generated by mass spectrometry studies of NPM-ALK, and use MSH2 and heat shock proteins as examples to illustrate the use of mass spectrometry data in stimulating new research in this field. In the third part, the evolving field of microRNA in the context of NPM-ALK biology is discussed.

  20. Synthesis of an aryloxy oxo pyrimidinone library that displays ALK-selective inhibition.

    PubMed

    Slavish, P Jake; Price, Jeanine E; Jiang, Qin; Cui, Xiaoli; Morris, Stephan W; Webb, Thomas R

    2011-08-01

    We report the synthesis of a pyrimidinone library that targets anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase. This library was generated in three steps from a versatile commercially available starting material. Some compounds within this library showed single digit micromolar inhibition of ALK in vitro, while showing minimal inhibition of other homologous insulin receptor family kinases including the human insulin receptor kinase (IRK), at the highest concentrations investigated. We also present initial ALK structure-activity relationships for this library. PMID:21708465

  1. ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes

    PubMed Central

    Parrilla Castellar, Edgardo R.; Jaffe, Elaine S.; Said, Jonathan W.; Swerdlow, Steven H.; Ketterling, Rhett P.; Knudson, Ryan A.; Sidhu, Jagmohan S.; Hsi, Eric D.; Karikehalli, Shridevi; Jiang, Liuyan; Vasmatzis, George; Gibson, Sarah E.; Ondrejka, Sarah; Nicolae, Alina; Grogg, Karen L.; Allmer, Cristine; Ristow, Kay M.; Wilson, Wyndham H.; Macon, William R.; Law, Mark E.; Cerhan, James R.; Habermann, Thomas M.; Ansell, Stephen M.; Dogan, Ahmet; Maurer, Matthew J.

    2014-01-01

    Anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a CD30-positive T-cell non-Hodgkin lymphoma that morphologically resembles ALK-positive ALCL but lacks chromosomal rearrangements of the ALK gene. The genetic and clinical heterogeneity of ALK-negative ALCL has not been delineated. We performed immunohistochemistry and fluorescence in situ hybridization on 73 ALK-negative ALCLs and 32 ALK-positive ALCLs and evaluated the associations among pathology, genetics, and clinical outcome. Chromosomal rearrangements of DUSP22 and TP63 were identified in 30% and 8% of ALK-negative ALCLs, respectively. These rearrangements were mutually exclusive and were absent in ALK-positive ALCLs. Five-year overall survival rates were 85% for ALK-positive ALCLs, 90% for DUSP22-rearranged ALCLs, 17% for TP63-rearranged ALCLs, and 42% for cases lacking all 3 genetic markers (P < .0001). Hazard ratios for death in these 4 groups after adjusting for International Prognostic Index and age were 1.0 (reference group), 0.58, 8.63, and 4.16, respectively (P = 7.10 × 10−5). These results were similar when restricted to patients receiving anthracycline-based chemotherapy, as well as to patients not receiving stem cell transplantation. Thus, ALK-negative ALCL is a genetically heterogeneous disease with widely disparate outcomes following standard therapy. DUSP22 and TP63 rearrangements may serve as predictive biomarkers to help guide patient management. PMID:24894770

  2. Treating patients with ALK-positive non-small cell lung cancer: latest evidence and management strategy

    PubMed Central

    Liao, Bin-Chi; Shih, Jin-Yuan; Yang, James Chih-Hsin

    2015-01-01

    Rearrangements in anaplastic lymphoma kinase (ALK) gene and echinoderm microtubule-associated protein-like 4 (EML4) gene were first described in a small portion of patients with non-small cell lung cancer (NSCLC) in 2007. Fluorescence in situ hybridization is used as the diagnostic test for detecting an EML4–ALK rearrangement. Crizotinib, an ALK inhibitor, is effective in treating advanced ALK-positive NSCLC, and the US Food and Drug Administration approved it for treating ALK-positive NSCLC in 2011. Several mechanisms of acquired resistance to crizotinib have recently been reported. Second-generation ALK inhibitors were designed to overcome these resistance mechanisms. Two of them, ceritinib and alectinib, were approved in 2014 for advanced ALK-positive NSCLC in the US and Japan, respectively. Heat shock protein 90 (Hsp90) inhibitors also showed activity against ALK-positive NSCLC. Here we review the recent development of crizotinib, ceritinib, alectinib and other second-generation ALK inhibitors as well as Hsp90 inhibitors. We also discuss management strategies for advanced ALK-positive NSCLC. PMID:26327925

  3. Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells.

    PubMed

    Oh, Se Jin; Noh, Kyung Hee; Lee, Young-Ho; Hong, Soon-Oh; Song, Kwon-Ho; Lee, Hyo-Jung; Kim, Soyeon; Kim, Tae Min; Jeon, Ju-Hong; Seo, Jae Hong; Kim, Dong-Wan; Kim, Tae Woo

    2015-11-24

    The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.

  4. Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

    PubMed

    Moe, Elin; Hall, David R; Leiros, Ingar; Monsen, Vivi Talstad; Timmins, Joanna; McSweeney, Sean

    2012-06-01

    3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.

  5. The analysis of ALK gene rearrangement by fluorescence in situ hybridization in non-small cell lung cancer patients

    PubMed Central

    Krawczyk, Paweł Adam; Ramlau, Rodryg Adam; Szumiło, Justyna; Kozielski, Jerzy; Kalinka-Warzocha, Ewa; Bryl, Maciej; Knopik-Dąbrowicz, Alina; Spychalski, Łukasz; Szczęsna, Aleksandra; Rydzik, Ewelina; Milanowski, Janusz

    2013-01-01

    Introduction ALK gene rearrangement is observed in a small subset (3–7%) of non-small cell lung cancer (NSCLC) patients. The efficacy of crizotinib was shown in lung cancer patients harbouring ALK rearrangement. Nowadays, the analysis of ALK gene rearrangement is added to molecular examination of predictive factors. Aim of the study The frequency of ALK gene rearrangement as well as the type of its irregularity was analysed by fluorescence in situ hybridisation (FISH) in tissue samples from NSCLC patients. Material and methods The ALK gene rearrangement was analysed in 71 samples including 53 histological and 18 cytological samples. The analysis could be performed in 56 cases (78.87%), significantly more frequently in histological than in cytological materials. The encountered problem with ALK rearrangement diagnosis resulted from the scarcity of tumour cells in cytological samples, high background fluorescence noises and fragmentation of cell nuclei. Results The normal ALK copy number without gene rearrangement was observed in 26 (36.62%) patients ALK gene polysomy without gene rearrangement was observed in 25 (35.21%) samples while in 3 (4.23%) samples ALK gene amplification was found. ALK gene rearrangement was observed in 2 (2.82%) samples from males, while in the first case the rearrangement coexisted with ALK amplification. In the second case, signet-ring tumour cells were found during histopathological examination and this patient was successfully treated with crizotinib with partial remission lasting 16 months. Conclusions FISH is a useful technique for ALK gene rearrangement analysis which allows us to specify the type of gene irregularities. ALK gene examination could be performed in histological as well as cytological (cellblocks) samples, but obtaining a reliable result in cytological samples depends on the cellularity of examined materials. PMID:24592134

  6. Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

    PubMed

    Moe, Elin; Hall, David R; Leiros, Ingar; Monsen, Vivi Talstad; Timmins, Joanna; McSweeney, Sean

    2012-06-01

    3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket. PMID:22683793

  7. The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

    SciTech Connect

    Varshney, Gaurav K.; Palmer, Ruth H. . E-mail: Ruth.Palmer@ucmp.umu.se

    2006-12-29

    During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function results in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.

  8. Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

    PubMed

    Young, Leah C; Bone, Kathleen M; Wang, Peng; Wu, Fang; Adam, Benjamin A; Hegazy, Samar; Gelebart, Pascal; Holovati, Jelena; Li, Liang; Andrew, Susan E; Lai, Raymond

    2011-07-01

    The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

  9. Design of novel quinazoline derivatives and related analogues as potent and selective ALK5 inhibitors

    SciTech Connect

    Gellibert, F.; Fouchet, M.-H.; Nguyen, V.-L.; Wang, R.; Krysa, G.; de Gouville, A.-C.; Huet, S.; Dodic, N.

    2009-07-23

    Starting from quinazoline 3a, we designed potent and selective ALK5 inhibitors over p38MAP kinase from a rational drug design approach based on co-crystal structures in the human ALK5 kinase domain. The quinazoline 3d exhibited also in vivo activity in an acute rat model of DMN-induced liver fibrosis when administered orally at 5 mg/kg (bid).

  10. Endoglin and Alk5 regulate epithelial-mesenchymal transformation during cardiac valve formation

    PubMed Central

    Mercado-Pimentel, Melania E.; Hubbard, Antony D.; Runyan, Raymond B.

    2007-01-01

    Endoglin is an accessory receptor for TGFß and can associate with Alk5 or Alk2. Although prior studies indicated that endoglin and Alk5 were not directly involved in epithelial-mesenchymal transformation (EMT) in the heart, the expression pattern of endoglin prompted a re-examination. We here show that loss of endoglin expression mediated by either antisense DNA or siRNA results in a direct perturbation of EMT and reduced expression of EMT markers including slug, runx2, RhoA, and latrophilin-2. An examination of BrdU incorporation shows that, while endoglin regulates proliferation at an early stage, reduced endothelial cell proliferation does not account for the loss of mesenchyme. As Alk5 interacts with endoglin, we utilized siRNA and a specific inhibitor, HTS466284 (HTS), to perturb this receptor as well. Alk5 inhibition produced similar effects to inhibition of endoglin. There was a reduction in mesenchymal cell formation and loss of EMT marker expression similar to that seen with endoglin. Alk5 kinase inhibition produced a similar loss of EMT marker expression but showed a contrasting upregulation of the proliferation and remodeling markers, Cyclin B2 and ß-catenin. Alk5 and endoglin both mediate endothelial cell proliferation in younger explants but, by stage 16, loss of endoglin no longer alters proliferation rates. These data show that both Alk5 and endoglin are directly involved in the process of EMT, that they interact with both TGFß-regulated activation and invasion pathways and that the roles of these receptors change during cardiac development. PMID:17250821

  11. ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH.

    PubMed

    Minca, Eugen C; Portier, Bryce P; Wang, Zhen; Lanigan, Christopher; Farver, Carol F; Feng, Yan; Ma, Patrick C; Arrossi, Valeria A; Pennell, Nathan A; Tubbs, Raymond R

    2013-05-01

    ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHC's routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases. PMID:23499337

  12. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    PubMed

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  13. Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis.

    PubMed

    Mussolin, L; Damm-Welk, C; Pillon, M; Zimmermann, M; Franceschetto, G; Pulford, K; Reiter, A; Rosolen, A; Woessmann, W

    2013-02-01

    We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ≤ 1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.

  14. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration.

    PubMed

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara; Boeri Erba, Elisabetta; Boccalatte, Francesco; Mohammed, Shabaz; Jensen, Ole N; Palestro, Giorgio; Inghirami, Giorgio; Chiarle, Roberto

    2007-05-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

  15. Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

    PubMed

    Paisse, Sandrine; Duran, Robert; Coulon, Frédéric; Goñi-Urriza, Marisol

    2011-11-01

    The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination. PMID:21660544

  16. L- and S-endoglin differentially modulate TGFbeta1 signaling mediated by ALK1 and ALK5 in L6E9 myoblasts.

    PubMed

    Velasco, Soraya; Alvarez-Muñoz, Patricia; Pericacho, Miguel; Dijke, Peter Ten; Bernabéu, Carmelo; López-Novoa, José M; Rodríguez-Barbero, Alicia

    2008-03-15

    TGFbeta regulates cellular processes by binding to type I and type II TGFbeta receptors (TbetaRI and TbetaRII, respectively). In addition to these signaling receptors, endoglin is an accessory TGFbeta receptor that regulates TGFbeta signaling. Although there are two different alternatively spliced isoforms of endoglin, L-endoglin (L, long) and S-endoglin (S, short), little is known about the effects of S-endoglin isoform on TGFbeta signaling. Here, we have analyzed the TGFbeta1 signaling pathways and the effects of L- and S-endoglin in endoglin-deficient L6E9 cells. We found that TGFbeta activates two distinct TbetaRI-Smad signaling pathways: ALK1-Smad1-Id1 and ALK5-Smad2-PAI1, in these cells. Interestingly, L-endoglin enhanced the ALK1-Id1 pathway, while S-endoglin promoted the ALK5-PAI1 route. These effects on signaling are supported by biological effects on TGFbeta1-induced collagen I expression and inhibition of cell proliferation. Thus, while L-endoglin decreased TGFbeta1-induced collagen I and CTGF expression and increased TGFbeta1-induced proliferation, S-endoglin strongly increased TGFbeta1-induced collagen I and CTGF expression, and reduced TGFbeta1-induced cell proliferation.

  17. Brain metastasis of ALK positive anaplastic large cell lymphoma after a long-term disease free survival in an old adult

    PubMed Central

    Wang, Cai-Xia; Wang, Hai; Li, Jie; Ma, Heng-Hui; Yu, Bo; Shi, Shan-Shan; Zhou, Xiao-Jun; Shi, Qun-Li

    2014-01-01

    Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma composed of CD30-positive cells and now recognized as three different entities: primary cutaneous ALCL, primary systemic anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL and primary ALK-negative (ALK-) ALCL. ALK+ ALCL is supposed to have a better prognosis than ALK- ALCL. It is rarely metastasized to other sites, especially to the central nervous system (CNS). Herein, we present a rare case of systemic ALK+ ALCL which metastasized to the brain after a long-term disease free survival in an adult. Neuroimaging revealed a well-enhanced mass in the left frontal lobe. And it was completely resected. The results of the pathological and immunohistochemical studies were consistent with the metastasized ALK+ ALCL. The clinical findings, pathologic characteristics and treatment are described. PMID:24696735

  18. Identification of multiple SNT-binding sites on NPM-ALK oncoprotein and their involvement in cell transformation.

    PubMed

    Chikamori, M; Fujimoto, J; Tokai-Nishizumi, N; Yamamoto, T

    2007-05-01

    The t(2;5) chromosomal translocation occurs in anaplastic large-cell lymphoma arising from activated T lymphocytes. This genomic rearrangement generates the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) oncoprotein that is a chimeric protein consisting of parts of the nuclear protein NPM and ALK receptor protein-tyrosine kinase. We used yeast two-hybrid screening to identify an adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT)-2 as a new partner that interacted with the cytoplasmic domain of ALK. Immunoprecipitation assay revealed that SNT-1 and SNT-2 interacted with NPM-ALK and kinase-negative NPM-ALK mutant. Y156, Y567 and a 19-amino-acid sequence (aa 631-649) of NPM-ALK were essential for this interaction. The interaction through Y156 and Y567 was dependent on phosphorylation of these tyrosines, whereas the interaction through the 19-amino-acid sequence was independent of phosphorylation. NPM-ALK mutant protein mutated at these three binding sites showed significantly reduced transforming activity. This transformation-defective NPM-ALK mutant still interacted with signal transducing proteins such as phospholipase C-gamma and phosphatidylinositol 3-kinase, which were previously reported to be relevant to NPM-ALK-dependent tumorigenesis. These observations indicate that the three SNT-binding sites of NPM-ALK are important for its transforming activity. This raises a possibility that SNT family proteins play significant roles in cellular transformation triggered by NPM-ALK, which though remains to be verified.

  19. The role of AlkB protein in repair of 1,N⁶-ethenoadenine in Escherichia coli cells.

    PubMed

    Maciejewska, Agnieszka M; Sokołowska, Beata; Nowicki, Adam; Kuśmierek, Jarosław T

    2011-05-01

    Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible. PMID:21193516

  20. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    PubMed

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  1. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

    SciTech Connect

    Sun, Hui-Yong; Ji, Feng-Qin

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

  2. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

    PubMed Central

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  3. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer.

    PubMed

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  4. Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK

    PubMed Central

    YOKOYAMA, NORIKO; MILLER, W. TODD

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling. PMID:25469238

  5. Targeting Oncogenic ALK and MET: A Promising Therapeutic Strategy for Glioblastoma

    PubMed Central

    Wallace, Gerald C; Dixon-Mah, Yaenette N; Vandergrift, W Alex; Ray, Swapan K; Haar, Catherine P; Mittendorf, Amber M; Patel, Sunil J; Banik, Naren L; Giglio, Pierre; Das, Arabinda

    2015-01-01

    Glioblastoma is the most common aggressive, highly glycolytic, and lethal brain tumor. In fact, it is among the most commonly diagnosed lethal malignancies, with thousands of new cases reported in the United States each year. Glioblastoma's lethality is derived from a number of factors including highly active pro-mitotic and pro-metastatic pathways. Two factors increasingly associated with the intracellular signaling and transcriptional machinery required for such changes are anaplastic lymphoma kinase (ALK) and the hepatocyte growth factor receptor (HGFR or, more commonly MET). Both receptors are members of the receptor tyrosine kinase (RTK) family, which has itself gained much attention for its role in modulating mitosis, migration, and survival in cancer cells. ALK was first described as a vital oncogene in lymphoma studies, but it has since been connected to many carcinomas, including non-small cell lung cancer and glioblastoma. As the receptor for HGF, MET has also been highly characterized and regulates numerous developmental and wound healing events which, when upregulated in cancer, can promote tumor progression. The wealth of information gathered over the last 30 years regarding these RTKs suggests three downstream cascades that depend upon activation of STAT3, Ras, and AKT. This review outlines the significance of ALK and MET as they relate to glioblastoma, explores the significance of STAT3, Ras, and AKT downstream of ALK/MET, and touches on the potential for new chemotherapeutics targeting ALK and MET to improve glioblastoma patient prognosis. PMID:23543207

  6. ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

    PubMed

    Ueda, T; Nakata, Y; Yamasaki, N; Oda, H; Sentani, K; Kanai, A; Onishi, N; Ikeda, K; Sera, Y; Honda, Z-I; Tanaka, K; Sata, M; Ogawa, S; Yasui, W; Saya, H; Takita, J; Honda, H

    2016-08-25

    Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors. PMID:26829053

  7. Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

    PubMed Central

    Desquesnes, Aurore; Le Gonidec, Sophie; AlSaati, Talal; Beau, Isabelle; Lamant, Laurence; Meggetto, Fabienne; Espinos, Estelle; Codogno, Patrice; Brousset, Pierre; Giuriato, Sylvie

    2015-01-01

    Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients. PMID:26338968

  8. ALK and crizotinib: after the honeymoon…what else? Resistance mechanisms and new therapies to overcome it.

    PubMed

    Rolfo, Christian; Passiglia, Francesco; Castiglia, Marta; Raez, Luis E; Germonpre, Paul; Gil-Bazo, Ignacio; Zwaenepoel, Karen; De Wilde, Annemieke; Bronte, Giuseppe; Russo, Antonio; Van Meerbeeck, Jan P; Van Schil, Paul; Pauwels, Patrick

    2014-08-01

    The last few decades have witnessed a silent revolution in the war against NSCLC, thanks to the discovery of "oncogenic drivers" and the subsequent development of targeted therapies. The discovery of the EML4-ALK fusion gene in a subgroup of patients with NSCLC and the subsequent clinical development of crizotinib has been an amazing success story in lung cancer translational-research, and its accelerated approval [only 4 years from the discovery of ALK rearrangement in NSCLC to the approval by the Food and Drug Administration (FDA)] marked the beginning of the new decade of targeted therapy. However, common to all targeted therapies, despite an initial benefit, patients inevitably experience tumor progression, due to the development of resistance. Several molecular mechanisms are responsible for acquired resistance, such as secondary mutations of ALK kinase domain or amplification of ALK fusion gene, or the activation of other oncogenic drivers, which may cause resistance independently of ALK genetic alterations. Pre-clinical data and early clinical trials showed the promising efficacy of a new class of ALK-inhibitors in overcoming acquired resistance. The inhibition of the molecular chaperone, HSP90, represents another promising strategy to overcome crizotinib resistance in ALK-rearranged NSCLC. Several molecules are currently under investigation in order to establish their specific role in the treatment of ALK-rearranged NSCLC.

  9. TKI sensitivity patterns of novel kinase-domain mutations suggest therapeutic opportunities for patients with resistant ALK+ tumors

    PubMed Central

    Rajan, Soumya S.; Gokhale, Vijay; Groysman, Matthew J.; Pongtornpipat, Praechompoo; Tapia, Edgar O.; Wang, Mengdie; Schatz, Jonathan H.

    2016-01-01

    The anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions. PMID:27009859

  10. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  11. Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Ito, Ken; Hong, Su Hyun; Hoffmann, Matthias; Pisani, Laura; Boudreau, Nancy; Gambhir, Sanjiv Sam; Coussens, Lisa M.

    2016-01-01

    Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies. PMID:27182558

  12. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond*

    PubMed Central

    Fedeles, Bogdan I.; Singh, Vipender; Delaney, James C.; Li, Deyu; Essigmann, John M.

    2015-01-01

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. PMID:26152727

  13. 3D-QSAR and molecular fragment replacement study on diaminopyrimidine and pyrrolotriazine ALK inhibitors

    NASA Astrophysics Data System (ADS)

    Ke, Zhipeng; Lu, Tao; Liu, Haichun; Yuan, Haoliang; Ran, Ting; Zhang, Yanmin; Yao, Sihui; Xiong, Xiao; Xu, Jinxing; Xu, Anyang; Chen, Yadong

    2014-06-01

    Over expression of anaplastic lymphoma kinase (ALK) has been found in many types of cancer, and ALK is a promising therapeutic target for the treatment of cancer. To obtain new potent inhibitors of ALK, we conducted lead optimization using 3D-QSAR modeling and molecular docking investigation of 2,4-diaminopyrimidines and 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine-based compounds. Three favorable 3D-QSAR models (CoMFA with q2, 0.555; r2, 0.939; CoMSIA with q2, 0.625; r2, 0.974; Topomer CoMFA with q2, 0.557; r2 0.756) have been developed to predict the biological activity of novel compounds. Topomer Search was utilized for virtual screening to obtain suitable fragments. The novel compounds generated by molecular fragment replacement (MFR) were evaluated by Topomer CoMFA prediction, Glide (docking) and further evaluated with CoMFA and CoMSIA prediction. 25 novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine derivatives as potential ALK inhibitors were finally obtained. In this paper, a combination of CoMFA, CoMSIA and Topomer CoMFA could obtain favorable 3D-QSAR models and suitable fragments for ALK inhibitors optimization. The work flow which comprised 3D-QSAR modeling, Topomer Search, MFR, molecular docking and evaluating criteria could be applied to de novo drug design and the resulted compounds initiate us to further optimize and design new potential ALK inhibitors.

  14. Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer.

    PubMed

    Tan, Chye Ling; Lim, Tse Hui; Lim, Tony Kh; Tan, Daniel Shao-Weng; Chua, Yong Wei; Ang, Mei Kim; Pang, Brendan; Lim, Chwee Teck; Takano, Angela; Lim, Alvin Soon-Tiong; Leong, Man Chun; Lim, Wan-Teck

    2016-04-26

    Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing. PMID:26993609

  15. Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer

    PubMed Central

    Lim, Tony KH; Tan, Daniel Shao-Weng; Chua, Yong Wei; Ang, Mei Kim; Pang, Brendan; Lim, Chwee Teck; Takano, Angela; Lim, Alvin Soon-Tiong; Leong, Man Chun; Lim, Wan-Teck

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3–15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0–2 cells/1.88 mL of blood). The latter range was validated as the ‘false positive’ cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing. PMID:26993609

  16. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  17. EML4-ALK induces epithelial–mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells

    SciTech Connect

    Guo, Fuchun; Liu, Xiaoke Qing, Qin Sang, Yaxiong Feng, Chengjun Li, Xiaoyu Jiang, Li Su, Pei Wang, Yongsheng

    2015-04-10

    The echinoderm microtubule-associated protein-like 4(EML4) – anaplastic lymphoma kinase (ALK) fusion gene has been identified as a driver mutation in non-small-cell lung cancer (NSCLC). However, the role of EML4-ALK in malignant transformation is not entirely clear. Here, for the first time, we showed that H1299 NSCLC cells stably expressing EML4-ALK acquire EMT phenotype, associated with enhanced invasive migration and increased expression of EMT-inducing transcription factors. H1299-EML4-ALK cells also displayed cancer stem cell-like properties with a concomitant up-regulation of CD133 and enhanced ability of mammospheres formation. Moreover, we found that inhibition of ERK1/2 reversed EMT induced by EML4-ALK in H1299 cells. Taken together, these results suggested that EML4-ALK induced ERK activation is mechanistically associated with EMT phenotype. Thus, inhibition of ERK signaling pathway could be a potential strategy in treatment of NSCLC patients with EML4-ALK translocation. - Highlights: • EML4-ALK induced epithelial–mesenchymal transition in H1299 cells. • Expression of EML4-ALK promotes invasion and migration in vitro. • EML4-ALK enhanced sphere formation and stem cell-like properties in H1299 cells. • Blockage of ERK1/2 reverse Epithelial–Mesenchymal transition induced by EML4-ALK.

  18. Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

    PubMed

    Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

    2015-04-01

    Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

  19. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  20. Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

    PubMed

    Sikora, Anna; Maciejewska, Agnieszka M; Poznański, Jarosław; Pilżys, Tomasz; Marcinkowski, Michał; Dylewska, Małgorzata; Piwowarski, Jan; Jakubczak, Wioletta; Pawlak, Katarzyna; Grzesiuk, Elżbieta

    2015-08-01

    An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

  1. Imaging Characteristics in ALK Fusion-Positive Lung Adenocarcinomas by Using HRCT

    PubMed Central

    Okumura, Sakae; Kuroda, Hiroaki; Uehara, Hirofumi; Mun, Mingyon; Takeuchi, Kengo; Nakagawa, Ken

    2014-01-01

    Objectives: We aimed to identify high-resolution computed tomography (HRCT) features useful to distinguish the anaplastic lymphoma kinase gene (ALK) fusion-positive and negative lung adenocarcinomas. Methods: We included 236 surgically resected adenocarcinoma lesions, which included 27 consecutive ALK fusion-positive (AP) lesions, 115 epidermal growth factor receptor mutation-positive lesions, and 94 double-negative lesions. HRCT parameters including size, air bronchograms, pleural indentation, spiculation, and tumor disappearance rate (TDR) were compared. In addition, prevalence of small lesions (≤20 mm) and solid lesions (TDR ≤20%) were compared. Results: AP lesions were significantly smaller and had lower TDR (%) than ALK fusion-negative (AN) lesions (tumor diameter: 20.7 mm ± 14.1 mm vs. 27.4 mm ± 13.8 mm, respectively, p <0.01; TDR: 22.8% ± 24.8% vs. 44.8% ± 33.2%, respectively, p <0.01). All AP lesions >20 mm (n = 7, 25.9%) showed a solid pattern. Among all small lesions, AP lesions had lower TDR and more frequent spiculation than AN lesions (p <0.01). Among solid lesions, AP lesions were smaller than AN lesions (p = 0.01). Conclusion: AP lung lesions were significantly smaller and had a lower TDR than AN lesions. Spiculation was more frequent in small lesions. Non-solid >20 mm lesions may be ALK fusion-negative. PMID:24899136

  2. Determining the contribution of NPM1 heterozygosity to NPM-ALK-induced lymphomagenesis.

    PubMed

    Mduff, Fiona K E; Hook, C Elizabeth; Tooze, Reuben M; Huntly, Brian J; Pandolfi, Pier Paolo; Turner, Suzanne D

    2011-09-01

    Heterozygous expression of Nucleophosmin (NPM1) predisposes to hematological malignancies in the mouse and cooperates with Myc in lymphomagenesis. NPM1 is therefore regarded as a haploinsufficient tumor suppressor. Heterozygous loss of NPM1 occurs as a result of the t(2;5), which generates the oncogenic fusion tyrosine kinase, NPM-anaplastic lymphoma kinase (ALK), a molecule underlying the pathogenesis of anaplastic large cell lymphoma (ALCL). Given the aforementioned role of NPM1 as a tumor suppressor, we hypothesized that NPM1 heterozygosity would cooperate with NPM-ALK in lymphomagenesis. In the event, we observed no difference in tumor latency, incidence or phenotype in NPM-ALK-transgenic mice heterozygous for NPM1 relative to transgenic mice expressing both NPM1 alleles. We propose that although the t(2;5) simultaneously reduces NPM1 allelic dosage and creates the NPM-ALK fusion protein, the two events do not cooperate in the pathogenesis of ALCL in our mouse model. These data indicate that a tumor-suppressive role for NPM1 may depend on cellular and/or genetic context.

  3. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-09-02

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway.

  4. Clinicopathologic characteristics of EGFR, KRAS, and ALK alterations in 6,595 lung cancers

    PubMed Central

    Lee, Boram; Lee, Taebum; Lee, Se-Hoon; Choi, Yoon-La; Han, Joungho

    2016-01-01

    Background EGFR, KRAS, and ALK alterations are major genetic changes found in non-small cell lung cancers (NSCLCs). Testing advanced lung adenocarcinoma tumors for these three genes is now standard care. The purpose of this study was to investigate the clinicopathologic expression pattern of these three genes in East Asian NSCLC patients. Patients and methods We conducted a retrospective study of all patients tested for mutations of these three genes at a single institute in Korea between 2006 and 2014. Study data were extracted from electronic medical records. Univariate and multivariate logistic regression analyses were used to measure associations between clinicopathologic features and alterations of EGFR, KRAS, and ALK. Results We detected 12 EGFR-mutated tumors with additional mutations in KRAS (N=6, 0.1%) or ALK (N=6, 0.1%). General clinicopathologic characteristics of tumors with EGFR, KRAS, or ALK mutations were similar to previous reports. Patients having EGFR L858R point mutations were older than patients having EGFR exon 19 deletions. EGFR G719X point mutations were more common in men and smokers than exon 19 deletions or L858R point mutations. Tumors having KRAS G12C mutations were less often of mucinous type than those with G12D or G12V, mutations. Conclusions This is the largest three gene molecular epidemiology study in East Asian NSCLC patients. Each genetic alteration was associated with distinct clinicopathologic characteristics. Furthermore, different age and sex are associated with different subtypes of EGFR and KRAS mutations. PMID:26992209

  5. An Unusual Case of Systemic Inflammatory Myofibroblastic Tumor with Successful Treatment with ALK-Inhibitor.

    PubMed

    Jacob, Sanjivini V; Reith, John D; Kojima, Angerika Y; Williams, William D; Liu, Chen; Vila Duckworth, Lizette

    2014-01-01

    Systemic inflammatory myofibroblastic tumor is an exceedingly rare entity. A 45-year-old Hispanic female presented with a 6-month history of left-sided thigh pain, low back pain, and generalized weakness. PET/CT scan revealed abnormal activity in the liver, adrenal gland, and pancreas. MRI of the abdomen demonstrated two 6-7 cm masses in the liver. MRI of the lumbar spine demonstrated lesions in the L2 to L4 spinous processes, paraspinal muscles, and subcutaneous tissues, as well as an 8 mm enhancing intradural lesion at T11, all thought to be metastatic disease. A biopsy of the liver showed portal tract expansion by a spindle cell proliferation rich in inflammation. Tumor cells showed immunoreactivity for smooth muscle actin and anaplastic lymphoma kinase 1 (ALK1). Tissue from the L5 vertebra showed a process histologically identical to that seen in the liver. FISH analysis of these lesions demonstrated an ALK (2p23) gene rearrangement. The patient was successfully treated with an ALK-inhibitor, Crizotinib, and is now in complete remission. We present the first reported case, to our knowledge, of inflammatory myofibroblastic tumor with systemic manifestations and ALK translocation. This case is a prime example of how personalized medicine has vastly improved patient care through the use of molecular-targeted therapy.

  6. An Unusual Case of Systemic Inflammatory Myofibroblastic Tumor with Successful Treatment with ALK-Inhibitor

    PubMed Central

    Jacob, Sanjivini V.; Reith, John D.; Kojima, Angerika Y.; Williams, William D.; Liu, Chen; Vila Duckworth, Lizette

    2014-01-01

    Systemic inflammatory myofibroblastic tumor is an exceedingly rare entity. A 45-year-old Hispanic female presented with a 6-month history of left-sided thigh pain, low back pain, and generalized weakness. PET/CT scan revealed abnormal activity in the liver, adrenal gland, and pancreas. MRI of the abdomen demonstrated two 6-7 cm masses in the liver. MRI of the lumbar spine demonstrated lesions in the L2 to L4 spinous processes, paraspinal muscles, and subcutaneous tissues, as well as an 8 mm enhancing intradural lesion at T11, all thought to be metastatic disease. A biopsy of the liver showed portal tract expansion by a spindle cell proliferation rich in inflammation. Tumor cells showed immunoreactivity for smooth muscle actin and anaplastic lymphoma kinase 1 (ALK1). Tissue from the L5 vertebra showed a process histologically identical to that seen in the liver. FISH analysis of these lesions demonstrated an ALK (2p23) gene rearrangement. The patient was successfully treated with an ALK-inhibitor, Crizotinib, and is now in complete remission. We present the first reported case, to our knowledge, of inflammatory myofibroblastic tumor with systemic manifestations and ALK translocation. This case is a prime example of how personalized medicine has vastly improved patient care through the use of molecular-targeted therapy. PMID:25045570

  7. ALK-positive inflammatory myofibroblastic tumor of the abdomen with widespread microscopic multifocality.

    PubMed

    Lorenzi, Luisa; Cigognetti, Marta; Medicina, Daniela; Pellegrini, Vilma; Balzarini, Piera; Cestari, Renzo; Facchetti, Fabio

    2014-10-01

    Inflammatory myofibroblastic tumor (IMT) is a locally aggressive neoplasm, most frequently occurring in the abdominal cavity as multiple recurrent nodules. We report a case of IMT in a 24-year-old male presenting as multiple nodules involving the omentum, the liver, and the colon. Spindle tumor cells expressed ALK with a cytoplasmic granular distribution, the CLTC-ALK fusion gene was demonstrated by reverse-transcriptase polymerase chain reaction analysis, and break-apart fluorescence in situ hybridization (FISH) probes for the ALK gene showed a pathological pattern (single red signal associated with 1/2 normal fused signals) highly suggestive for combined gene fusion and deletion. To reduce the surgically unresectable liver mass, the patient was treated with crizotinib, and after 4 months of treatment the disease was defined stable according to RECIST criteria. Interestingly, ALK and FISH/FICTION analysis revealed that tumor cells were widely dispersed as multiple microscopic foci or as single cells beneath the omental mesothelium. These findings indicate that IMT multifocality might result either from dissemination from the main tumor mass or development of multiple independent neoplastic foci; furthermore, they underline the need of omentectomy in abdominal IMT to obtain surgical radicality.

  8. ClogP(alk): a method for predicting alkane/water partition coefficient.

    PubMed

    Kenny, Peter W; Montanari, Carlos A; Prokopczyk, Igor M

    2013-05-01

    Alkane/water partition coefficients (P(alk)) are less familiar to the molecular design community than their 1-octanol/water equivalents and access to both data and prediction tools is much more limited. A method for predicting alkane/water partition coefficient from molecular structure is introduced. The basis for the ClogP(alk) model is the strong (R² = 0.987) relationship between alkane/water partition coefficient and molecular surface area (MSA) that was observed for saturated hydrocarbons. The model treats a molecule as a perturbation of a saturated hydrocarbon molecule with the same MSA and uses increments defined for functional groups to quantify the extent to which logP(alk) is perturbed by the introduction each functional group. Interactions between functional groups, such as intramolecular hydrogen bonds are also parameterized within a perturbation framework. The functional groups and interactions between them are specified substructurally in a transparent and reproducible manner using SMARTS notation. The ClogP(alk) model was parameterized using data measured for structurally prototypical compounds that dominate the literature on alkane/water partition coefficients and then validated using an external test set of 100 alkane/water logP measurements, the majority of which were for drugs.

  9. Atractylodin Inhibits Interleukin-6 by Blocking NPM-ALK Activation and MAPKs in HMC-1.

    PubMed

    Chae, Hee-Sung; Kim, Young-Mi; Chin, Young-Won

    2016-01-01

    Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway. PMID:27598116

  10. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified...

  11. 40 CFR 721.435 - Alkylphenylpolyether-alk-a-nol-a-mines (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.435 Alkylphenylpolyether-alk-a-nol-a-mines (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substances identified...

  12. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; El-Salem, M; Halasa, K; Raghunath, P N; Bucki, R; Wlodarski, P; Wasik, M A

    2007-08-16

    The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

  13. Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

    PubMed

    Marzec, M; Kasprzycka, M; Liu, X; Raghunath, P N; Wlodarski, P; Wasik, M A

    2007-02-01

    The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

  14. ALK Positive Lung Cancer: Clinical Profile, Practice and Outcomes in a Developing Country

    PubMed Central

    Chougule, Anuradha; Kane, Subhadha; Kumar, Rajiv; Mahajan, Abhishek; Janu, Amit

    2016-01-01

    Objectives To evaluate the performance and treatment profile of advanced EML4—ALK positive Non-small cell lung cancer (NSCLC) patients in a developing country with potentially restricted access to Crizotinib. Materials and Methods A retrospective analysis of advanced ALK positive NSCLC patients who were treated from June 2012 to September 2015 was conducted. The primary goal was to evaluate outcomes of advanced ALK positive NSCLC in our practice and examine the logistic constraints in procuring Crizotinib. Results 94 patients were available for analysis. 21 (22.3%) patients were started on Crizotinib upfront, 60 (63.8%) on chemotherapy, 10 (10.6%) on Tyrosine kinase inhibitors (in view of poor PS) and 3 (3.2%) patients were offered best supportive care. Reasons for not starting Crizotinib upfront included symptomatic patients needing early initiation of therapy (23.3%), ALK not tested upfront (23.3%) and financial constraints (21.9%). 69 patients (73.4%) received Crizotinib at some stage during treatment. Dose interruptions (> 1 week) with Crizotinib were seen in 20 patients (29%), with drug toxicity being the commonest reason (85%). Median Progression free survival (PFS) on first line therapy for the entire cohort was 10 months, with a significant difference between patients receiving Crizotinib and those who did not ever receive Crizotinib (10 months vs. 2 months, p = 0.028). Median Overall Survival (OS) was not reached for the entire cohort, with 1 year survival being 81.2%. Patients with an ECOG Performance Status (PS) of >2 had a significantly reduced PFS compared to patients with PS < = 2 (1.5 months vs. 11 months, p< 0.001). 47 patients with financial constraints (68.1%) received Crizotinib completely free via various extramural support schemes. Conclusion A majority of our ALK positive NSCLC patients were exposed to Crizotinib through the help of various support mechanisms and these patients had similar outcomes to that reported from previously published

  15. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    PubMed

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

  16. Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

    PubMed

    Moore, Nathan F; Azarova, Anna M; Bhatnagar, Namrata; Ross, Kenneth N; Drake, Lauren E; Frumm, Stacey; Liu, Qinsong S; Christie, Amanda L; Sanda, Takaomi; Chesler, Louis; Kung, Andrew L; Gray, Nathanael S; Stegmaier, Kimberly; George, Rani E

    2014-09-30

    Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

  17. Detection of EML4-ALK fusion gene and features associated with EGFR mutations in Chinese patients with non-small-cell lung cancer

    PubMed Central

    Wen, Miaomiao; Wang, Xuejiao; Sun, Ying; Xia, Jinghua; Fan, Liangbo; Xing, Hao; Zhang, Zhipei; Li, Xiaofei

    2016-01-01

    Purpose Echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). Methods We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. Results Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ2 test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. Conclusion EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent. PMID:27103824

  18. In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

    PubMed

    Nanjo, Shigeki; Nakagawa, Takayuki; Takeuchi, Shinji; Kita, Kenji; Fukuda, Koji; Nakada, Mitsutoshi; Uehara, Hisanori; Nishihara, Hiroshi; Hara, Eiji; Uramoto, Hidetaka; Tanaka, Fumihiro; Yano, Seiji

    2015-03-01

    EML4-ALK lung cancer accounts for approximately 3-7% of non-small-cell lung cancer cases. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. In this study, we found that the lung adenocarcinoma cell line A925L expresses an EML4-ALK gene fusion (variant 5a, E2:A20) and is sensitive to the ALK inhibitors crizotinib and alectinib. We further established highly tumorigenic A925LPE3 cells, which also have the EML4-ALK gene fusion (variant 5a) and are sensitive to ALK inhibitors. By using A925LPE3 cells with luciferase gene transfection, we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, whereas alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

  19. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming.

    PubMed

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y; Cheng, Mangeng; Baldwin, Donald; Tobias, John W; Schuster, Stephen J; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D; Ødum, Niels; Wasik, Mariusz A

    2013-12-15

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.

  20. Molecular Characterization of Inflammatory Myofibroblastic Tumors with Frequent ALK and ROS1 Fusions and Rare Novel RET Gene Rearrangement

    PubMed Central

    Antonescu, Cristina R; Suurmeijer, Albert JH; Zhang, Lei; Sung, Yun-Shao; Jungbluth, Achim A; Travis, William D; Al-Ahmadie, Hikmat; Fletcher, Christopher DM; Alaggio, Rita

    2015-01-01

    Approximately 50% of conventional IMTs harbor ALK gene rearrangement and overexpress ALK. Recently gene fusions involving other kinases have been implicated in the pathogenesis of IMT, including ROS1 and in one patient PDGFRB. However, it remains uncertain if the emerging genotypes correlate with clinicopathologic characteristics of IMT. In this study we expand the molecular investigation of IMT in a large cohort of different clinical presentations and analyze for potential genotype-phenotype associations. Criteria for inclusion in the study were typical morphology and tissue availability for molecular studies. The lack of ALK immunoreactivity was not an excluding factor. As overlapping gene fusions involving actionable kinases are emerging in both IMT and lung cancer, we set out to evaluate abnormalities in ALK, ROS1, PDGFRB, NTRK1 and RET by FISH. Additionally, next generation paired-end RNA sequencing and FusionSeq algorithm was applied in 4 cases, which identified EML4-ALK fusions in 2 cases. Of the 62 IMTs (25 children and 37 adults), 35 (56%) showed ALK gene rearrangement. Of note, EML4-ALK inversion was noted in 7 (20%) cases, seen mainly in the lung and soft tissue of young children including 2 lesions from newborns. There were 6 (10%) ROS1 rearranged IMTs, all except one presenting in children, mainly in the lung and intra-abdominal and showed a distinctive fascicular growth of spindle cells with long cell processes, often positive for ROS1 IHC. Two of the cases showed TFG-ROS1 fusions. Interestingly, one adult IMT revealed a RET gene rearrangement, a previously unreported finding. Our results show that 42/62 (68%) of IMTs are characterized by kinase fusions, offering a rationale for targeted therapeutic strategies. Interestingly 90% of fusion negative IMT were seen in adults, while >90% of pediatric IMT showed gene rearrangements.EML4-ALK inversion and ROS1 fusions emerge as common fusion abnormalities in IMT, closely recapitulating the pattern seen in

  1. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  2. Redundant and nonredundant roles for Cdc42 and Rac1 in lymphomas developed in NPM-ALK transgenic mice.

    PubMed

    Choudhari, Ramesh; Minero, Valerio Giacomo; Menotti, Matteo; Pulito, Roberta; Brakebusch, Cord; Compagno, Mara; Voena, Claudia; Ambrogio, Chiara; Chiarle, Roberto

    2016-03-10

    Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In vitro studies have shown that Cdc42 and Rac1 control rather similar phenotypes of ALCL biology such as the proliferation, survival, and migration of lymphoma cells. However, their role and possible redundancy in ALK-driven lymphoma development in vivo are still undetermined. We genetically deleted Cdc42 or Rac1 in a mouse model of ALK-rearranged ALCL to show that either Cdc42 or Rac1 deletion impaired lymphoma development, modified lymphoma morphology, actin filament distribution, and migration properties of lymphoma cells. Cdc42 or Rac1 deletion primarily affected survival rather than proliferation of lymphoma cells. Apoptosis of lymphoma cells was equally induced following Cdc42 or Rac1 deletion, was associated with upregulation of the proapoptotic molecule Bid, and was blocked by Bcl2 overexpression. Remarkably, Cdc42/Rac1 double deletion, but not Cdc42 or Rac1 single deletions, completely prevented NPM-ALK lymphoma dissemination in vivo. Thus, Cdc42 and Rac1 have nonredundant roles in controlling ALK-rearranged lymphoma survival and morphology but are redundant for lymphoma dissemination, suggesting that targeting both GTPases could represent a preferable therapeutic option for ALCL treatment.

  3. Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas.

    PubMed

    Thornber, K; Colomba, A; Ceccato, L; Delsol, G; Payrastre, B; Gaits-Iacovoni, F

    2009-07-23

    The chimera nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the tyrosine kinase activity of which is constitutively upregulated, is the causative agent of 75% of the anaplastic large-cell lymphomas (ALCLs). We have demonstrated that NPM-ALK induces the production of reactive oxygen species (ROS) by a pathway involving the arachidonic acid-metabolizing enzymes of the lipoxygenase (LOX) family. The use of the LOX inhibitor nordihydroguaiaretic acid (NDGA) and of the anti-oxidant N-acetylcysteine (NAC) demonstrated that ROS are important in maintaining the ALK kinase active. Consistent with this, NDGA treatment resulted in the inhibition of key pathways, such as Akt, signal transducer and activator of transcription factor 3 (STAT3) and extracellular signal-regulated kinase (ERK), which are involved in NPM-ALK antiapoptotic and pro-mitogenic functions. Conversely, the stress-activated kinase p38, described in some instances as a mediator of apoptosis, was activated. Interestingly, 5-LOX, an isoform involved in many cancers, was found to be activated in NPM-ALK(+) cells. Functional studies have shown that transforming properties, namely proliferation and resistance to apoptosis, were abrogated by treatment with either NDGA or the 5-LOX inhibitor (N-(3-phenoxycinnamyl)-acetohydroxamic acid) (BW A4C). Together, these data point to the ROS/LOX pathway as a potential new target for therapy in NPM-ALK-positive tumors.

  4. PD-1 Axis Inhibitors in EGFR- and ALK-Driven Lung Cancer: Lost Cause?

    PubMed

    Gettinger, Scott; Politi, Katerina

    2016-09-15

    Programmed death axis 1 (PD-1) inhibitors have ushered in a new error of cancer immunotherapeutics for advanced smoking-associated non-small cell lung cancer. Their role in treating EGFR-mutant and ALK-rearranged lung cancer has yet to be determined. Clin Cancer Res; 22(18); 4539-41. ©2016 AACRSee related article by Gainor et al., p. 4585. PMID:27470969

  5. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

    PubMed

    Shahid, Mohd; Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A; Leyton, Patricio A; Cheng, Juan; Tainsh, Robert E T; Mayeur, Claire; Rhee, David K; Wu, Mei X; Scherrer-Crosbie, Marielle; Buys, Emmanuel S; Zapol, Warren M; Bloch, Kenneth D; Bloch, Donald B

    2016-04-15

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis. PMID:26873969

  6. An ALK translocation positive carcinoma of the lung presenting as uremia due to bilateral renal obstruction.

    PubMed

    Rosenberg, Shilo; Katz, Ran; Pode, Dov; Gofrit, N Ofer; Pizov, Galina; Hovav, Nechushtan

    2013-01-01

    We describe an unusual presentation of metastatic lung adenocarcinoma as malignant retroperitoneal fibrosis (MRPF). The diagnostic challenge, due to the small solitary lung mass and absence of a discrete retroperitoneal mass, was overcome by diagnostic laparoscopy. Molecular analysis of tissue acquired was positive for ALK gene rearrangement. Treatment of the patient with crizotinib reversed MRPF. He was weaned off the nephrostomy tubes and is with stable renal function 11 months after diagnosis. PMID:23914266

  7. Rapamycin reverses NPM-ALK-induced glucocorticoid resistance in lymphoid tumor cells by inhibiting mTOR signaling pathway, enhancing G1 cell cycle arrest and apoptosis.

    PubMed

    Gu, L; Gao, J; Li, Q; Zhu, Y P; Jia, C S; Fu, R Y; Chen, Y; Liao, Q K; Ma, Z

    2008-11-01

    The anaplastic lymphoma kinase (ALK) is an oncogene product involved in hematopoietic and non-hematopoietic malignancies. Recent studies have demonstrated that nucleophosmin (NPM)-ALK, originated from the fusion of NPM and ALK genes, causes cell transformation through diverse mechanisms. Here, we show a novel mechanism by which NPM-ALK transforms lymphoid tumor cells to become resistant to glucocorticoid (GC) or dexamethasone (Dex) treatment. Transformed BaF3 cells by NPM-ALK were much more resistant to Dex compared with their parental cells, and concurrently had a constitutive activation of mammalian target of rapamycin (mTOR) signaling, as evidenced by hyperphosphorylation of its downstream effectors, p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The mTOR inhibitor rapamycin suppressed activation of p70S6K in BaF3/NPM-ALK cells and reversed GC resistance by synergistically inhibiting mTOR signaling pathway, enhancing cell cycle arrest at G(1) phase and promoting apoptotic cell death. In conclusion, our data indicate that the ALK fusion kinase, NPM-ALK, induces GC resistance by activating mTOR signaling, and addition of mTOR inhibitors to the chemotherapeutic regimen of ALK+ lymphomas may improve the prognosis.

  8. Three Years Sustained Complete Remission Achieved in a Primary Refractory ALK-Positive Anaplastic T Large Cell Lymphoma Treated with Crizotinib

    PubMed Central

    Mahuad, Carolina Valeria; Repáraz, María de los Ángeles Vicente; Zerga, Marta E.; Aizpurua, María Florencia; Casali, Claudia; Garate, Gonzalo

    2016-01-01

    The prognosis of the primary refractory anaplastic lymphoma kinase (ALK+) anaplastic T large cell lymphoma is ominous. The identification of molecular targets with potential to drive oncogenesis remains a cornerstone for the designing of new selective cancer therapies. Crizotinib is a selective ATP-competitive inhibitor for ALK, approved for its use in lung cancer with rearrangements on ALK gene. The reported cases describe the use of crizotinib as a bridging strategy prior to allotransplantation; there are no reported prolonged survivals under monotherapy with Crizotinib. We report a case of a primary refractory ALK+ anaplastic large-cell lymphoma that sustains complete response after 3 years of crizotinib monotherapy. PMID:27441079

  9. Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs.

    PubMed

    Li, Jianzong; Liu, Wei; Luo, Hao; Bao, Jinku

    2016-09-01

    Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified. PMID:27585676

  10. Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

    PubMed

    Fujimoto, Mai; Ohte, Satoshi; Shin, Masashi; Yoneyama, Katsumi; Osawa, Kenji; Miyamoto, Arei; Tsukamoto, Sho; Mizuta, Takato; Kokabu, Shoichiro; Machiya, Aiko; Okuda, Akihiko; Suda, Naoto; Katagiri, Takenobu

    2014-12-12

    Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-β1 (TGF-β1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

  11. Three Years Sustained Complete Remission Achieved in a Primary Refractory ALK-Positive Anaplastic T Large Cell Lymphoma Treated with Crizotinib.

    PubMed

    Mahuad, Carolina Valeria; Repáraz, María de Los Ángeles Vicente; Zerga, Marta E; Aizpurua, María Florencia; Casali, Claudia; Garate, Gonzalo

    2016-06-28

    The prognosis of the primary refractory anaplastic lymphoma kinase (ALK+) anaplastic T large cell lymphoma is ominous. The identification of molecular targets with potential to drive oncogenesis remains a cornerstone for the designing of new selective cancer therapies. Crizotinib is a selective ATP-competitive inhibitor for ALK, approved for its use in lung cancer with rearrangements on ALK gene. The reported cases describe the use of crizotinib as a bridging strategy prior to allotransplantation; there are no reported prolonged survivals under monotherapy with Crizotinib. We report a case of a primary refractory ALK+ anaplastic large-cell lymphoma that sustains complete response after 3 years of crizotinib monotherapy. PMID:27441079

  12. The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes.

    PubMed

    Zhang, Qian; Wei, Fang; Wang, Hong Yi; Liu, Xiaobin; Roy, Darshan; Xiong, Qun-Bin; Jiang, Shuguang; Medvec, Andrew; Danet-Desnoyers, Gwenn; Watt, Christopher; Tomczak, Ewa; Kalos, Michael; Riley, James L; Wasik, Mariusz A

    2013-12-01

    With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

  13. Regulation of the ALK1 ligands, BMP9 and BMP10.

    PubMed

    Li, Wei; Salmon, Richard M; Jiang, He; Morrell, Nicholas W

    2016-08-15

    Bone morphogenetic protein (BMP)9 and BMP10 are high affinity ligands for activin receptor-like kinase 1 (ALK1), a type I BMP receptor mainly expressed on vascular endothelial cells (ECs). ALK1-mediated BMP9/BMP10 signalling pathways have emerged as essential in EC biology and in angiogenesis. Several genetic mutations in the genes encoding the ligands and receptors of this pathway have been reported in two cardiovascular diseases, pulmonary arterial hypertension (PAH) and hereditary haemorrhagic telangiectasia (HHT). Administration of recombinant BMP9 reverses experimental PAH in preclinical rodent models. Dalantercept, an Fc-fusion protein of the extracellular domain of ALK1 and a ligand trap for BMP9 and BMP10, is in phase II clinical trials for anti-tumour angiogenesis. Understanding the regulation of BMP9 and BMP10, at both gene and protein levels, under physiological and pathological conditions, will reveal essential information and potential novel prognostic markers for the BMP9/BMP10-targeted therapies. PMID:27528761

  14. Quantitative determination of S-alk(en)ylcysteine-S-oxides by micellar electrokinetic capillary chromatography.

    PubMed

    Kubec, Roman; Dadáková, Eva

    2008-11-28

    A novel method for determination of S-alk(en)ylcysteine-S-oxides by capillary electrophoresis has been developed and validated. The method is based on extraction of these sulfur amino acids by methanol, their derivatization by fluorenylmethyl chloroformate and subsequent separation by micellar electrokinetic capillary chromatography. Main advantages of the new method are simplicity, sensitivity, high specificity and very low running costs, making it suitable for routine analysis of a large number of samples. Employing this method, the content of S-alk(en)ylcysteine-S-oxides was determined in 12 commonly consumed alliaceous and cruciferous vegetables (e.g. garlic, onion, leek, chive, cabbage, radish, cauliflower and broccoli). The total content of these amino acids in the Allium species evaluated varied between 0.59 and 12.3mg g(-1) fresh weight. Whereas alliin was found only in garlic, isoalliin was the major S-alk(en)ylcysteine-S-oxide in onion, leek, chive and shallot. On the other hand, the cruciferous species analyzed contained only methiin in the range of 0.06-2.45mg g(-1) fresh weight. PMID:18952220

  15. Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva.

    PubMed

    Chaikuad, Apirat; Alfano, Ivan; Kerr, Georgina; Sanvitale, Caroline E; Boergermann, Jan H; Triffitt, James T; von Delft, Frank; Knapp, Stefan; Knaus, Petra; Bullock, Alex N

    2012-10-26

    Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

  16. Fatal Haemoptysis Associated with Dramatic Response to Crizotinib in an ALK-Rearranged Lung Adenocarcinoma

    PubMed Central

    Mussat, Elodie; Giraud, Violaine; Julie, Catherine; Chinet, Thierry

    2016-01-01

    The presence of an ALK (Anaplastic Lymphoma Kinase) rearrangement is a rare molecular feature in Non-Small Cell Lung Carcinoma (NSCLC), and concerns mainly non- or light smokers, young patients, with adenocarcinoma histological type. These tumours are particularly sensitive to Alk-targeted therapies, as crizotinib. Crizotinib is usually well-tolerated. We report a case of fatal haemoptysis associated with dramatic response to crizotinib in a patient with an ALK-rearranged lung adenocarcinoma. The patient presented a mediastinal invasion with tracheal involvement and compression of the right pulmonary artery. The initial evolution under crizotinib was good with tumour response. At 6 weeks of crizotinib the patient presented a massive haemoptysis with a tracheobronchial fistula and pneumomediastinum. She died of acute respiratory failure. Our case is the first to report a fatal effect of crizotinib associated with tumour necrosis and good tumour response on a massive mediastinal infiltration. Precautions are recommended with the use of crizotinib in proximal lung tumours with vascular invasion. PMID:27134984

  17. Identification of ALK5 inhibitor via structure-based virtual screening and ADMET prediction.

    PubMed

    Lin, Aibin; Cai, Zhongqi; Hu, Guoping; Li, Qiang

    2015-01-01

    TGF-β plays a critical role in the initiation and progression of fibrosis in various organ systems such as kidney, heart, lung and liver. TGF-β and its receptors (ALK5 and TβR II) are able to control the cellular growth and promote several biological responses. To date, many pharmaceutical companies have employed virtual screening to identify potent inhibitors against ALK5. Nevertheless, none of these studies had involved the in silico ADMET evaluation and Raccoon filtering. In our experiment, all 57423 molecules were downloaded from TCM database and were filtered and converted to PDBQT formats by Raccoon software. Then 24 189 structures were run through AutoDock Vina in PyRx 0.8, 164 molecules were selected and further evaluated by ADMET Predictor 6.5, and 56 structures were selected and docked by Glide 6.2. Finally, the top 10 hits were identified as promising oral ALK5 inhibitors according to their Glide scores. The Glide scores of the best two compounds, 40686 and 33534, were -10.75 and -10.30 kcal/mol, respectively. This research provides a set of combined and detailed virtual screening protocol and is helpful for explaining the mechanism of receptor-ligand interactions.

  18. ALK5-dependent TGF-β signaling is a major determinant of late stage adult neurogenesis

    PubMed Central

    He, Yingbo; Zhang, Hui; Yung, Andrea; Villeda, Saul A; Jaeger, Philipp A; Olayiwola, Oluwatobi; Fainberg, Nina; Wyss-Coray, Tony

    2014-01-01

    The transforming growth factor-β (TGF-β) signaling pathway serves critical functions in central nervous system (CNS) development, but apart from its proposed neuroprotective actions, its physiological role in the adult brain is unclear. We observed a prominent activation of TGF-β signaling in the adult dentate gyrus and expression of downstream Smad proteins in this neurogenic zone. Consistent with a function of TGF-β signaling in adult neurogenesis, genetic deletion of the TGF-β receptor ALK5 reduced the number, migration, and dendritic arborization of newborn neurons. Conversely, constitutive activation of neuronal ALK5 in forebrain caused a striking increase in these aspects of neurogenesis and was associated with higher expression of c-fos in newborn neurons and with stronger memory function. Our findings describe a new and unexpected role for ALK5-dependent TGF-β signaling as a regulator of the late stages of adult hippocampal neurogenesis which may have implications for changes in neurogenesis during aging and disease. PMID:24859199

  19. Primary structure of chicken muscle pyruvate kinase mRNA.

    PubMed Central

    Lonberg, N; Gilbert, W

    1983-01-01

    We have determined the cDNA sequence corresponding to chicken muscle pyruvate kinase mRNA; the predicted coding region spans 529 amino acids and establishes the complete amino acid sequence for the vertebrate enzyme. We demonstrate that the level of mRNA for this enzyme is under developmental control and suggest a structural model for the protein kinase-mediated regulation of the mammalian liver isozyme. We report a method for the direct analysis of, and the preparation of cDNA probes from, mRNA which has been fractionated on methylmercury/agarose gels. Images PMID:6574503

  20. Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain

    PubMed Central

    Chen, Wanqiu; Guo, Yi; Walker, Espen J.; Shen, Fanxia; Jun, Kristine; Oh, S. Paul; Degos, Vincent; Lawton, Michael T.; Tihan, Tarik; Davalos, Dimitrios; Akassoglou, Katerina; Nelson, Jeffrey; Pile-Spellman, John; Su, Hua; Young, William L.

    2013-01-01

    Objective Vessels in brain arteriovenous malformations (bAVM) are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 (HHT2) patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell-coverage. Methods and Results Adult Alk11f/2f mice (loxP sites flanking exons 4-6) and wild-type (WT) mice were injected with 2×107 PFU Ad-Cre and 2×109 genome copies of AAV-VEGF to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed eight weeks later. Compared to WT mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 vs. 40±13×103, P=0.001), iron deposition (508±506 pixels/mm2 vs. 6 ±49, P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 vs. 240±104 Iba1+, P=0.001) after VEGF stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-SMA- vessels (52±9% vs. 12±7%, P<0.001), fewer vascular associated pericytes (503±179/mm2 vs. 931±115, P<0.001), and reduced PDGFR-β expression (26±9%, P<0.001). Conclusion Reduction of mural cell coverage in response to VEGF stimulation is a potential mechanism for the impairment of vessel wall integrity in HHT2-associated bAVM. PMID:23241407

  1. Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

    PubMed Central

    Kim, Bu-Yeo; Jeong, SangKyun; Lee, Seo-Young; Lee, So Min; Gweon, Eun Jeong; Ahn, Hyunjun; Kim, Janghwan; Chung, Sun-Ku

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs. PMID:27256111

  2. Clinical and Pathologic Findings of Spitz Nevi and Atypical Spitz Tumors with ALK Fusions

    PubMed Central

    Busam, Klaus J; Kutzner, Heinz; Cerroni, Lorenzo; Wiesner, Thomas

    2016-01-01

    Spitz tumors represent a group of melanocytic neoplasms that typically affects young individuals. Microscopically the lesions are composed of cytologically distinct spindle and epithelioid melanocytes, with a range in the architectural display or the cells, their nuclear features, and secondary epidermal or stromal changes. Recently, kinase fusions have been documented in a subset of Spitz tumors, but there is limited information on the clinical and pathologic features associated with those lesions. Here, we report a series of 17 patients (9 male, 8 female) with spitzoid neoplasms showing ALK fusions (5 Spitz nevi and 12 atypical Spitz tumors). The patients’ ages ranged from 2 years to 35 years (mean = 17; median = 16). Most lesions were located on the lower extremities and presented clinically as polypoid nodules. All tumors were compound melanocytic proliferations with a predominant intradermal growth. Tumor thickness ranged from 1.1 to 6 mm (mean = 2.9 mm; median = 2.5 mm). The most characteristic histopathologic feature of the tumors (seen in all but two lesions) was a plexiform dermal growth of intersecting fascicles of fusiform melanocytes. All but two tumors were amelanotic. All tumors were strongly immunoreactive for ALK. The ALK rearrangements were confirmed in all cases by fluorescence in situ hybridization (FISH) and the fusion partner was determined by quantitative polymerase chain reaction as TPM3 (tropomyosin 3) in 11 cases and DCTN1 (dynactin 1) in 6 cases. None of the eight tumors, which were analyzed by FISH for copy number changes of 6p, 6q, 9p, or 11q met criteria for melanoma. Two patients underwent a sentinel lymph node biopsy, and in both cases melanocytes nests were found in the subcapsular sinus of the node. Array comparative genomic hybridization of these two tumors revealed no chromosomal gains or losses. In conclusion, our study revealed that Spitz nevi/tumors with ALK rearrangement show a characteristic plexiform morphology and that

  3. ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes.

    PubMed

    Passoni, Lorena; Scardino, Antonio; Bertazzoli, Carla; Gallo, Barbara; Coluccia, Addolorata M L; Lemonnier, François A; Kosmatopoulos, Konstadinos; Gambacorti-Passerini, Carlo

    2002-03-15

    Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201-restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89- and p375-86-induced peptide-specific CTL lines were able to specifically release interferon-gamma (IFN-gamma) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus-transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I-restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.

  4. Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

    PubMed

    Takeoka, Kayo; Okumura, Atsuko; Maesako, Yoshitomo; Akasaka, Takashi; Ohno, Hitoshi

    2015-03-01

    This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

  5. Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non-Small Cell Lung Cancer Cells.

    PubMed

    Dong, Xuyuan; Fernandez-Salas, Ester; Li, Enxiao; Wang, Shaomeng

    2016-03-01

    Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non-small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.

  6. Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non–Small Cell Lung Cancer Cells

    PubMed Central

    Dong, Xuyuan; Fernandez-Salas, Ester; Li, Enxiao; Wang, Shaomeng

    2016-01-01

    Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non–small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC. PMID:26992917

  7. Rearranged Anaplastic Lymphoma Kinase (ALK) Gene in Adult-Onset Papillary Thyroid Cancer Amongst Atomic Bomb Survivors

    PubMed Central

    Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

    2012-01-01

    Background We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAFV600E) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. Methods The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (5′ RACE). Results We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK

  8. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    PubMed

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  9. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

    PubMed

    Ambrogio, Chiara; Martinengo, Cinzia; Voena, Claudia; Tondat, Fabrizio; Riera, Ludovica; di Celle, Paola Francia; Inghirami, Giorgio; Chiarle, Roberto

    2009-11-15

    Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

  10. Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family

    PubMed Central

    Augustine, Rehna; Bisht, Naveen C.

    2015-01-01

    Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 μmoles g−1 DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops. PMID:26657321

  11. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

    PubMed Central

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

  12. Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

    PubMed Central

    Buetti-Dinh, Antoine; O’Hare, Thomas

    2016-01-01

    A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance. PMID:27669408

  13. Identification of a new subclass of ALK-negative ALCL expressing aberrant levels of ERBB4 transcripts.

    PubMed

    Scarfò, Irene; Pellegrino, Elisa; Mereu, Elisabetta; Kwee, Ivo; Agnelli, Luca; Bergaggio, Elisa; Garaffo, Giulia; Vitale, Nicoletta; Caputo, Manuel; Machiorlatti, Rodolfo; Circosta, Paola; Abate, Francesco; Barreca, Antonella; Novero, Domenico; Mathew, Susan; Rinaldi, Andrea; Tiacci, Enrico; Serra, Sara; Deaglio, Silvia; Neri, Antonino; Falini, Brunangelo; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio; Piva, Roberto

    2016-01-14

    Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions. PMID:26463425

  14. Alectinib induced CNS radiation necrosis in an ALK+NSCLC patient with a remote (7 years) history of brain radiation.

    PubMed

    Ou, Sai-Hong Ignatius; Weitz, Michael; Jalas, John R; Kelly, Daniel F; Wong, Vanessa; Azada, Michele C; Quines, Oliver; Klempner, Samuel J

    2016-06-01

    Alectinib is a second generation ALK inhibitor that has significant clinical activity in central nervous system (CNS) metastases in anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Pseudoprogression (PsP) due to radiation necrosis during alecitnib treatment of central nervous system (CNS) metastases from ALK-rearranged NSCLC as been reported. Hence, distinguishing radiation-related PsP from alectinib-induced radiographic changes is important to avoid erroneous early trial discontinuation and abandonment of an effective treatment. However, it remains difficult to assess casuality of radiation necrosis is related to recent direct radiation or induced by alectinib treatment or both. It is also unknown how long from previous radiation can alectinib still induce radiation necrosis. Here we reported a crizotinib-refractory ALK-positive NSCLC patient who develop radiation necrosis in one of his metastatic CNS lesions after approximately 12 months of alectinib treatment who otherwise had on-going CNS response on alectinib. His most recent radiation to his CNS metastases was 7 years prior to the start of alectinib. This case illustrates that in the setting of pror CNS radiation, given the significant clinical activity of alectinib in CNS metastases in ALK-positive NSCLC patients the risk of CNS radiation necrosis remains long after previous radiation to the CNS metastases has been completed and can occur after durable response of treatment. PMID:27133743

  15. Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family.

    PubMed

    Augustine, Rehna; Bisht, Naveen C

    2015-01-01

    Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 μmoles g(-1) DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops. PMID:26657321

  16. Downregulation of NPM-ALK by siRNA causes anaplastic large cell lymphoma cell growth inhibition and augments the anti cancer effects of chemotherapy in vitro.

    PubMed

    Hsu, Faye Yuan-yi; Zhao, Yi; Anderson, W French; Johnston, Patrick B

    2007-06-01

    The fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), results from the chromosome translocation t(2;5)(p23;q25) and is present in 50-70 percent of anaplastic large-cell lymphomas (ALCLs). NPM-ALK is a constitutively activated kinase that transforms cells through stimulating several mitogenic signaling pathways. To examine if the NPM-ALK is a potential therapeutic target in ALCL, we used siRNA to specifically downregulate the expression of the NPM-ALK in ALCL cell lines. In this report, we demonstrated viability loss in t(2;5)-positive ALCL cell lines, SUDHL-1 and Karpas 299 cells, but not in lymphoma cell lines without the chromosome translocation, Jurkat and Granta 519 cells. Further study demonstrated that the downregulation of NPM-ALK resulted in decreased cell proliferation and increased cell apoptosis. When used in combination with chemotherapeutic agents, such as doxorubicin, the inhibition of the NPM-ALK augments the chemosensitivity of the tumor cells. These results revealed the importance of continuous expression of NPM-ALK in maintaining the growth of ALCL cells. Our data also suggested that the repression of the fusion gene might be a potential novel therapeutic strategy for NPM-ALK positive ALCLs.

  17. Crizotinib (PF-2341066) induces apoptosis due to downregulation of pSTAT3 and BCL-2 family proteins in NPM-ALK(+) anaplastic large cell lymphoma.

    PubMed

    Hamedani, Farid Saei; Cinar, Munevver; Mo, Zhicheng; Cervania, Melissa A; Amin, Hesham M; Alkan, Serhan

    2014-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product with tyrosine kinase activity and is expressed in substantial subset of anaplastic large cell lymphomas (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3). Although NPM-ALK(+) ALCL overall shows a better prognosis, there is a sub-group of patients who relapses and is resistant to conventional chemotherapeutic regimens. NPM-ALK is a potential target for small molecule kinase inhibitors. Crizotinib (PF-2341066) is a small, orally bioavailable molecule that inhibits growth of tumors with ALK activity as shown in a subgroup of non-small lung cancer patients with EML4-ALK expression. In this study, we have investigated the in vitro effects of Crizotinib in ALCL cell line with NPM-ALK fusion. Crizotinib induced marked downregulation of STAT3 phosphorylation, which was associated with significant apoptotic cell death. Apoptosis induction was attributed to caspase-3 cleavage and marked downregulation of the Bcl-2 family of proteins including MCL-1. These findings implicate that Crizotinib has excellent potential to treat patients with NPM-ALK(+) ALCL through induction of apoptotic cell death and downregulation of major oncogenic proteins in this aggressive lymphoma.

  18. Involvement of Grb2 adaptor protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and anaplastic large cell lymphoma growth.

    PubMed

    Riera, Ludovica; Lasorsa, Elena; Ambrogio, Chiara; Surrenti, Nadia; Voena, Claudia; Chiarle, Roberto

    2010-08-20

    Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

  19. Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas.

    PubMed

    Colomba, A; Courilleau, D; Ramel, D; Billadeau, D D; Espinos, E; Delsol, G; Payrastre, B; Gaits-Iacovoni, F

    2008-04-24

    The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.

  20. Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

    PubMed

    Wang, Peng; Wu, Fang; Zhang, Jingdong; McMullen, Todd; Young, Leah C; Ingham, Robert J; Li, Liang; Lai, Raymond

    2011-02-01

    It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

  1. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE. PMID:25432418

  2. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    EPA Science Inventory

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  3. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE.

  4. Structure–Activity Relationship of 3,5-Diaryl-2-aminopyridine ALK2 Inhibitors Reveals Unaltered Binding Affinity for Fibrodysplasia Ossificans Progressiva Causing Mutants

    PubMed Central

    2015-01-01

    There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure–activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG. PMID:25101911

  5. Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants.

    PubMed

    Mohedas, Agustin H; Wang, You; Sanvitale, Caroline E; Canning, Peter; Choi, Sungwoon; Xing, Xuechao; Bullock, Alex N; Cuny, Gregory D; Yu, Paul B

    2014-10-01

    There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure-activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG.

  6. ETV6-NTRK3 Is Expressed in a Subset of ALK-Negative Inflammatory Myofibroblastic Tumors.

    PubMed

    Alassiri, Ali H; Ali, Rola H; Shen, Yaoqing; Lum, Amy; Strahlendorf, Caron; Deyell, Rebecca; Rassekh, Rod; Sorensen, Poul H; Laskin, Janessa; Marra, Marco; Yip, Stephen; Lee, Cheng-Han; Ng, Tony L

    2016-08-01

    Inflammatory myofibroblastic tumor (IMT) is a genetically heterogenous tumor of the viscera and soft tissues, with multiple molecular features having been demonstrated in this tumor type. About 50% of cases harbor an anaplastic lymphoma kinase (ALK) gene rearrangement, and recent studies have described novel fusions involving the ROS1 and PDGFRβ genes in a subset of ALK-negative cases. However, the molecular features of the remaining subset of cases are not yet defined. We report a case of a large, highly aggressive IMT of the lung in a 17-year-old girl. This case was molecularly characterized through whole-genome and transcriptome sequencing. Subsequently, we investigated a cohort of 15 ALK-negative IMTs of various anatomic sites. All cases were screened using fluorescence in situ hybridization (FISH) for rearrangement of the ETV6 locus and with reverse transcription polymerase chain reaction (RT-PCR) for the ETV6-NTRK3 fusion transcript. Whole-genome and transcriptome sequencing revealed an ETV6-NTRK3 fusion transcript in our index case. This was confirmed by FISH studies for ETV6 gene rearrangement, as well as by RT-PCR. In addition, 2 additional cases in our cohort demonstrated ETV6 rearrangement by FISH. The presence of ETV6-NTRK3 fusion transcript was demonstrated by RT-PCR in one of these additional cases. In summary, we demonstrate the expression of the ETV6-NTRK3 fusion oncogene in a small subset of IMTs, lending further support to the role of oncogenic tyrosine kinases in the pathophysiology of this tumor type. Our data also further expand the growing spectrum of tumor types expressing the ETV6-NTRK3 fusion. PMID:27259007

  7. An integrated molecular modeling approach for in silico design of new tetracyclic derivatives as ALK inhibitors.

    PubMed

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2016-10-01

    Anaplastic lymphoma kinase (ALK), a promising therapeutic target for treatment of human cancers, is a receptor tyrosine kinase that instigates the activation of several signal transduction pathways. In the present study, in silico methods have been employed in order to explore the structural features and functionalities of a series of tetracyclic derivatives displaying potent inhibitory activity toward ALK. Initially docking was performed using GLIDE 5.6 to probe the bioactive conformation of all the compounds and to understand the binding modes of inhibitors. The docking results revealed that ligand interaction with Met 1199 plays a crucial role in binding of inhibitors to ALK. Further to establish a robust 3D-QSAR model using CoMFA and CoMSIA methods, the whole dataset was divided into three splits. Model obtained from Split 3 showed high accuracy ([Formula: see text] of 0.700 and 0.682, [Formula: see text] of 0.971 and 0.974, [Formula: see text] of 0.673 and 0.811, respectively for CoMFA and CoMSIA). The key structural requirements for enhancing the inhibitory activity were derived from CoMFA and CoMSIA contours in combination with site map analysis. Substituting small electronegative groups at Position 8 by replacing either morpholine or piperidine rings and maintaining hydrophobic character at Position 9 in tetracyclic derivatives can enhance the inhibitory potential. Finally, we performed molecular dynamics simulations in order to investigate the stability of protein ligand interactions and MM/GBSA calculations to compare binding free energies of co-crystal ligand and newly designed molecule N1. Based on the coherence of outcome of various molecular modeling studies, a set of 11 new molecules having potential predicted inhibitory activity were designed. PMID:26758803

  8. An integrated molecular modeling approach for in silico design of new tetracyclic derivatives as ALK inhibitors.

    PubMed

    Peddi, Saikiran Reddy; Sivan, Sree Kanth; Manga, Vijjulatha

    2016-10-01

    Anaplastic lymphoma kinase (ALK), a promising therapeutic target for treatment of human cancers, is a receptor tyrosine kinase that instigates the activation of several signal transduction pathways. In the present study, in silico methods have been employed in order to explore the structural features and functionalities of a series of tetracyclic derivatives displaying potent inhibitory activity toward ALK. Initially docking was performed using GLIDE 5.6 to probe the bioactive conformation of all the compounds and to understand the binding modes of inhibitors. The docking results revealed that ligand interaction with Met 1199 plays a crucial role in binding of inhibitors to ALK. Further to establish a robust 3D-QSAR model using CoMFA and CoMSIA methods, the whole dataset was divided into three splits. Model obtained from Split 3 showed high accuracy ([Formula: see text] of 0.700 and 0.682, [Formula: see text] of 0.971 and 0.974, [Formula: see text] of 0.673 and 0.811, respectively for CoMFA and CoMSIA). The key structural requirements for enhancing the inhibitory activity were derived from CoMFA and CoMSIA contours in combination with site map analysis. Substituting small electronegative groups at Position 8 by replacing either morpholine or piperidine rings and maintaining hydrophobic character at Position 9 in tetracyclic derivatives can enhance the inhibitory potential. Finally, we performed molecular dynamics simulations in order to investigate the stability of protein ligand interactions and MM/GBSA calculations to compare binding free energies of co-crystal ligand and newly designed molecule N1. Based on the coherence of outcome of various molecular modeling studies, a set of 11 new molecules having potential predicted inhibitory activity were designed.

  9. Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

    PubMed Central

    Nilsson, R. Jonas A.; Karachaliou, Niki; Berenguer, Jordi; Gimenez-Capitan, Ana; Schellen, Pepijn; Teixido, Cristina; Tannous, Jihane; Kuiper, Justine L.; Drees, Esther; Grabowska, Magda; van Keulen, Marte; Heideman, Danielle A.M.; Thunnissen, Erik; Dingemans, Anne-Marie C.; Viteri, Santiago; Tannous, Bakhos A.; Drozdowskyj, Ana; Rosell, Rafael; Smit, Egbert F.; Wurdinger, Thomas

    2016-01-01

    Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone. PMID:26544515

  10. Overcoming acquired resistance to kinase inhibition: the cases of EGFR, ALK and BRAF.

    PubMed

    Giroux, Simon

    2013-01-15

    In the past decade, several kinase inhibitors have been approved based on their clinical benefit for cancer patients. Unfortunately, in many cases, patients develop resistance to these agents via secondary mutations and alternative mechanisms. This review will focus on the cases of acquired resistance to EGFR and ALK inhibitors for non-small cell lung cancer patients and BRAF inhibitors for melanoma patients. I will overview the main causes of acquired resistance, and explore the chemical scaffolds as well as combination of drugs, used to tackle these major causes of resistance. PMID:23245516

  11. Connections Underlying Translation and mRNA Stability.

    PubMed

    Radhakrishnan, Aditya; Green, Rachel

    2016-09-11

    Gene expression and regulation in organisms minimally depends on transcription by RNA polymerase and on the stability of the RNA product (for both coding and non-coding RNAs). For coding RNAs, gene expression is further influenced by the amount of translation by the ribosome and by the stability of the protein product. The stabilities of these two classes of RNA, non-coding and coding, vary considerably: tRNAs and rRNAs tend to be long lived while mRNAs tend to be more short lived. Even among mRNAs, however, there is a considerable range in stability (ranging from seconds to hours in bacteria and up to days in metazoans), suggesting a significant role for stability in the regulation of gene expression. Here, we review recent experiments from bacteria, yeast and metazoans indicating that the stability of most mRNAs is broadly impacted by the actions of ribosomes that translate them. Ribosomal recognition of defective mRNAs triggers "mRNA surveillance" pathways that target the mRNA for degradation [Shoemaker and Green (2012) ]. More generally, even the stability of perfectly functional mRNAs appears to be dictated by overall rates of translation by the ribosome [Herrick et al. (1990), Presnyak et al. (2015) ]. Given that mRNAs are synthesized for the purpose of being translated into proteins, it is reassuring that such intimate connections between mRNA and the ribosome can drive biological regulation. In closing, we consider the likelihood that these connections between protein synthesis and mRNA stability are widespread or whether other modes of regulation dominate the mRNA stability landscape in higher organisms. PMID:27261255

  12. Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-alpha-hydroxyethanocytosine.

    PubMed

    Maciejewska, Agnieszka M; Ruszel, Karol P; Nieminuszczy, Jadwiga; Lewicka, Joanna; Sokołowska, Beata; Grzesiuk, Elzbieta; Kuśmierek, Jarosław T

    2010-02-01

    Etheno (epsilon) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate epsilon-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of epsilon-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac(+) revertants, the latter arose by GC-->AT or GC-->TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of varepsilonC proceeds via a relatively stable intermediate, 3,N(4)-alpha-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and microg, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA(+), alkB(+), and microg(+) controls. Considering the levels of CAA-induced Lac(+) revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of epsilonC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs epsilonC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and epsilonC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein. PMID:19941873

  13. Targeted Therapies in Non-Small Cell Lung Cancer-Beyond EGFR and ALK.

    PubMed

    Rothschild, Sacha I

    2015-01-01

    Systemic therapy for non-small cell lung cancer (NSCLC) has undergone a dramatic paradigm shift over the past decade. Advances in our understanding of the underlying biology of NSCLC have revealed distinct molecular subtypes. A substantial proportion of NSCLC depends on oncogenic molecular aberrations (so-called "driver mutations") for their malignant phenotype. Personalized therapy encompasses the strategy of matching these subtypes with effective targeted therapies. EGFR mutations and ALK translocation are the most effectively targeted oncogenes in NSCLC. EGFR mutations and ALK gene rearrangements are successfully being targeted with specific tyrosine kinase inhibitors. The number of molecular subgroups of NSCLC continues to grow. The scope of this review is to discuss recent data on novel molecular targets as ROS1, BRAF, KRAS, HER2, c-MET, RET, PIK3CA, FGFR1 and DDR2. Thereby the review will focus on therapeutic strategies targeting these aberrations. Moreover, the emerging challenge of acquired resistance to initially effective therapies will be discussed. PMID:26018876

  14. The type I BMP receptor ACVR1/ALK2 is required for chondrogenesis during development

    PubMed Central

    Rigueur, Diana; Brugger, Sean; Anbarchian, Teni; Kim, Jong Kil; Lee, Yoo Jin; Lyons, Karen

    2014-01-01

    Bone morphogenetic proteins (BMPs) are crucial regulators of chondrogenesis. BMPs transduce their signals through three type I receptors: BMPR1A, BMPR1B, and ACVR1/ALK2. Fibrodysplasia ossificans progressiva (FOP), a rare disorder characterized by progressive ossification of connective tissue, is caused by an activating mutation in Acvr1 (the gene that encodes ACVR1/ALK2). However, there are few developmental defects associated with FOP. Thus, the role of ACVR1 in chondrogenesis during development is unknown. Here we report the phenotype of mice lacking ACVR1 in cartilage. Acvr1CKO mice are viable but exhibit defects in the development of cranial and axial structures. Mutants exhibit a shortened cranial base, and cervical vertebrae are hypoplastic. Acvr1CKO adult mice develop progressive kyphosis. These morphological defects were associated with decreased levels of Smad1/5 and p38 activation, and with reduced rates of chondrocyte proliferation in vertebral cartilage. We also tested whether ACVR1 exerts coordinated functions with BMPR1A and BMPR1B through analysis of double mutants. Acvr1/Bmpr1a and Acvr1/Bmpr1b mutant mice exhibited generalized perinatal lethal chondrodysplasia that was much more severe than in any of the corresponding mutant strains. These findings demonstrate that ACVR1 is required for chondrocyte proliferation and differentiation, particularly in craniofacial and axial elements, but exerts coordinated functions with both BMPR1A and BMPR1B throughout the developing endochondral skeleton. PMID:25413979

  15. mRNA transcript therapy.

    PubMed

    Weissman, Drew

    2015-02-01

    mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery. PMID:25359562

  16. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    PubMed

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  17. Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

    PubMed Central

    Bonzheim, Irina; Irmler, Martin; Klier-Richter, Margit; Steinhilber, Julia; Anastasov, Nataša; Schäfer, Sabine; Adam, Patrick; Beckers, Johannes; Raffeld, Mark; Fend, Falko; Quintanilla-Martinez, Leticia

    2013-01-01

    C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. PMID:23741337

  18. Novel targeted therapies for resistant ALK-rearranged non-small-cell lung cancer: ceritinib and beyond.

    PubMed

    Kanaan, Zeyad; Kloecker, Goetz H; Paintal, Ajit; Perez, Cesar A

    2015-01-01

    Lung cancer is the leading cause of cancer-related mortality in both sexes, accounting for over one quarter of cancer deaths. Non-small-cell lung cancer (NSCLC) comprises 85%-90% of lung cancer diagnoses and despite advances in multimodality therapies, 5-year survival rates remain dismal with a median survival for patients with metastatic disease of 1 year. The positive outcomes of targeted therapies against the kinase domain of epidermal growth factor receptor in NSCLC triggered consistent efforts to identify the so-called driver mutations as other potential targets. Anaplastic large-cell kinase (ALK) gene rearrangements were identified and targeted resulting in promising response rates in early studies. Unfortunately, most of the patients treated with crizotinib, the first-generation ALK inhibitor, progressed within 9 months. Ceritinib is a second-generation ALK inhibitor that has demonstrated activity in crizotinib-resistant patients, becoming a promising treatment option in this population. Furthermore, additional novel ALK inhibitors and agents targeting alternative pathways have been recruited to rechallenge this evasive disease post-crizotinib resistance.

  19. Novel targeted therapies for resistant ALK-rearranged non-small-cell lung cancer: ceritinib and beyond

    PubMed Central

    Kanaan, Zeyad; Kloecker, Goetz H; Paintal, Ajit; Perez, Cesar A

    2015-01-01

    Lung cancer is the leading cause of cancer-related mortality in both sexes, accounting for over one quarter of cancer deaths. Non-small-cell lung cancer (NSCLC) comprises 85%–90% of lung cancer diagnoses and despite advances in multimodality therapies, 5-year survival rates remain dismal with a median survival for patients with metastatic disease of 1 year. The positive outcomes of targeted therapies against the kinase domain of epidermal growth factor receptor in NSCLC triggered consistent efforts to identify the so-called driver mutations as other potential targets. Anaplastic large-cell kinase (ALK) gene rearrangements were identified and targeted resulting in promising response rates in early studies. Unfortunately, most of the patients treated with crizotinib, the first-generation ALK inhibitor, progressed within 9 months. Ceritinib is a second-generation ALK inhibitor that has demonstrated activity in crizotinib-resistant patients, becoming a promising treatment option in this population. Furthermore, additional novel ALK inhibitors and agents targeting alternative pathways have been recruited to rechallenge this evasive disease post-crizotinib resistance. PMID:25945060

  20. In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

    PubMed

    Matturro, Bruna; Frascadore, Emanuela; Cappello, Simone; Genovese, Mariella; Rossetti, Simona

    2016-09-15

    This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes. PMID:27315756

  1. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  2. Metachronous primary uterine cancer surgically resected during Crizotinib treatment in a ALK-rearranged advanced lung adenocarcinoma

    PubMed Central

    Misino, Andrea; Scattone, Anna; Caldarola, Lucia; Petroni, Stella; Logroscino, Antonio; Montagna, Elisabetta Sara; Serio, Gabriella; Simone, Giovanni; Galetta, Domenico

    2016-01-01

    Rearrangements of the anaplastic lymphoma kinase (ALK) gene are present in 3% to 7% of non-small-cell lung cancers (NSCLCs). Patients harboring ALK rearrangements show very favourable outcomes if treated with targeted agents, among which crizotinib is the first and best studied. Crizotinib, an oral small-molecule tyrosine kinase inhibitor of ALK, MET, and ROS1 kinases, is a very active and well tolerated drug. Nevertheless, the optimal therapy management with this new drug is still partially unknown, especially with regard to the safety of combined treatments. Recently, the integration of locoregional treatments has been proposed as a feasible multimodality strategy in selected patients with good clinical conditions and slow-growing or oligoprogressive disease. In this report, a case of advanced lung adenocarcinoma, progressed after first line chemotherapy and re-biopsied detecting ALK rearrangement, is described. During crizotinib treatment the primary lung tumor showed an excellent regression; meanwhile a major surgery for a metachronous uterine cancer was safely and successfully carried out. PMID:26958511

  3. Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells

    PubMed Central

    Aveic, Sanja; Pantile, Marcella; Seydel, Anke; Esposito, Maria Rosaria; Zanon, Carlo; Li, Gary; Tonini, Gian Paolo

    2016-01-01

    Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib. PMID:26735175

  4. Hereditary haemorrhagic telangiectasia: a questionnaire based study to delineate the different phenotypes caused by endoglin and ALK1 mutations

    PubMed Central

    Berg, J; Porteous, M; Reinhardt, D; Gallione, C; Holloway, S; Umasunthar, T; Lux, A; McKinnon, W; Marchuk, D; Guttmacher, A

    2003-01-01

    Background: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia characterised by mucocutaneous telangiectasis, epistaxis, gastrointestinal haemorrhage, and arteriovenous malformations in the lung and brain. Causative mutations for HHT have been identified in two genes, endoglin and ALK1, which encode proteins involved in serine-threonine kinase signalling in the endothelial cell. Methods: A number of people affected with HHT had completed a postal questionnaire as part of an international study to delineate the HHT phenotype. We identified questionnaires completed by subjects in whom we had identified a mutation in endoglin or ALK1. Further questionnaires were sent to families with known mutations. Data were only included from questionnaires returned by people known to carry disease causing mutations. Results: Questionnaires were completed by 83 subjects with known mutations. Of these, 49 had endoglin mutations (HHT1) and 34 had ALK1 mutations (HHT2). Subjects with HHT1 reported an earlier onset of epistaxis (p=0.01) and telangiectasis (p=0.0001) than those with HHT2. Pulmonary arteriovenous malformations were only reported in the endoglin mutation group in our study (p<0.001). Conclusions: Our questionnaire based study provides evidence that the HHT phenotype caused by mutations in endoglin (HHT1) is distinct from, and more severe than, HHT caused by mutations in ALK1 (HHT2). This has significant implications for diagnosis, screening, and treatment in the two different forms of HHT, as well as for understanding the pathogenesis of the disease. PMID:12920067

  5. NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair.

    PubMed

    Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

    2015-05-15

    The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography-mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2(Y238F) mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2(Y238F) into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2(Y238F) abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2(Y238F) into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR.

  6. NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of JNK/cJun signaling in anaplastic large-cell lymphoma.

    PubMed

    Leventaki, Vasiliki; Drakos, Elias; Medeiros, L Jeffrey; Lim, Megan S; Elenitoba-Johnson, Kojo S; Claret, Francois X; Rassidakis, George Z

    2007-09-01

    Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K>R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK(+) ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK(+) ALCL cells. cJun phosphorylation in NPM-ALK(+) ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

  7. Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons.

    PubMed

    Wallisch, Stefanie; Gril, Tjasa; Dong, Xia; Welzl, Gerd; Bruns, Christian; Heath, Ester; Engel, Marion; Suhadolc, Marjetka; Schloter, Michael

    2014-01-01

    Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and

  8. Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons

    PubMed Central

    Wallisch, Stefanie; Gril, Tjasa; Dong, Xia; Welzl, Gerd; Bruns, Christian; Heath, Ester; Engel, Marion; Suhadolc, Marjetka; Schloter, Michael

    2014-01-01

    Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and

  9. ALK-positive anaplastic large cell lymphoma limited to the skin: clinical, histopathological and molecular analysis of 6 pediatric cases. A report from the ALCL99 study.

    PubMed

    Oschlies, Ilske; Lisfeld, Jasmin; Lamant, Laurence; Nakazawa, Atsuko; d'Amore, Emanuele S G; Hansson, Ulrika; Hebeda, Konnie; Simonitsch-Klupp, Ingrid; Maldyk, Jadwiga; Müllauer, Leonhard; Tinguely, Marianne; Stücker, Markus; Ledeley, Marie-Cecile; Siebert, Reiner; Reiter, Alfred; Brugières, Laurence; Klapper, Wolfram; Woessmann, Wilhelm

    2013-01-01

    Anaplastic large cell lymphomas are peripheral T-cell lymphomas that are characterized by a proliferation of large anaplastic blasts expressing CD30. In children, systemic anaplastic large cell lymphomas often present at advanced clinical stage and harbor translocations involving the anaplastic lymphoma kinase (ALK) gene leading to the expression of chimeric anaplastic lymphoma kinase (ALK)-fusion proteins. Primary cutaneous anaplastic large cell lymphoma is regarded as an ALK-negative variant confined to the skin and is part of the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders. Thirty-three of 487 pediatric patients registered within the Anaplastic Large Cell Lymphoma-99 trial (1999 to 2006) presented with a skin limited CD30-positive lympho-proliferative disorder. In 23 of the 33 patients, material for international histopathological review was available, and the cases were studied for histopathological, immunophenotypical and clinical features as well as for breaks within the ALK gene. Five of 23 cases and one additional case (identified after closure of the trial) expressed ALK-protein. Complete staging excluded any other organ involvement in all children. Expression of ALK proteins was demonstrated by immunohistochemistry in all cases and the presence of breaks of the ALK gene was genetically confirmed in 5 evaluable cases. The histopathological and clinical picture of these skin-restricted ALK-positive lymphomas was indistinguishable from that of cutaneous anaplastic large cell lymphoma. Five children presented with a single skin lesion that was completely resected in 4 and incompletely resected in one. Three of these patients received no further therapy, 2 additional local radiotherapy, and one chemotherapy. All children remain in complete remission with a median follow up of seven years (range 1-8 years). We present 6 pediatric cases of ALK-positive primary cutaneous anaplastic large cell lymphomas. After thorough

  10. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    PubMed

    Wang, Jinghui; Cai, Yiran; Dong, Yujie; Nong, Jingying; Zhou, Lijuan; Liu, Guimei; Su, Dan; Li, Xi; Wu, Shafei; Chen, Xuejing; Qin, Na; Zeng, Xuan; Zhang, Haiqing; Zhang, Zongde; Zhang, Shucai

    2014-01-01

    EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  11. TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling.

    PubMed

    Remst, Dennis F G; Blaney Davidson, Esmeralda N; Vitters, Elly L; Bank, Ruud A; van den Berg, Wim B; van der Kraan, Peter M

    2014-01-01

    Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

  12. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  13. Alectinib's activity against CNS metastases from ALK-positive non-small cell lung cancer: a single institution case series.

    PubMed

    Metro, Giulio; Lunardi, Gianluigi; Bennati, Chiara; Chiarini, Pietro; Sperduti, Isabella; Ricciuti, Biagio; Marcomigni, Luca; Costa, Cinzia; Crinò, Lucio; Floridi, Piero; Gori, Stefania; Chiari, Rita

    2016-09-01

    In the present study we assessed the activity of the next-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (-TKI) alectinib, in patients with ALK-postive, advanced non-small cell lung cancer (NSCLC) and central nervous system (CNS) metastases. NSCLCs with ALK-positive disease, as assessed by fluorescence in situ hybridization, and CNS metastases were treated with alectinib 600 mg BID. Included patients were followed prospectively in order to evaluate the efficacy of the drug, with particular emphasis on activity in the CNS. Eleven consecutive patients were enrolled. The majority of them were pretreated with crizotinib (n = 10, 90.9 %), and cranial radiotherapy (n = 8, 72.7 %). Six of the seven patients with measurable CNS disease experienced a CNS response, including three patients who were naïve for cranial radiation. Median duration of response was 8 months. For the whole population, median CNS-progression-free survival (-PFS), systemic-PFS, overall-PFS, overall survival, and 1-year survival were 8, 11, 8, 13 months, and 31.1 %, respectively. Two patients experiencing a CNS response were assessed for alectinib's concentrations in serum and cerebro-spinal fluid (CSF), and showed a CSF-to-serum ratio ranging from 0.001 to 0.003 ng/mL. Alectinib is highly active against CNS metastases from ALK-positive NSCLCs, irrespective of prior treatment(s) with ALK-TKI(s) and/or cranial radiotherapy. The low CSF-to-serum ratio of alectinib suggests that measuring the concentrations of the drug in the CSF may not be a reliable surrogate of its distribution into the CNS. PMID:27324494

  14. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases

    PubMed Central

    Lee, Taebum; Lee, Boram; Choi, Yoon-La; Han, Joungho; Ahn, Myung-Ju; Um, Sang-Won

    2016-01-01

    Background: Although epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and anaplastic lymphoma kinase (ALK) mutations in non-small cell lung cancer (NSCLC) were thought to be mutually exclusive, some tumors harbor concomitant mutations. Discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. Methods: In 6,637 patients with NSCLC, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. Clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease-free survival. Histologic features included dominant patterns, nuclear and cytoplasmic features. Results: All patients were diagnosed with adenocarcinoma and had an EGFR mutation. Six patients had concomitant KRAS mutations and the other six had KRAS mutations. Five of six EGFR-KRAS mutation patients showed papillary and acinar histologic patterns with hobnail cells. Three of six received EGFR tyrosine kinase inhibitor (TKI) and showed partial response for 7–29 months. All six EGFR-ALK mutation patients showed solid or cribriform patterns and three had signet ring cells. Five of six EGFR-ALK mutation patients received EGFR TKI and/or ALK inhibitor and four showed partial response or stable disease, except for one patient who had acquired an EGFR mutation. Conclusions: EGFR and ALK mutations play an important role as driver mutations in double mutated NSCLC, and morphologic analysis can be used to predict treatment response. PMID:27086595

  15. Decoding Tumor Phenotypes for ALK, ROS1, and RET Fusions in Lung Adenocarcinoma Using a Radiomics Approach

    PubMed Central

    Yoon, Hyun Jung; Sohn, Insuk; Cho, Jong Ho; Lee, Ho Yun; Kim, Jae-Hun; Choi, Yoon-La; Kim, Hyeseung; Lee, Genehee; Lee, Kyung Soo; Kim, Jhingook

    2015-01-01

    Abstract Quantitative imaging using radiomics can capture distinct phenotypic differences between tumors and may have predictive power for certain phenotypes according to specific genetic mutations. We aimed to identify the clinicoradiologic predictors of tumors with ALK (anaplastic lymphoma kinase), ROS1 (c-ros oncogene 1), or RET (rearranged during transfection) fusions in patients with lung adenocarcinoma. A total of 539 pathologically confirmed lung adenocarcinomas were included in this retrospective study. The baseline clinicopathologic characteristics were retrieved from the patients’ medical records and the ALK/ROS1/RET fusion status was reviewed. Quantitative computed tomography (CT) and positron emission tomography imaging characteristics were evaluated using a radiomics approach. Significant features for the fusion-positive tumor prediction model were extracted from all of the clinicoradiologic features, and were used to calculate diagnostic performance for predicting 3 fusions’ positivity. The clinicoradiologic features were compared between ALK versus ROS1/RET fusion-positive tumors to identify the clinicoradiologic similarity between the 2 groups. The fusion-positive tumor prediction model was a combination of younger age, advanced tumor stage, solid tumor on CT, higher values for SUVmax and tumor mass, lower values for kurtosis and inverse variance on 3-voxel distance than those of fusion-negative tumors (sensitivity and specificity, 0.73 and 0.70, respectively). ALK fusion-positive tumors were significantly different in tumor stage, central location, SUVmax, homogeneity on 1-, 2-, and 3-voxel distances, and sum mean on 2-voxel distance compared with ROS1/RET fusion-positive tumors. ALK/ROS1/RET fusion-positive lung adenocarcinomas possess certain clinical and imaging features that enable good discrimination of fusion-positive from fusion-negative lung adenocarcinomas. PMID:26469915

  16. Crizotinib for the Treatment of ALK-Rearranged Non-Small Cell Lung Cancer: A Success Story to Usher in the Second Decade of Molecular Targeted Therapy in Oncology

    PubMed Central

    Bartlett, Cynthia Huang; Mino-Kenudson, Mari; Cui, Jean; Iafrate, A. John

    2012-01-01

    Crizotinib, an ALK/MET/ROS1 inhibitor, was approved by the U.S. Food and Drug Administration for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) in August 2011, merely 4 years after the first publication of ALK-rearranged NSCLC. The crizotinib approval was accompanied by the simultaneous approval of an ALK companion diagnostic fluorescent in situ hybridization assay for the detection of ALK-rearranged NSCLC. Crizotinib continued to be developed as an ALK and MET inhibitor in other tumor types driven by alteration in ALK and MET. Crizotinib has recently been shown to be an effective ROS1 inhibitor in ROS1-rearranged NSCLC, with potential future clinical applications in ROS1-rearranged tumors. Here we summarize the heterogeneity within the ALK- and ROS1-rearranged molecular subtypes of NSCLC. We review the past and future clinical development of crizotinib for ALK-rearranged NSCLC and the diagnostic assays to detect ALK-rearranged NSCLC. We highlight how the success of crizotinib has changed the paradigm of future drug development for targeted therapies by targeting a molecular-defined subtype of NSCLC despite its rarity and affected the practice of personalized medicine in oncology, emphasizing close collaboration between clinical oncologists, pathologists, and translational scientists. PMID:22989574

  17. The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

    PubMed Central

    Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H.

    2014-01-01

    Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo. PMID:25380037

  18. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Park, Jangchul; Kobayashi, Yoshihisa; Urayama, Kevin Y.; Yamaura, Hidekazu; Yatabe, Yasushi; Hida, Toyoaki

    2016-01-01

    This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB–IV) and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups’ characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159), KRAS mutations (n = 55), or ALK rearrangements (n = 51). Among the three groups, we evaluated only patients with stage IIIB–IV lung adenocarcinoma who had EGFR mutations (n = 126), KRAS mutations (n = 35), or ALK rearrangements (n = 47). We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009). Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003). Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively). Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049). Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively). Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively). In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  19. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB.

    PubMed

    Li, Deyu; Delaney, James C; Page, Charlotte M; Yang, Xuedong; Chen, Alvin S; Wong, Cintyu; Drennan, Catherine L; Essigmann, John M

    2012-05-30

    The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo. PMID:22512456

  20. A large-scale cross-sectional study of ALK rearrangements and EGFR mutations in non-small-cell lung cancer in Chinese Han population.

    PubMed

    Hong, Shaodong; Fang, Wenfeng; Hu, Zhihuang; Zhou, Ting; Yan, Yue; Qin, Tao; Tang, Yanna; Ma, Yuxiang; Zhao, Yuanyuan; Xue, Cong; Huang, Yan; Zhao, Hongyun; Zhang, Li

    2014-01-01

    The predictive power of age at diagnosis and smoking history for ALK rearrangements and EGFR mutations in non-small-cell lung cancer (NSCLC) remains not fully understood. In this cross-sectional study, 1160 NSCLC patients were prospectively enrolled and genotyped for EML4-ALK rearrangements and EGFR mutations. Multivariate logistic regression analysis was performed to explore the association between clinicopathological features and these two genetic aberrations. Receiver operating characteristic (ROC) curves methodology was applied to evaluate the predictive value. We showed that younger age at diagnosis was the only independent variable associated with EML4-ALK rearrangements (odds ratio (OR) per 5 years' increment, 0.68; p < 0.001), while lower tobacco exposure (OR per 5 pack-years' increment, 0.88; p < 0.001), adenocarcinoma (OR, 6.61; p < 0.001), and moderate to high differentiation (OR, 2.05; p < 0.001) were independently associated with EGFR mutations. Age at diagnosis was a very strong predictor of ALK rearrangements but poorly predicted EGFR mutations, while smoking pack-years may predict the presence of EGFR mutations and ALK rearrangements but with rather limited power. These findings should assist clinicians in assessing the likelihood of EML4-ALK rearrangements and EGFR mutations and understanding their biological implications in NSCLC.

  1. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    PubMed

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  2. In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

    PubMed

    Mathivet, Thomas; Mazot, Pierre; Vigny, Marc

    2007-12-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.

  3. Evidence that the lung adenocarcinoma EML4-ALK fusion gene is not caused by exposure to secondhand tobacco smoke during childhood

    PubMed Central

    Jen, Jin; Yi, Eunhee S.; Olivo-Marston, Susan; Yang, Ping; Harris, Curtis C.

    2014-01-01

    Background The EML4-ALK fusion gene is more frequently found in younger, never smoking, lung cancer patients. Meanwhile, never smokers exposed to secondhand tobacco smoke (SHS) during childhood are diagnosed at a younger age compared with never smoking lung cancer patients that are not exposed. We therefore hypothesized that SHS, which can induce DNA damage, is associated with the EML4-ALK fusion gene. Methods We compared the frequency of the EML4-ALK fusion gene among 197 never smoker lung cancer patients with and without a history of exposure to SHS during childhood at Mayo Clinic. Results The EML4-ALK fusion gene was detected in 33% of cases from never smokers with a history of SHS exposure during childhood, while 47% of never smoking lung cancer cases without a history of childhood SHS exposure tested positive for the fusion gene. Conclusions The EML4-ALK fusion gene is not enriched in tumors from individuals exposed to SHS during childhood. Impact These data suggest that childhood exposure to SHS is not a significant etiologic cause of the EML4-ALK fusion gene in lung cancer. PMID:24755712

  4. Analysis and in vitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA.

    PubMed

    Rana, A P; Tuck, M T

    1990-08-25

    A T7 RNA transcript coding for mouse dihydrofolate reductase (DHFR) was utilized as a substrate for the N6-methyladenosine mRNA methyltransferase isolated from HeLa cell nuclei. This transcript acted as a 3 fold better substrate than either prolactin mRNA or a synthetic RNA substrate which contained multiple methylation consensus sequences. Formation of internal N6-methyladenine (m6A) residues in the DHFR transcript was shown to increase slightly by the absence of a 7-methylguanine-2'-O-methyl cap structure. Using T7 transcripts from different regions of the DHFR gene, the majority of the m6A residues were localized to the coding region and a segment of the transcript just 3' to the coding region. This data suggests that DHFR mRNA contains multiple methylation sites with most of these sites concentrated in the coding region of the transcript. PMID:2395644

  5. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  6. Uplink Coding

    NASA Technical Reports Server (NTRS)

    Pollara, Fabrizio; Hamkins, Jon; Dolinar, Sam; Andrews, Ken; Divsalar, Dariush

    2006-01-01

    This viewgraph presentation reviews uplink coding. The purpose and goals of the briefing are (1) Show a plan for using uplink coding and describe benefits (2) Define possible solutions and their applicability to different types of uplink, including emergency uplink (3) Concur with our conclusions so we can embark on a plan to use proposed uplink system (4) Identify the need for the development of appropriate technology and infusion in the DSN (5) Gain advocacy to implement uplink coding in flight projects Action Item EMB04-1-14 -- Show a plan for using uplink coding, including showing where it is useful or not (include discussion of emergency uplink coding).

  7. Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

    PubMed

    Liu, Zheng; Hirani, Arvind H; McVetty, Peter B E; Daayf, Fouad; Quiros, Carlos F; Li, Genyi

    2012-05-01

    The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

  8. Expression of growth differentiation factor 9 (GDF9), ALK5, and claudin-11 in adult alpaca testis.

    PubMed

    Guo, Qing Yun; Gao, Zhen Zhen; Zhao, Li; He, Jun Ping; Dong, Cheng Sheng

    2013-01-01

    Growth differentiation factor 9 (GDF9) is an oocyte-derived factor critical for folliculogenesis. Recently, in vitro data showed that GDF9 inhibited the localization of tight junction (TJ) proteins, suggesting that GDF9 could potentially regulate spermatogenesis in vivo, via inhibition of Sertoli cell TJ function. The purpose of the present study was to determine the expression and localization of GDF9, its receptor, ALK5, and its latent target protein, claudin-11 (one of TJ proteins) in adult alpaca testis using Western blot and immunohistochemistry. Western blotting results demonstrated that GDF9, ALK5 and claudin-11 were expressed in the adult alpaca testis. Immunohistochemistry revealed that GDF9 was expressed stage-specifically in the cytoplasm of pachytene spermatocytes and round spermatids of the adult alpaca seminiferous epithelium. Type I receptor, ALK5 was mainly localized in the cytoplasm of round spermatids and Leydig cells, and to a lesser extent in the cytoplasm of pachytene spermatocytes and Sertoli cells. Its latent target protein, claudin-11, was perpendicular or parallel to the basal lamina in the basal part of Sertoli cells. These results indicated that GDF9, as a paracrine and autocrine growth factor derived from round spermatids and pachytene spermatocytes, is involved in regulating spermatogenesis via action on germ cells or somatic cells (i.e. Leydig cells, Sertoli cells).

  9. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    PubMed

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. PMID:26495981

  10. Fluorescence in situ hybridization analysis of the ALK gene in 2,045 non-small cell lung cancer patients from North-Western Spain (Galicia)

    PubMed Central

    Sánchez-Ares, María; Cameselle-Teijeiro, José M.; Vázquez-Estévez, Sergio; Lázaro-Quintela, Martín; Vázquez-Boquete, Ángel; Afonso-Afonso, Francisco J.; Casal-Rubio, Joaquín; González-Piñeiro, Ana L.; Rico-Rodríguez, Yolanda; Fírvida-Pérez, José L.; Ruíz-Bañobre, Juan; Couso, Elena; Santomé, Lucía; Pérez-Becerra, Raquel; García-Campelo, Rosario; Amenedo, Margarita; Azpitarte-Raposeiras, Cristina; Antúnez, José; Abdulkader, Ihab

    2016-01-01

    Identification of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements is a standard diagnostic test in patients with advanced non-small cell lung cancer (NSCLC). The current study describes the experience of ALK rearrangement detection of a referral center in the public health care system of Galicia in North-Western Spain. The fluorescence in situ hybridization (FISH) patterns of the ALK gene and the clinical and pathological features of these patients are reported. This study is also of interest for comparative purposes due to the relative geographical isolation of the area, which could have contributed to particular genetic features. A total of 2,045 tissue samples from NSCLC patients were collected between October 2010 and July 2015 and tested for ALK rearrangements by FISH. Examination of 1,686 paraffin-embedded tissue specimens and 395 cytological samples (306 cell block preparations and 53 cytological smears) was conducted, and any associations between the FISH results and clinicopathological features were assessed. The rate of successful evaluation was marginally higher in tissue samples than in cytological samples (92.9% vs. 84.1%); this difference was not significant. ALK rearrangements were identified in 82 patients(4%): 65 (79.3%) in tissue specimens, 15 (18.3%) in cell block samples and 2 (2.4%) in cytological smears. This genetic translocation appeared to be associated with a non-smoking history, younger age, female gender, stage IV and adenocarcinoma histological type. The findings demonstrate that ALK evaluation by FISH is feasible in tissue and cytological samples. The clinical and pathological features of the ALK-positive series of patients are similar to those previously reported in the literature. PMID:27446444

  11. Sharing code.

    PubMed

    Kubilius, Jonas

    2014-01-01

    Sharing code is becoming increasingly important in the wake of Open Science. In this review I describe and compare two popular code-sharing utilities, GitHub and Open Science Framework (OSF). GitHub is a mature, industry-standard tool but lacks focus towards researchers. In comparison, OSF offers a one-stop solution for researchers but a lot of functionality is still under development. I conclude by listing alternative lesser-known tools for code and materials sharing.

  12. Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer

    PubMed Central

    Nakaoku, Takashi; Tsuta, Koji; Tsuchihara, Katsuya; Matsumoto, Shingo; Yoh, Kiyotaka; Goto, Koichi

    2015-01-01

    Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions. PMID:25870798

  13. Role of the TGF-β/Alk5 Signaling Pathway in Monocrotaline-induced Pulmonary Hypertension

    PubMed Central

    Zaiman, Ari L.; Podowski, Megan; Medicherla, Satya; Gordy, Kimberley; Xu, Fang; Zhen, Lijie; Shimoda, Larissa A.; Neptune, Enid; Higgins, Linda; Murphy, Alison; Chakravarty, Sarvajit; Protter, Andrew; Sehgal, Pravin B.; Champion, Hunter C.; Tuder, Rubin M.

    2008-01-01

    Rationale: Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-β receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-β family signaling in PH and pulmonary vascular remodeling remains unclear. Objectives: To determine whether inhibition of TGF-β signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). Methods: We have used an orally active small-molecule TGF-β receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. Measurements and Main Results: The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-β signaling as documented by psmad2 expression. Inhibition of TGF-β signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-β signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. Conclusions: These findings provide evidence that increased TGF-β signaling participates in the pathogenesis of experimental severe PH. PMID:18202349

  14. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    PubMed

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  15. Early assessment of minimal residual disease identifies patients at very high relapse risk in NPM-ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Damm-Welk, Christine; Mussolin, Lara; Zimmermann, Martin; Pillon, Marta; Klapper, Wolfram; Oschlies, Ilske; d'Amore, Emanuele S G; Reiter, Alfred; Woessmann, Wilhelm; Rosolen, Angelo

    2014-01-16

    Detection of minimal disseminated disease (MDD) at diagnosis correlates with relapse risk in children with anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL). We investigated whether minimal residual disease (MRD) positivity by qualitative reverse-transcriptase polymerase chain reaction (RT-PCR) for Nucleophosmin (NPM)-ALK during treatment identifies patients at the highest relapse risk. Blood and/or bone marrow of 180 patients with NPM-ALK-positive ALCL treated with Berlin-Frankfurt-Münster-type protocols were screened for NPM-ALK transcripts at diagnosis; 103 were found to be MDD-positive. MRD before the second therapy course could be evaluated in 52 MDD-positive patients. MRD positivity correlated with uncommon histology. The cumulative incidence of relapses (CIR) of 26 MDD-positive/MRD-positive patients (81% ± 8%) was significantly higher than the CIR of 26 MDD-positive/MRD-negative (31% ± 9%) and 77 MDD-negative patients (15% ± 5%) (P < .001). Five-year survival of MDD-negative and MDD-positive/MRD-negative patients was 91% ± 3% and 92% ± 5%, respectively, compared with 65% ± 9% of MDD-positive/MRD-positive patients (P < .001). Early evaluation of MRD in NPM-ALK-positive ALCL identifies patients with a very high relapse risk and inferior survival.

  16. Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI-TOF Mass Spectrometry

    PubMed Central

    Li, Deyu; Delaney, James C.; Page, Charlotte M.; Chen, Alvin S.; Wong, Cintyu; Drennan, Catherine L.; Essigmann, John M.

    2010-01-01

    DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N 6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N 4-ethenocytosine are reported for the first time. It is also demonstrated that O 6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro. PMID:21048928

  17. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C.

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK. PMID:19887368

  18. ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

    PubMed

    Han, Arum; Zhao, Hu; Li, Jingyuan; Pelikan, Richard; Chai, Yang

    2014-08-01

    The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

  19. Molecular cloning of seal myoglobin mRNA.

    PubMed Central

    Wood, D; Blanchetot, A; Jeffreys, A J

    1982-01-01

    Grey seal skeletal muscle containing high levels of myoglobin was used to prepare poly(A)+ RNA. In vitro translation of this RNA produced a range of polypeptides including myoglobin. cDNA was prepared by reverse transcription of muscle poly(A)+ RNA and cloned into the plasmid pAT 153. 4% of cDNA recombinants were shown to contain myoglobin cDNA inserts. DNA sequence analysis of one clone (pSM 178) which contained a relatively large myoglobin cDNA insert showed an incomplete cDNA comprising the terminal 293 nucleotides of 3' non-translated mRNA sequences. Hybridization experiments using this myoglobin cDNA indicated that seal myoglobin is coded by a single gene which is transcribed to give a 1400 nucleotide mRNA considerably longer than related haemoglobin mRNAs. Images PMID:6185919

  20. A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

    PubMed

    Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A; Eichman, Brandt F

    2016-09-14

    DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair. PMID:27571247

  1. Overcoming resistance to first/second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene-addicted advanced non-small cell lung cancer

    PubMed Central

    Romanidou, Ourania; Landi, Lorenza; Cappuzzo, Federico; Califano, Raffaele

    2016-01-01

    Epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) gene rearrangement in advanced non-small cell lung cancer (NSCLC) represent the two oncogenic events with an impact on current clinical practice. EGFR tyrosine kinase inhibitors (TKIs) and crizotinib are the standard of care for the treatment of EGFR mutant and ALK gene rearranged advanced NSCLC patients. Unfortunately, despite initial clinical benefit, acquired resistance to EGFR-TKIs or crizotinib usually develops after an average of 10–12 months of treatment. The aim of this review is to describe the mechanisms of resistance to first/second generation EGFR-TKIs and crizotinib. In particular, we focus on strategies to overcome resistance due to secondary EGFR T790M mutation and mutations of the ALK domain. PMID:27239236

  2. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.

    PubMed

    Zhang, Chuanzhao; Samanta, Debangshu; Lu, Haiquan; Bullen, John W; Zhang, Huimin; Chen, Ivan; He, Xiaoshun; Semenza, Gregg L

    2016-04-01

    N(6)-methyladenosine (m(6)A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m(6)A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m(6)A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m(6)A residue in the 3'-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3'-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment. PMID:27001847

  3. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    NASA Astrophysics Data System (ADS)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  4. Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents.

    PubMed

    Rose, Peter; Whiteman, Matt; Moore, Philip K; Zhu, Yi Zhun

    2005-06-01

    S-Alk(en)yl cysteine sulfoxides are odourless, non-protein sulfur amino acids typically found in members of the family Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomically important genus Allium. Traditionally, Allium species, particularly the onion (Allium cepa) and garlic (A. sativum), have been used for centuries in European, Asian and American folk medicines for the treatment of numerous human pathologies, however it is only recently that any significant progress has been made in determining their mechanisms of action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from the combination of several academic disciplines including botany, biochemistry and nutrition. During tissue damage, S-alk(en)yl cysteine sulfoxides are converted to their respective thiosulfinates or propanethial-S-oxide by the action of the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinates can decompose to form additional sulfur constituents including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, the vinyldithiins and (E)- and (Z)-ajoene. Recent reports have shown onion and garlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes like glutathione-S-transferases (EC 2.5.1.18) and quinone reductase (QR) NAD(P)H: (quinine acceptor) oxidoreductase (EC 1.6.99.2) in mammalian tissues, as well as also influencing cell cycle arrest and apoptosis in numerous in vitro cancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds in cardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxide metabolites in the context of their biochemical and pharmacological mechanisms.

  5. Extended Survival and Prognostic Factors for Patients With ALK-Rearranged Non–Small-Cell Lung Cancer and Brain Metastasis

    PubMed Central

    Johung, Kimberly L.; Yeh, Norman; Desai, Neil B.; Williams, Terence M.; Lautenschlaeger, Tim; Arvold, Nils D.; Ning, Matthew S.; Attia, Albert; Lovly, Christine M.; Goldberg, Sarah; Beal, Kathryn; Yu, James B.; Kavanagh, Brian D.; Chiang, Veronica L.; Camidge, D. Ross

    2016-01-01

    Purpose We performed a multi-institutional study to identify prognostic factors and determine outcomes for patients with ALK-rearranged non–small-cell lung cancer (NSCLC) and brain metastasis. Patients and Methods A total of 90 patients with brain metastases from ALK-rearranged NSCLC were identified from six institutions; 84 of 90 patients received radiotherapy to the brain (stereotactic radiosurgery [SRS] or whole-brain radiotherapy [WBRT]), and 86 of 90 received tyrosine kinase inhibitor (TKI) therapy. Estimates for overall (OS) and intracranial progression-free survival were determined and clinical prognostic factors were identified by Cox proportional hazards modeling. Results Median OS after development of brain metastases was 49.5 months (95% CI, 29.0 months to not reached), and median intracranial progression-free survival was 11.9 months (95% CI, 10.1 to 18.2 months). Forty-five percent of patients with follow-up had progressive brain metastases at death, and repeated interventions for brain metastases were common. Absence of extracranial metastases, Karnofsky performance score ≥ 90, and no history of TKIs before development of brain metastases were associated with improved survival (P = .003, < .001, and < .001, respectively), whereas a single brain metastasis or initial treatment with SRS versus WBRT were not (P = .633 and .666, respectively). Prognostic factors significant by multivariable analysis were used to describe four patient groups with 2-year OS estimates of 33%, 59%, 76%, and 100%, respectively (P < .001). Conclusion Patients with brain metastases from ALK-rearranged NSCLC treated with radiotherapy (SRS and/or WBRT) and TKIs have prolonged survival, suggesting that interventions to control intracranial disease are critical. The refinement of prognosis for this molecular subtype of NSCLC identifies a population of patients likely to benefit from first-line SRS, close CNS observation, and treatment of emergent CNS disease. PMID:26438117

  6. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Parsons, Zachary D; Yuen, Philip K; David, Sheila S; Igarashi, Yasuhiro; Eichman, Brandt F

    2015-11-12

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision. PMID:26524531

  7. Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

    PubMed

    Singh, Vipender; Fedeles, Bogdan I; Li, Deyu; Delaney, James C; Kozekov, Ivan D; Kozekova, Albena; Marnett, Lawrence J; Rizzo, Carmelo J; Essigmann, John M

    2014-09-15

    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

  8. Management of Brain Metastases in ALK-Positive Non-Small-Cell Lung Cancer.

    PubMed

    Rusthoven, Chad G; Doebele, Robert C

    2016-08-20

    The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors' suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice.A 54-year-old man with a former 15-pack-year smoking history presents with cough and dyspnea. Initial work-up with imaging demonstrates a right suprahilar mass measuring 4.7 cm as well as several enlarged hilar and ipsilateral mediastinal lymph nodes. Bronchoscopy with biopsy reveals adenocarcinoma consistent with a lung primary. Staging with positron emission tomography/computed tomography (PET/CT) reidentifies the primary mass and lymph nodes and shows several PET-avid bone metastases. Brain magnetic resonance imaging (MRI) demonstrates a 1.6-cm right parietal mass with mild vasogenic edema and four additional brain metastases measuring 4 to 9 mm in size. Molecular testing is positive for an anaplastic lymphoma kinase (ALK) gene rearrangement using fluorescence in situ hybridization and negative for EGFR, ROS1, RET, BRAF, KRAS, and other oncogenes. The patient denies any neurologic symptoms and has no significant findings on neurologic exam. He is referred to you for management options for newly diagnosed stage IV (T2aN2M1b) lung adenocarcinoma. PMID:27298405

  9. ALK evaluation in the world of multiplex testing: Network Genomic Medicine (NGM): the Cologne model for implementing personalised oncology.

    PubMed

    Heydt, C; Kostenko, A; Merkelbach-Bruse, S; Wolf, J; Büttner, R

    2016-09-01

    Comprehensive molecular genotyping of lung cancers has become a key requirement for guiding therapeutic decisions. As a paradigm model of implementing next-generation comprehensive diagnostics, Network Genomic Medicine (NGM) has established central diagnostic and clinical trial platforms for centralised testing and decentralised personalised treatment in clinical practice. Here, we describe the structures of the NGM network and give a summary of technologies to identify patients with anaplastic lymphoma kinase (ALK) fusion-positive lung adenocarcinomas. As unifying test platforms will become increasingly important for delivering reliable, quick and affordable tests, the NGM diagnostic platform is currently implementing a comprehensive hybrid capture-based parallel sequencing pan-cancer assay.

  10. ALK evaluation in the world of multiplex testing: Network Genomic Medicine (NGM): the Cologne model for implementing personalised oncology.

    PubMed

    Heydt, C; Kostenko, A; Merkelbach-Bruse, S; Wolf, J; Büttner, R

    2016-09-01

    Comprehensive molecular genotyping of lung cancers has become a key requirement for guiding therapeutic decisions. As a paradigm model of implementing next-generation comprehensive diagnostics, Network Genomic Medicine (NGM) has established central diagnostic and clinical trial platforms for centralised testing and decentralised personalised treatment in clinical practice. Here, we describe the structures of the NGM network and give a summary of technologies to identify patients with anaplastic lymphoma kinase (ALK) fusion-positive lung adenocarcinomas. As unifying test platforms will become increasingly important for delivering reliable, quick and affordable tests, the NGM diagnostic platform is currently implementing a comprehensive hybrid capture-based parallel sequencing pan-cancer assay. PMID:27573753

  11. GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-βRII heterodimer.

    PubMed

    Artz, Annette; Butz, Stefan; Vestweber, Dietmar

    2016-07-28

    Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1. PMID:27235139

  12. The bicistronic nature of lens alpha-crystallin 14S mRNA.

    PubMed Central

    Chen, J H; Spector, A

    1977-01-01

    The A2 and B2 polypeptide chains of calf lens alpha-crystallin are synthesized on a 14S, 1500-nucleotide mRNA and a 10S, 735-nucleotide mRNA, respectively. The 10S mRNA is theoretically compatible with the size of the B2 chain, but the 14S mRNA contains approximately twice the required number of nucleotides necessary for A2 chain synthesis. This fact raises the question of the function of the additional nucleotide sequence in the 14S mRNA. The following observations on 14S mRNA suggest that it may contain an additional cistron. (i) Under a number of denaturing conditions, 14S mRNA continues to retain its initial size characteristics. (ii) In addition to synthesis of the A2 chain, 14S mRNA directs the synthesis of another polypeptide with the same electrophoretic mobility as that of the B2 chain. (iii) Molecular hybridization of the 14S mRNA with the cDNA produced from the 10S mRNA suggests that 2 mol of the cDNA bind to 1 mol of the 14S mRNA. (iv) Examination of the nucleotide sequences of the 10S and 14S mRNAs by two-dimensional maps of RNase A and T1 digests indicates marked similarity. The overall data suggest that the additional cistronic component may carry coding information for an alpha-crystallin polypeptide or a closely related polypeptide species. Images PMID:271967

  13. n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

    SciTech Connect

    Kogure, Takahisa; Takagi, Masamichi; Ohta, Akinori . E-mail: aaohta@mail.ecc.u-tokyo.ac.jp

    2005-04-01

    The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.

  14. Speech coding

    SciTech Connect

    Ravishankar, C., Hughes Network Systems, Germantown, MD

    1998-05-08

    Speech is the predominant means of communication between human beings and since the invention of the telephone by Alexander Graham Bell in 1876, speech services have remained to be the core service in almost all telecommunication systems. Original analog methods of telephony had the disadvantage of speech signal getting corrupted by noise, cross-talk and distortion Long haul transmissions which use repeaters to compensate for the loss in signal strength on transmission links also increase the associated noise and distortion. On the other hand digital transmission is relatively immune to noise, cross-talk and distortion primarily because of the capability to faithfully regenerate digital signal at each repeater purely based on a binary decision. Hence end-to-end performance of the digital link essentially becomes independent of the length and operating frequency bands of the link Hence from a transmission point of view digital transmission has been the preferred approach due to its higher immunity to noise. The need to carry digital speech became extremely important from a service provision point of view as well. Modem requirements have introduced the need for robust, flexible and secure services that can carry a multitude of signal types (such as voice, data and video) without a fundamental change in infrastructure. Such a requirement could not have been easily met without the advent of digital transmission systems, thereby requiring speech to be coded digitally. The term Speech Coding is often referred to techniques that represent or code speech signals either directly as a waveform or as a set of parameters by analyzing the speech signal. In either case, the codes are transmitted to the distant end where speech is reconstructed or synthesized using the received set of codes. A more generic term that is applicable to these techniques that is often interchangeably used with speech coding is the term voice coding. This term is more generic in the sense that the

  15. Overcoming the resistance to crizotinib in patients with non-small cell lung cancer harboring EML4/ALK translocation.

    PubMed

    Perez, Cesar A; Velez, Michel; Raez, Luis E; Santos, Edgardo S

    2014-05-01

    The large knowledge learned in molecular biology specifically in the oncology field during the last ten years has resulted in fruitful results for the treatment of non-small cell lung cancer. The first pathway to be effectively targeted in lung cancer was the epidermal growth factor receptor. The acceptance of epidermal growth factor receptor mutation as a strong predictive biomarker in non-small cell lung carcinoma has encouraged the search for more targets. In 2011, regulatory entities granted conditional approval to an anaplastic lymphoma kinase inhibitor (crizotinib) based on an impressive overall response rate in previously treated non-small cell lung cancer patients whose tumors harbored EML4/ALK translocations. The landmark approval of crizotinib based on early promising clinical data highlights the remarkable success of molecular medicine in lung cancer therapeutics. The cumulative data developed after that approval has confirmed the appropriateness of this decision as recently reported phase III has now demonstrated. Unfortunately, resistance to this agent invariably develops and we now face the challenge of understanding several resistance pathways and overcoming them with new and more potent compounds. New agents in clinical development such as alectinib, LDK378, AP26113, and AUY922 have not only demonstrated promising activity in crizotinib resistant patients, but also crossing new pharmacokinetic boundaries in ALK inhibition as potent CNS penetration.

  16. Quantum mechanics/molecular mechanics study on the oxygen binding and substrate hydroxylation step in AlkB repair enzymes.

    PubMed

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N(1) -methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)-oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ- and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.

  17. Overcoming the resistance to crizotinib in patients with non-small cell lung cancer harboring EML4/ALK translocation.

    PubMed

    Perez, Cesar A; Velez, Michel; Raez, Luis E; Santos, Edgardo S

    2014-05-01

    The large knowledge learned in molecular biology specifically in the oncology field during the last ten years has resulted in fruitful results for the treatment of non-small cell lung cancer. The first pathway to be effectively targeted in lung cancer was the epidermal growth factor receptor. The acceptance of epidermal growth factor receptor mutation as a strong predictive biomarker in non-small cell lung carcinoma has encouraged the search for more targets. In 2011, regulatory entities granted conditional approval to an anaplastic lymphoma kinase inhibitor (crizotinib) based on an impressive overall response rate in previously treated non-small cell lung cancer patients whose tumors harbored EML4/ALK translocations. The landmark approval of crizotinib based on early promising clinical data highlights the remarkable success of molecular medicine in lung cancer therapeutics. The cumulative data developed after that approval has confirmed the appropriateness of this decision as recently reported phase III has now demonstrated. Unfortunately, resistance to this agent invariably develops and we now face the challenge of understanding several resistance pathways and overcoming them with new and more potent compounds. New agents in clinical development such as alectinib, LDK378, AP26113, and AUY922 have not only demonstrated promising activity in crizotinib resistant patients, but also crossing new pharmacokinetic boundaries in ALK inhibition as potent CNS penetration. PMID:24598368

  18. ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

    PubMed Central

    Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew D.; Robinson, Eva H.; Phizicky, Eric M.; Lowe, Todd M.

    2015-01-01

    High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules. PMID:26237225

  19. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    PubMed

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  20. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

    PubMed Central

    Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V.

    2014-01-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

  1. MCNP code

    SciTech Connect

    Cramer, S.N.

    1984-01-01

    The MCNP code is the major Monte Carlo coupled neutron-photon transport research tool at the Los Alamos National Laboratory, and it represents the most extensive Monte Carlo development program in the United States which is available in the public domain. The present code is the direct descendent of the original Monte Carlo work of Fermi, von Neumaum, and Ulam at Los Alamos in the 1940s. Development has continued uninterrupted since that time, and the current version of MCNP (or its predecessors) has always included state-of-the-art methods in the Monte Carlo simulation of radiation transport, basic cross section data, geometry capability, variance reduction, and estimation procedures. The authors of the present code have oriented its development toward general user application. The documentation, though extensive, is presented in a clear and simple manner with many examples, illustrations, and sample problems. In addition to providing the desired results, the output listings give a a wealth of detailed information (some optional) concerning each state of the calculation. The code system is continually updated to take advantage of advances in computer hardware and software, including interactive modes of operation, diagnostic interrupts and restarts, and a variety of graphical and video aids.

  2. QR Codes

    ERIC Educational Resources Information Center

    Lai, Hsin-Chih; Chang, Chun-Yen; Li, Wen-Shiane; Fan, Yu-Lin; Wu, Ying-Tien

    2013-01-01

    This study presents an m-learning method that incorporates Integrated Quick Response (QR) codes. This learning method not only achieves the objectives of outdoor education, but it also increases applications of Cognitive Theory of Multimedia Learning (CTML) (Mayer, 2001) in m-learning for practical use in a diverse range of outdoor locations. When…

  3. Chromoplectic TPM3–ALK rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib

    PubMed Central

    Mansfield, A. S.; Murphy, S. J.; Harris, F. R.; Robinson, S. I.; Marks, R. S.; Johnson, S. H.; Smadbeck, J. B.; Halling, G. C.; Yi, E. S.; Wigle, D.; Vasmatzis, G.; Jen, J.

    2016-01-01

    Background Inflammatory myofibroblastic tumors (IMTs) are rare sarcomas that can occur at any age. Surgical resection is the primary treatment for patients with localized disease; however, these tumors frequently recur. Less commonly, patients with IMTs develop or present with metastatic disease. There is no standard of care for these patients and traditional cytotoxic therapy is largely ineffective. Most IMTs are associated with oncogenic ALK, ROS1 or PDGFRβ fusions and may benefit from targeted therapy. Patient and methods We sought to understand the genomic abnormalities of a patient who presented for management of metastatic IMT after progression of disease on crizotinib and a significant and durable partial response to the more potent ALK inhibitor ceritinib. Results The residual IMT was resected based on the recommendations of a multidisciplinary tumor sarcoma tumor board and analyzed by whole-genome mate pair sequencing. Analysis of the residual, resected tumor identified a chromoplectic TPM3–ALK rearrangement that involved many other known oncogenes and was confirmed by rtPCR. Conclusions In our analysis of the treatment-resistant, residual IMT, we identified a complex pattern of genetic rearrangements consistent with chromoplexy. Although it is difficult to know for certain if these chromoplectic rearrangements preceded treatment, their presence suggests that chromoplexy has a role in the oncogenesis of IMTs. Furthermore, this patient's remarkable response suggests that ceritinib should be considered as an option after progression on crizotinib for patients with metastatic or unresectable IMT and ALK mutations. PMID:27742657

  4. Monitoring the alkane monooxygenase gene alkB in different soil interfaces during plant litter degradation of C3 and C4 plants

    NASA Astrophysics Data System (ADS)

    Schulz, S.; Munch, J. C.; Schloter, M.

    2009-04-01

    Hydrocarbons like n-alkanes are ubiquitous in the environment as a result of anthropogenic contamination (e.g. oil spills) as well as a part of an ecosystem's biomass. For example n-alkanes become released during plant litter degradation; consequently they become a high abundant carbon source for microorganism. One possibility for the prokaryotic hydrocarbon metabolisation is an aerobic degradation pathway where the initial step is catalysed by the membrane bound alkane monooxygenase alkB. We analysed the influence of alkanes on the abundance of the alkB gene in different interfaces of the litter-soil system during the degradation of maize and pea litter. Therefore soil samples of a sandy and a loamy soil have been incubated with straw of maize and pea plants up to 30 weeks with constant soil moisture and temperature. Using quantitative real-time PCR we were able to monitor the changes of the abundance and the expression rates of alkB. In our experiments we focused on the straw layer, the litter/soil interface and the soil 1 cm below this interface (bulk soil). Our results clearly demonstrate time and space dependent abundance patterns of alkB genes and transcripts in the different layers studied, which are additionally shaped by the soil type used.

  5. Case report: A unique pediatric case of a primary CD8 expressing ALK-1 positive anaplastic large cell lymphoma of skeletal muscle.

    PubMed

    Gaiser, Timo; Geissinger, Eva; Schattenberg, Torsten; Scharf, Hanns-Peter; Dürken, Matthias; Dinter, Dietmar; Rosenwald, Andreas; Marx, Alexander

    2012-01-01

    Primary involvement of skeletal muscle is a very rare event in ALK-1 positive anaplastic large cell lymphoma (ALCL). We describe a case of a 10-year old boy presenting with a three week history of pain and a palpable firm swelling at the dorsal aspect of the left thigh. Histological examination of the lesion revealed a tumoral and diffuse polymorphic infiltration of the muscle by large lymphoid cells. Tumor cells displayed eccentric, lobulated "horse shoe" or "kidney-shape" nuclei. The cells showed immunohistochemical positivity for CD30, ALK-1, CD2, CD3, CD7, CD8, and Perforin. Fluorescence in situ hybridization analysis revealed a characteristic rearrangement of the ALK-1 gene in 2p23 leading to the diagnosis of ALK-1 positive ALCL. Chemotherapy according to the ALCL-99-NHL-BFM protocol was initiated and resulted in a complete remission after two cycles. This case illustrates the unusual presentation of a pediatric ALCL in soft tissue with a good response to chemotherapy.

  6. Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

    PubMed

    Merz, Hartmut; Kaehler, Christian; Hoefig, Kai P; Branke, Biggi; Uckert, Wolfgang; Nadrowitz, Roger; Cerny-Reiterer, Sabine; Herrmann, Harald; Feller, Alfred C; Valent, Peter

    2010-06-01

    Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.

  7. Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

    PubMed

    Merz, Hartmut; Kaehler, Christian; Hoefig, Kai P; Branke, Biggi; Uckert, Wolfgang; Nadrowitz, Roger; Cerny-Reiterer, Sabine; Herrmann, Harald; Feller, Alfred C; Valent, Peter

    2010-06-01

    Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30. PMID:21297223

  8. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  9. Biomaterials for mRNA delivery.

    PubMed

    Islam, Mohammad Ariful; Reesor, Emma K G; Xu, Yingjie; Zope, Harshal R; Zetter, Bruce R; Shi, Jinjun

    2015-12-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  10. Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review)

    PubMed Central

    DI LIEGRO, CARLO MARIA; SCHIERA, GABRIELLA; DI LIEGRO, ITALIA

    2014-01-01

    Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5′ or 3′ to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3′-untranslated (3′-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs. PMID:24452120

  11. Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

    PubMed

    Horne-Badovinac, Sally; Bilder, David

    2008-01-01

    Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

  12. The life of an mRNA in space and time.

    PubMed

    Ben-Ari, Ya'ara; Brody, Yehuda; Kinor, Noa; Mor, Amir; Tsukamoto, Toshiro; Spector, David L; Singer, Robert H; Shav-Tal, Yaron

    2010-05-15

    Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed beta-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic beta-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the beta-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

  13. Pol II-directed short RNAs suppress the nuclear export of mRNA.

    PubMed

    Komarova, Tatiana V; Schwartz, Anton M; Frolova, Olga Y; Zvereva, Anna S; Gleba, Yuri Y; Citovsky, Vitaly; Dorokhov, Yuri L

    2010-12-01

    The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA. PMID:20953971

  14. BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

    PubMed

    Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

    2015-11-01

    Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

  15. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    SciTech Connect

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-05-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.

  16. Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA

    PubMed Central

    Stoddard, Thomas J.; Clasen, Benjamin M.; Baltes, Nicholas J.; Demorest, Zachary L.; Voytas, Daniel F.; Zhang, Feng; Luo, Song

    2016-01-01

    Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were <10bp; in contrast, 88% of insertions generated through DNA delivery were >10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing. PMID:27176769

  17. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  18. Analysis of mRNA nuclear export kinetics in mammalian cells by microinjection.

    PubMed

    Gueroussov, Serge; Tarnawsky, Stefan P; Cui, Xianying A; Mahadevan, Kohila; Palazzo, Alexander F

    2010-12-04

    In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes and genetically tractable organisms such as yeast and the Drosophila derived S2 cell line, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR). In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

  19. Quantitative PCR detection of NPM/ALK fusion gene and CD30 gene expression in patients with anaplastic large cell lymphoma--residual disease monitoring and a correlation with the disease status.

    PubMed

    Kalinova, Marketa; Krskova, Lenka; Brizova, Helena; Kabickova, Edita; Kepak, Tomas; Kodet, Roman

    2008-01-01

    Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of malignant lymphoproliferative diseases with a consistent expression of the cytokine receptor CD30. ALCL is frequently associated with a NPM/ALK fusion gene which is found in up to 75% of pediatric ALCLs. Real-time quantitative RT-PCR (RQ-RT-PCR) of NPM/ALK and CD30 gene expression was employed to analyze minimal residual disease (MRD) in 10 patients with NPM/ALK positive ALCL in 79 follow-up bone marrow (BM) and/or peripheral blood (PB) samples. In all BM samples from relapses and/or closely before a relapse, BM samples revealed NPM/ALK and CD30 positivity in at least one of the iliac BM trephines. Five out of nine relapses were preceded or were accompanied by minimally half log increased NPM/ALK levels in the BM. We found that RQ-RT-PCR of the CD30 expression is not suitable for MRD detection--only two relapses were accompanied by an increase of the CD30 level above a level which was detected in BM/PB samples from healthy individuals. RQ-RT-PCR of NPM/ALK expression is a promising and rapid approach for monitoring MRD.

  20. Pre-Mrna Introns as a Model for Cryptographic Algorithm:. Theory and Experiments

    NASA Astrophysics Data System (ADS)

    Regoli, Massimo

    2010-01-01

    The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. In particular the RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

  1. Coupling mRNA Synthesis and Decay

    PubMed Central

    Braun, Katherine A.

    2014-01-01

    What has been will be again, what has been done will be done again; there is nothing new under the sun.—Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus—capping, splicing, and polyadenylation—are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm—mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process. PMID:25154419

  2. Neuropeptide Y in black seabream Acanthopagrus schlegelii: identification, distribution and mRNA expression responses to ghrelin.

    PubMed

    Ma, X L; Zheng, L W; Mao, L T; Zhou, L B; Wang, A L

    2013-04-01

    The coding region of neuropeptide Y (NPY) complementary (c)DNA was cloned from the hypothalamus RNA of black seabream Acanthopagrus schlegelii, including 297 bp coding for prepro-NPY of 98 amino acids. Real-time reverse transcriptase-polymerase chain reaction was performed to determine A. schlegelii npy gene expression; NPY messenger RNA (mRNA) was expressed highly in the brain and stomach. Treatment with acylated ghrelin significantly up-regulated NPY mRNA level in the hypothalamus, suggesting that NPY may be involved in regulating food intake of A. schlegelii.

  3. Bilateral breast adenocarcinomas with EML4–ALK fusion in a patient with multiple metastases successfully treated with crizotinib: is lung the primary site?

    PubMed Central

    Liu, Chao; Ding, Lijuan; Sun, Bing; Wu, Shikai

    2016-01-01

    Breast metastases from non-mammary cancers are rare, especially when they appear synchronously. Clinically, it is vitally important to accurately diagnose these patients, as this will directly influence their treatment and survival. We present a very rare and complex case of bilateral breast adenocarcinomas with an EML4–ALK fusion, which was diagnosed as bilateral breast metastases of non-small-cell lung cancer by immunohistochemistry and comprehensive genomic investigation. The patient was successfully treated with an ALK inhibitor (crizotinib); symptoms improved quickly after initiation of crizotinib therapy, and a partial response was observed after 3 months. The experience of diagnosis and treatment of this case indicates the importance and necessity of genomic investigations in such patients, and suggests that we need to consider the rare possibility of this kind of metastasis in order to provide optimal treatment. PMID:27366096

  4. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

  5. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. PMID:25620012

  6. Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

    PubMed

    Kiss, Izabella; Unger, Christine; Huu, Chi Nguyen; Atanasov, Atanas Georgiev; Kramer, Nina; Chatruphonprasert, Waranya; Brenner, Stefan; McKinnon, Ruxandra; Peschel, Andrea; Vasas, Andrea; Lajter, Ildiko; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

    2015-01-28

    An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this

  7. The atomic resolution structure of human AlkB homolog 7 (ALKBH7), a key protein for programmed necrosis and fat metabolism.

    PubMed

    Wang, Guoqiang; He, Qingzhong; Feng, Chong; Liu, Yang; Deng, Zengqin; Qi, Xiaoxuan; Wu, Wei; Mei, Pinchao; Chen, Zhongzhou

    2014-10-01

    ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … Xn … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the "nucleotide recognition lid" which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.

  8. Transforming growth factor-beta type I receptor/ALK5 contributes to doxazosin-induced apoptosis in H9C2 cells.

    PubMed

    Yang, Yi-Fan; Wu, Chau-Chung; Chen, Wen-Pin; Su, Ming-Jai

    2009-12-01

    The mechanism of doxazosin-induced apoptosis through alpha(1)-adrenoceptor-independent pathway has been reported in various types of cell models. However, the molecular events involved in this effect are still not fully discovered. In present study, we proposed that the transforming growth factor-beta type I receptor (TbetaRI/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell viability, apoptotic nuclei, and caspase-3 activity, we found that doxazosin induced concentration- and time-dependent apoptosis in H9C2 cells. The cell apoptosis induced by 30 muM doxazosin was exacerbated by the addition of 10 ng/ml transforming growth factor-beta1 (TGF-beta1). Doxazosin or TGF-beta1 alone respectively elevated p38 mitogen-activated protein kinases (MAPK) and Smad3 protein phosphorylation in H9C2 cells. However, the cotreatment of doxazosin and TGF-beta1 attenuated the TGF-beta1-induced Smad3 protein phosphorylation and increased doxazosin-induced p38 MAPK protein phosphorylation. Furthermore, inhibitors of TbetaRI/ALK5 (SB431542) and p38 MAPK (SB202190) or TbetaRI/ALK5 knockdown all dramatically reduced the doxazosin-induced apoptosis in H9C2 cells. In conclusion, our results demonstrated that TbetaRI/ALK5-p38 MAPK phosphorylation signaling pathway could contribute to doxazosin-induced cell apoptosis, which could be further enhanced by TGF-beta1 in association with attenuating Smad3 phosphorylation in H9C2 cells.

  9. Quantitative determination of allergenic 5-alk(en)ylresorcinols in mango (Mangifera indica L.) peel, pulp, and fruit products by high-performance liquid chromatography.

    PubMed

    Knödler, Matthias; Reisenhauer, Katharina; Schieber, Andreas; Carle, Reinhold

    2009-05-13

    Despite a number of serious case reports of mango dermatitis, no attempts at the identification and quantification of allergenic 5-alk(en)ylresorcinols in mango fruits have so far been made. Therefore, total alk(en)ylresorcinol content and relative homologue composition in 13 mango peel samples and 7 samples of mango pulp were determined by HPLC and LC-MS/MS analyses. Furthermore, mango puree and nectar prepared on pilot plant scale were also analyzed and compared with commercially available thermally preserved products. Depending on cultivar, alk(en)ylresorcinol contents ranged from 79.3 to 1850.5 mg/kg of dry matter (DM) in mango peels and from 4.9 to 187.3 mg/kg of DM in samples of mango pulp. The profile of alk(en)ylresorcinols was found to be highly characteristic, with an average homologue composition of C15:0 (6.1%), C15:1 (1.7%), C17:0 (1.1%), C17:1 (52.5%), C17:2 (33.4%), C17:3 (2.4%), C19:1 (2.1%), and C19:2 (0.8%). Mango puree samples prepared from peeled and unpeeled fruits revealed contents of 3.8 and 12.3 mg/kg of fresh weight, respectively. Content and homologue composition were not significantly affected during puree processing and thermal preservation. In nectar samples prepared from peeled and unpeeled fruits, contents of 1.4 and 4.6 mg/L, respectively, were found.

  10. The ALK inhibitor ASP3026 eradicates NPM-ALK⁺ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model.

    PubMed

    George, Suraj Konnath; Vishwamitra, Deeksha; Manshouri, Roxsan; Shi, Ping; Amin, Hesham M

    2014-07-30

    NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.

  11. Towards a biological coding theory discipline.

    SciTech Connect

    May, Elebeoba Eni

    2003-09-01

    How can information required for the proper functioning of a cell, an organism, or a species be transmitted in an error-introducing environment? Clearly, similar to engineering communication systems, biological systems must incorporate error control in their information transmissino processes. if genetic information in the DNA sequence is encoded in a manner similar to error control encoding, the received sequence, the messenger RNA (mRNA) can be analyzed using coding theory principles. This work explores potential parallels between engineering communication systems and the central dogma of genetics and presents a coding theory approach to modeling the process of protein translation initiation. The messenger RNA is viewed as a noisy encoded sequence and the ribosoe as an error control decoder. Decoding models based on chemical and biological characteristics of the ribosome and the ribosome binding site of the mRNA are developed and results of applying the models to the Escherichia coli K-12 are presented.

  12. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  13. Expression and regulation of Icer mRNA in the Syrian hamster pineal gland.

    PubMed

    Diaz, Elena; Garidou, Marie-Laure; Dardente, Hugues; Salingre, Anthony; Pévet, Paul; Simonneaux, Valérie

    2003-04-10

    Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

  14. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems.

    PubMed

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5'-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3'>5'direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.

  15. Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

    PubMed Central

    Quaresma, Alexandre Jose Christino; Sievert, Rachel; Nickerson, Jeffrey A.

    2013-01-01

    UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5′ end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs. PMID:23427269

  16. Reduced secreted mu mRNA synthesis in selective IgM deficiency of Bloom's syndrome.

    PubMed Central

    Kondo, N; Ozawa, T; Kato, Y; Motoyoshi, F; Kasahara, K; Kameyama, T; Orii, T

    1992-01-01

    Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Bloom's syndrome. Although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were markedly reduced. The membrane-bound mu (microns) and secreted mu (microseconds) mRNAs are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of microseconds mRNA synthesis depends on the addition of poly(A) to microseconds C-terminal segment. In both patients, mu mRNA was well detected but microseconds C-terminal mRNA was scarcely detected, suggesting that microns mRNA was well transcribed but microseconds mRNA was not. There was, at least, no mutation or deletion in the microseconds C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of microseconds C-terminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Bloom's syndrome is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells and a failure of microseconds mRNA synthesis. Moreover, reduced microseconds mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the mu gene. Images Fig. 2 PMID:1563106

  17. Homological stabilizer codes

    SciTech Connect

    Anderson, Jonas T.

    2013-03-15

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev's toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev's toric code or to the topological color codes. - Highlights: Black-Right-Pointing-Pointer We show that Kitaev's toric codes are equivalent to homological stabilizer codes on 4-valent graphs. Black-Right-Pointing-Pointer We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. Black-Right-Pointing-Pointer We find and classify all 2D homological stabilizer codes. Black-Right-Pointing-Pointer We find optimal codes among the homological stabilizer codes.

  18. Non coding RNA in muscle differentiation and disease.

    PubMed

    Morlando, Mariangela; Rosa, Alessandro; Caffarelli, Elisa; Fatica, Alessandro; Bozzoni, Irene

    2013-01-01

    Non coding RNAs have provided in the last decades a very exciting research field with the discovery that a largely unexplored fraction of our genome encodes for RNA without protein coding activity. Here we revise the current knowledge of how non coding RNAs impact on muscle differentiation and homeostasis in normal and disease conditions and how they can provide powerful tools for therapeutic interventions and disease diagnosis. Moreover, we discuss new insights into additional mechanisms of post-transcriptional regulation involving a new class of long non coding RNAs shown to impact on the distribution of microRNA molecules on their mRNA targets.

  19. Gene and genon concept: coding versus regulation

    PubMed Central

    2007-01-01

    We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various

  20. Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli.

    PubMed

    Jaspers, M C; Meier, C; Zehnder, A J; Harms, H; van der Meer, J R

    2001-08-01

    Diffusion of octane from oily droplets in different microscale settings was measured using Escherichia coli expressing the stable green fluorescent protein (GFP) from the alkB promoter of Pseudomonas oleovorans. GFP fluorescence intensities were determined quantitatively at the single-cell level after 1.0 or 2.5 h incubation and compared with different calibration series using known concentrations of octane. By immobilizing the E. coli sensor cells on the bottom glass plate of a microscope flow chamber, it was possible to monitor the diffusion process for octane in aqueous solution as a function of time and distance from non-aqueous phase droplets of octane alone or oily octane mixtures. When a gas phase was included in the flow chambers, octane transport could be demonstrated from the oily mixtures to the cells through both gas and liquid phase. Assays of non-immobilized sensor cells in microdroplets in the presence or absence of soil particles incubated with octane through the vapour phase revealed a slight reduction in the total amount of induced E. coli cells in the presence of soil. Our results indicate the power of using GFP-marked single-cell biosensors in determining microscale bioavailability of organic pollutants. PMID:11578312

  1. The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

    1995-07-01

    The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

  2. Coding of Neuroinfectious Diseases.

    PubMed

    Barkley, Gregory L

    2015-12-01

    Accurate coding is an important function of neurologic practice. This contribution to Continuum is part of an ongoing series that presents helpful coding information along with examples related to the issue topic. Tips for diagnosis coding, Evaluation and Management coding, procedure coding, or a combination are presented, depending on which is most applicable to the subject area of the issue. PMID:26633789

  3. Model Children's Code.

    ERIC Educational Resources Information Center

    New Mexico Univ., Albuquerque. American Indian Law Center.

    The Model Children's Code was developed to provide a legally correct model code that American Indian tribes can use to enact children's codes that fulfill their legal, cultural and economic needs. Code sections cover the court system, jurisdiction, juvenile offender procedures, minor-in-need-of-care, and termination. Almost every Code section is…

  4. Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation.

    PubMed Central

    Kornitzer, D; Teff, D; Altuvia, S; Oppenheim, A B

    1989-01-01

    The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression. Images PMID:2523380

  5. Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo.

    PubMed

    Mahony, J B; Brown, I R

    1979-11-22

    Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.

  6. Prognostic significance of circulating tumor cells in bone marrow or peripheral blood as detected by qualitative and quantitative PCR in pediatric NPM-ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Damm-Welk, Christine; Busch, Kerstin; Burkhardt, Birgit; Schieferstein, Jutta; Viehmann, Susanne; Oschlies, Ilske; Klapper, Wolfram; Zimmermann, Martin; Harbott, Jochen; Reiter, Alfred; Woessmann, Willi

    2007-07-15

    Clinical and histopathological characteristics have limited prognostic value for children with anaplastic large-cell lymphoma (ALCL). We evaluated the presence, extent, and prognostic impact of circulating tumor cells in bone marrow (BM) and peripheral blood (PB) of children and adolescents with NPM-ALK-positive ALCL at diagnosis using qualitative and quantitative polymerase chain reaction (PCR) for NPM-ALK. Numbers of NPM-ALK transcripts were normalized to 10(4) copies ABL (NCNs). BM was analyzed from 80 patients and PB from 52. BM was positive for NPM-ALK in 47.5% of patients, and positivity was significantly correlated with clinical stage, mediastinal or visceral involvement, microscopic BM involvement, and histologic subtype. Qualitative and quantitative PCR results in BM and PB strongly correlated. BM PCR was associated with the cumulative incidence of relapses (CI-Rs): CI-R was 50% +/- 10% for 38 PCR-positive and 15% +/- 7% for 42 PCR-negative patients (P < .001). Sixteen patients with more than 10 NCNs NPM-ALK in BM had a CI-R of 71% +/- 14% compared with a CI-R of 18% +/- 6% for 59 patients with 10 or fewer NCNs (P < .001). PB PCR results led to a similar grouping. Thus, quantitative PCR in BM or PB allows identification of 20% of patients experiencing 60% of all relapses with an event-free survival of 20%.

  7. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  8. To Code or Not To Code?

    ERIC Educational Resources Information Center

    Parkinson, Brian; Sandhu, Parveen; Lacorte, Manel; Gourlay, Lesley

    1998-01-01

    This article considers arguments for and against the use of coding systems in classroom-based language research and touches on some relevant considerations from ethnographic and conversational analysis approaches. The four authors each explain and elaborate on their practical decision to code or not to code events or utterances at a specific point…

  9. cncRNAs: Bi-functional RNAs with protein coding and non-coding functions

    PubMed Central

    Kumari, Pooja; Sampath, Karuna

    2015-01-01

    For many decades, the major function of mRNA was thought to be to provide protein-coding information embedded in the genome. The advent of high-throughput sequencing has led to the discovery of pervasive transcription of eukaryotic genomes and opened the world of RNA-mediated gene regulation. Many regulatory RNAs have been found to be incapable of protein coding and are hence termed as non-coding RNAs (ncRNAs). However, studies in recent years have shown that several previously annotated non-coding RNAs have the potential to encode proteins, and conversely, some coding RNAs have regulatory functions independent of the protein they encode. Such bi-functional RNAs, with both protein coding and non-coding functions, which we term as ‘cncRNAs’, have emerged as new players in cellular systems. Here, we describe the functions of some cncRNAs identified from bacteria to humans. Because the functions of many RNAs across genomes remains unclear, we propose that RNAs be classified as coding, non-coding or both only after careful analysis of their functions. PMID:26498036

  10. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    SciTech Connect

    Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

    1986-03-01

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

  11. Bare Code Reader

    NASA Astrophysics Data System (ADS)

    Clair, Jean J.

    1980-05-01

    The Bare code system will be used, in every market and supermarket. The code, which is normalised in US and Europe (code EAN) gives informations on price, storage, nature and allows in real time the gestion of theshop.

  12. Effect of carbamates on mRNA encoding lipid enzymes in hamster flank organs.

    PubMed

    López-Lezama, Juan C; Cabeza, Marisa; Mayorga, Israel; Soriano, Juan; Sainz, Teresita; Bratoeff, Eugene

    2014-05-01

    Flank organs are an androgen-dependent pilosebaceous complex present in male and female hamsters. These organs have been used for the evaluation of antiandrogenic drugs, which could be used for the treatment of androgen-dependent afflictions. This study demonstrated the role of four different steroidal carbamates 7-10 in the expression of mRNAs coding for different enzymes involved in the lipid metabolism in flank organs. To determine the biological effects of compounds 7-10 on the expression of mRNA coding for lipid enzymes, steroids 7-10, testosterone (T), progesterone (P), and/or 7-10 were applied on the flank organs. Later, the mRNA expression for the enzymes was determined by polymerase chain reaction. The binding of 8 and 9 to the progesterone receptor (PR) as well as their effects on the activity of 5α-reductase were also evaluated. Treatments with T, P, and 7-10 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase (PI-S), and squalene-synthase (SQ-S). However, the combined treatments with P + 7-10 decreased the expression of GPAT, HMG-CoA-S, and HMG-CoA-R. Expression of mRNA for all enzymes was variable under treatment with T + 7-10. Data showed that these carbamates did not bind to the PR, but inhibited the activity of 5α-reductase. Carbamates 7-10 changed the mRNA expression model induced by T and P in flank organs.

  13. Novel Ciliate Genetic Code Variants Including the Reassignment of All Three Stop Codons to Sense Codons in Condylostoma magnum

    PubMed Central

    Heaphy, Stephen M.; Mariotti, Marco; Gladyshev, Vadim N.; Atkins, John F.; Baranov, Pavel V.

    2016-01-01

    mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3′ end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state. PMID:27501944

  14. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  15. The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway.

    PubMed

    Martinelli, Paola; Bonetti, Paola; Sironi, Cristina; Pruneri, Giancarlo; Fumagalli, Caterina; Raviele, Paola Rafaniello; Volorio, Sara; Pileri, Stefano; Chiarle, Roberto; McDuff, Fiona Kate Elizabeth; Tusi, Betsabeh Khoramian; Turner, Suzanne D; Inghirami, Giorgio; Pelicci, Pier Giuseppe; Colombo, Emanuela

    2011-06-16

    Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

  16. miR-135b mediates NPM-ALK-driven oncogenicity and renders IL-17-producing immunophenotype to anaplastic large cell lymphoma.

    PubMed

    Matsuyama, Hironori; Suzuki, Hiroshi I; Nishimori, Hikaru; Noguchi, Masaaki; Yao, Takashi; Komatsu, Norio; Mano, Hiroyuki; Sugimoto, Koichi; Miyazono, Kohei

    2011-12-22

    Many transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-driven oncogenicity and empowers IL-17-producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b-mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this "Th17 mimic" pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.

  17. Plant litter and soil type drive abundance, activity and community structure of alkB harbouring microbes in different soil compartments

    PubMed Central

    Schulz, Stephan; Giebler, Julia; Chatzinotas, Antonis; Wick, Lukas Y; Fetzer, Ingo; Welzl, Gerhard; Harms, Hauke; Schloter, Michael

    2012-01-01

    Alkanes are major constituents of plant-derived waxy materials. In this study, we investigated the abundance, community structure and activity of bacteria harbouring the alkane monooxygenase gene alkB, which catalyses a major step in the pathway of aerobic alkane degradation in the litter layer, the litter–soil interface and in bulk soil at three time points during the degradation of maize and pea plant litter (2, 8 and 30 weeks) to improve our understanding about drivers for microbial performance in different soil compartments. Soil cores of different soil textures (sandy and silty) were taken from an agricultural field and incubated at constant laboratory conditions. The abundance of alkB genes and transcripts (by qPCR) as well as the community structure (by terminal restriction fragment polymorphism fingerprinting) were measured in combination with the concentrations and composition of alkanes. The results obtained indicate a clear response pattern of all investigated biotic and abiotic parameters depending on the applied litter material, the type of soil used, the time point of sampling and the soil compartment studied. As expected the distribution of alkanes of different chain length formed a steep gradient from the litter layer to the bulk soil. Mainly in the two upper soil compartments community structure and abundance patterns of alkB were driven by the applied litter type and its degradation. Surprisingly, the differences between the compartments in one soil were more pronounced than the differences between similar compartments in the two soils studied. This indicates the necessity for analysing processes in different soil compartments to improve our mechanistic understanding of the dynamics of distinct functional groups of microbes. PMID:22402403

  18. Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells, leading to EMT and differentiation.

    PubMed

    Yoo, Young A; Kang, Myoung Hee; Kim, Byung Soo; Kim, Jun Suk; Seo, Jae Hong

    2009-06-01

    The interaction between mammary epithelial cells and their surrounding microenvironment are important in the development of the mammary gland. Thus, mesenchymal stem cells (MSCs), which retain pluripotency for various mesenchymal lineages, may provide a permissive environment for the morphologic alteration and differentiation of mammary epithelial cells. To this end, we investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal-alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, alpha-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway. PMID:19375841

  19. Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

    PubMed Central

    Richards, Mark W.; Law, Edward W. P.; Rennalls, La’Verne P.; Busacca, Sara; O’Regan, Laura; Fry, Andrew M.; Fennell, Dean A.; Bayliss, Richard

    2014-01-01

    Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners. PMID:24706829

  20. The anti-fibrotic effect of inhibition of TGFβ-ALK5 signalling in experimental pulmonary fibrosis in mice is attenuated in the presence of concurrent γ-herpesvirus infection.

    PubMed

    Smoktunowicz, Natalia; Alexander, Robert E; Franklin, Linda; Williams, Andrew E; Holman, Beverley; Mercer, Paul F; Jarai, Gabor; Scotton, Chris J; Chambers, Rachel C

    2015-09-01

    TGFβ-ALK5 pro-fibrotic signalling and herpesvirus infections have been implicated in the pathogenesis and exacerbation of pulmonary fibrosis. In this study we addressed the role of TGFβ-ALK5 signalling during the progression of fibrosis in a two-hit mouse model of murine γ-herpesvirus 68 (MHV-68) infection on the background of pre-existing bleomycin-induced pulmonary fibrosis. Assessment of total lung collagen levels in combination with ex vivo micro-computed tomography (µCT) analysis of whole lungs demonstrated that MHV-68 infection did not enhance lung collagen deposition in this two-hit model but led to a persistent and exacerbated inflammatory response. Moreover, µCT reconstruction and analysis of the two-hit model revealed distinguishing features of diffuse ground-glass opacities and consolidation superimposed on pre-existing fibrosis that were reminiscent of those observed in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Virally-infected murine fibrotic lungs further displayed evidence of extensive inflammatory cell infiltration and increased levels of CCL2, TNFα, IL-1β and IL-10. Blockade of TGFβ-ALK5 signalling attenuated lung collagen accumulation in bleomycin-alone injured mice, but this anti-fibrotic effect was reduced in the presence of concomitant viral infection. In contrast, inhibition of TGFβ-ALK5 signalling in virally-infected fibrotic lungs was associated with reduced inflammatory cell aggregates and increased levels of the antiviral cytokine IFNγ. These data reveal newly identified intricacies for the TGFβ-ALK5 signalling axis in experimental lung fibrosis, with different outcomes in response to ALK5 inhibition depending on the presence of viral infection. These findings raise important considerations for the targeting of TGFβ signalling responses in the context of pulmonary fibrosis.

  1. Breast implant-associated ALK-negative anaplastic large cell lymphoma: a case report and discussion of possible pathogenesis

    PubMed Central

    George, Eva V; Pharm, John; Houston, Courtney; Al-Quran, Semar; Brian, Grey; Dong, Huijia; Hai, Wang; Reeves, Westley; Yang, Li-Jun

    2013-01-01

    Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a recently recognized clinical entity, with only 39 well-documented cases reported worldwide, including 3 fatalities. Because of its rarity, the clinical and pathologic features of this malignancy have yet to be fully defined. Moreover, the pathogenesis of ALCL in association with textured silicone gel breast implants is poorly understood. Here we report a case of BIA-ALCL arising in a 67-year-old woman with a mastectomy due to breast cancer followed by implantation of textured silicone gel breast prosthesis. The patient presented with breast enlargement and tenderness 8 years following reconstructive surgery. MRI revealed a fluid collection surrounding the affected breast implant. Pathologic examination confirmed the presence of malignant ALCL T cells that were CD30+, CD8+, CD15+, HLA-DR+, CD25+ ALK- and p53. A diagnosis of indolent BIA-ALCL was made since tumor cells were not found outside of the capsule. Interestingly, an extensive mixed lymphocytic infiltrate and ectopic lymphoid tissue (lymphoid neogenesis) adjacent to the fibrous implant capsule were present. The patient was treated with capsulectomy and implantation of new breast prostheses. Six months later, the patient was found to have BIA-ALCL involvement of an axillary lymph node with cytogenetic evolutionof the tumor. To our knowledge, this is the sixth reported case of aggressive BIA-ALCL. Unique features of this case include the association with lymphoid neogenesis and the in vivo cytogenetic progression of the tumor. This case provides insight into the potential role of chronic inflammation and genetic instability in the pathogenesis of BIA-ALCL. PMID:23923082

  2. Breast implant-associated ALK-negative anaplastic large cell lymphoma: a case report and discussion of possible pathogenesis.

    PubMed

    George, Eva V; Pharm, John; Houston, Courtney; Al-Quran, Semar; Brian, Grey; Dong, Huijia; Hai, Wang; Reeves, Westley; Yang, Li-Jun

    2013-01-01

    Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a recently recognized clinical entity, with only 39 well-documented cases reported worldwide, including 3 fatalities. Because of its rarity, the clinical and pathologic features of this malignancy have yet to be fully defined. Moreover, the pathogenesis of ALCL in association with textured silicone gel breast implants is poorly understood. Here we report a case of BIA-ALCL arising in a 67-year-old woman with a mastectomy due to breast cancer followed by implantation of textured silicone gel breast prosthesis. The patient presented with breast enlargement and tenderness 8 years following reconstructive surgery. MRI revealed a fluid collection surrounding the affected breast implant. Pathologic examination confirmed the presence of malignant ALCL T cells that were CD30+, CD8+, CD15+, HLA-DR+, CD25+ ALK- and p53. A diagnosis of indolent BIA-ALCL was made since tumor cells were not found outside of the capsule. Interestingly, an extensive mixed lymphocytic infiltrate and ectopic lymphoid tissue (lymphoid neogenesis) adjacent to the fibrous implant capsule were present. The patient was treated with capsulectomy and implantation of new breast prostheses. Six months later, the patient was found to have BIA-ALCL involvement of an axillary lymph node with cytogenetic evolution of the tumor. To our knowledge, this is the sixth reported case of aggressive BIA-ALCL. Unique features of this case include the association with lymphoid neogenesis and the in vivo cytogenetic progression of the tumor. This case provides insight into the potential role of chronic inflammation and genetic instability in the pathogenesis of BIA-ALCL. PMID:23923082

  3. Alterations in genes other than EGFR/ALK/ROS1 in non-small cell lung cancer: trials and treatment options

    PubMed Central

    Desai, Arpita; Menon, Smitha P.; Dy, Grace K.

    2016-01-01

    During the last decade, we have seen tremendous progress in the therapy of lung cancer. Discovery of actionable mutations in EGFR and translocations in ALK and ROS1 have identified subsets of patients with excellent tumor response to oral targeted agents with manageable side effects. In this review, we highlight treatment options including corresponding clinical trials for oncogenic alterations affecting the receptor tyrosine kinases MET, FGFR, NTRK, RET, HER2, HER3, and HER4 as well as components of the RAS-RAF-MEK signaling pathway. PMID:27144064

  4. Discussion on LDPC Codes and Uplink Coding

    NASA Technical Reports Server (NTRS)

    Andrews, Ken; Divsalar, Dariush; Dolinar, Sam; Moision, Bruce; Hamkins, Jon; Pollara, Fabrizio

    2007-01-01

    This slide presentation reviews the progress that the workgroup on Low-Density Parity-Check (LDPC) for space link coding. The workgroup is tasked with developing and recommending new error correcting codes for near-Earth, Lunar, and deep space applications. Included in the presentation is a summary of the technical progress of the workgroup. Charts that show the LDPC decoder sensitivity to symbol scaling errors are reviewed, as well as a chart showing the performance of several frame synchronizer algorithms compared to that of some good codes and LDPC decoder tests at ESTL. Also reviewed is a study on Coding, Modulation, and Link Protocol (CMLP), and the recommended codes. A design for the Pseudo-Randomizer with LDPC Decoder and CRC is also reviewed. A chart that summarizes the three proposed coding systems is also presented.

  5. Manually operated coded switch

    DOEpatents

    Barnette, Jon H.

    1978-01-01

    The disclosure relates to a manually operated recodable coded switch in which a code may be inserted, tried and used to actuate a lever controlling an external device. After attempting a code, the switch's code wheels must be returned to their zero positions before another try is made.

  6. Parafermion stabilizer codes

    NASA Astrophysics Data System (ADS)

    Güngördü, Utkan; Nepal, Rabindra; Kovalev, Alexey A.

    2014-10-01

    We define and study parafermion stabilizer codes, which can be viewed as generalizations of Kitaev's one-dimensional (1D) model of unpaired Majorana fermions. Parafermion stabilizer codes can protect against low-weight errors acting on a small subset of parafermion modes in analogy to qudit stabilizer codes. Examples of several smallest parafermion stabilizer codes are given. A locality-preserving embedding of qudit operators into parafermion operators is established that allows one to map known qudit stabilizer codes to parafermion codes. We also present a local 2D parafermion construction that combines topological protection of Kitaev's toric code with additional protection relying on parity conservation.

  7. Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution.

    PubMed

    Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

    2013-09-01

    Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.

  8. Two Seemingly Homologous Noncoding RNAs Act Hierarchically to Activate glmS mRNA Translation

    PubMed Central

    Urban, Johannes H; Vogel, Jörg

    2008-01-01

    Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3′ end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms. PMID:18351803

  9. N6-methyladenosine in mRNA disrupts tRNA selection and translation elongation dynamics

    PubMed Central

    Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan; Chen, Jin; Petrov, Alexey; Prabhakar, Arjun; O'Leary, Seán E.; Dominissini, Dan; Rechavi, Gideon; Soltis, S. Michael; Ehrenberg, Måns

    2016-01-01

    N6-methylation of adenosine (m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base pairs with uridine during decoding as shown by x-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements employing an Escherichia coli translation system revealed that m6A modification of mRNA can act as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, as delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics. PMID:26751643

  10. Upstream AUGs in embryonic proinsulin mRNA control its low translation level

    PubMed Central

    Hernández-Sánchez, Catalina; Mansilla, Alicia; de la Rosa, Enrique J.; Pollerberg, G.Elisabeth; Martínez-Salas, Encarna; de Pablo, Flora

    2003-01-01

    Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5′ extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal. PMID:14532130

  11. Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

    PubMed

    Wong, Queenie Wing-Lei; Vaz, Candida; Lee, Qian Yi; Zhao, Tian Yun; Luo, Raymond; Archer, Stuart K; Preiss, Thomas; Tanavde, Vivek; Vardy, Leah A

    2016-01-01

    The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

  12. ARA type protograph codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush (Inventor); Abbasfar, Aliazam (Inventor); Jones, Christopher R. (Inventor); Dolinar, Samuel J. (Inventor); Thorpe, Jeremy C. (Inventor); Andrews, Kenneth S. (Inventor); Yao, Kung (Inventor)

    2008-01-01

    An apparatus and method for encoding low-density parity check codes. Together with a repeater, an interleaver and an accumulator, the apparatus comprises a precoder, thus forming accumulate-repeat-accumulate (ARA codes). Protographs representing various types of ARA codes, including AR3A, AR4A and ARJA codes, are described. High performance is obtained when compared to the performance of current repeat-accumulate (RA) or irregular-repeat-accumulate (IRA) codes.

  13. QR Codes 101

    ERIC Educational Resources Information Center

    Crompton, Helen; LaFrance, Jason; van 't Hooft, Mark

    2012-01-01

    A QR (quick-response) code is a two-dimensional scannable code, similar in function to a traditional bar code that one might find on a product at the supermarket. The main difference between the two is that, while a traditional bar code can hold a maximum of only 20 digits, a QR code can hold up to 7,089 characters, so it can contain much more…

  14. Preproglucagon mRNA expression in adult rat submandibular glands.

    PubMed

    Egéa, J C; Hirtz, C; Deville de Périère, D

    2003-04-01

    Salivary glands of various animal species have been reported to contain and suggested to produce glucagon or glucagon-like material, but the origin and the nature of this salivary peptide are still doubtful. The present study was undertaken to ascertain whether the glucagon gene is expressed in rat submandibular glands and in an immortalized murine cell line derived from salivary glands (SCA-9 cell line). For this purpose, total RNA was isolated from submandibular glands or cultured cells and submitted to reverse transcription. The cDNAs obtained were amplified by a nested polymerase chain reaction using preproglucagon primers. The results showed that the preproglucagon mRNA was expressed in adult rat submandibular glands but not in the SCA-9 cell line. Determination of cyclic DNA (cDNA) sequence established identity with the coding regions of rat pancreatic pre-proglucagon gene. In conclusion, these results strongly support the idea that rat submandibular glands could represent a source of extrapancreatic glucagon or of its precursor's peptide.

  15. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  16. Vitamin D and the RNA transcriptome: more than mRNA regulation

    PubMed Central

    Campbell, Moray J.

    2014-01-01

    The GRCh37.p13 primary assembly of the human genome contains 20805 protein coding mRNA, and 37147 non-protein coding genes and pseudogenes that as a result of RNA processing and editing generate 196501 gene transcripts. Given the size and diversity of the human transcriptome, it is timely to revisit what is known of VDR function in the regulation and targeting of transcription. Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approximate size, and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. With the discovery of microRNA, investigators also considered VDR regulation of these non-protein coding RNA. Again, cell and time dependency has emerged. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. As the awareness of the diversity of non-coding RNA increases, it is increasingly likely it will be revealed that VDR actions are mediated through these molecules also. Key knowledge gaps remain over the VDR transcriptome. The causes for the cell and type dependent transcriptional heterogenetiy remain enigmatic. ChIP-Seq approaches have confirmed that VDR binding choices differ very significantly by cell type, but as yet the underlying causes distilling VDR binding choices are unclear. Similarly, it is clear that many of the VDR binding sites are non-canonical in nature but again the mechanisms underlying these interactions are unclear. Finally, although alternative splicing is clearly a very significant process in cellular transcriptional control, the lack of RNA-Seq data centered on VDR function are currently limiting the global assessment of the VDR transcriptome. VDR

  17. Nonbinary Quantum Convolutional Codes Derived from Negacyclic Codes

    NASA Astrophysics Data System (ADS)

    Chen, Jianzhang; Li, Jianping; Yang, Fan; Huang, Yuanyuan

    2015-01-01

    In this paper, some families of nonbinary quantum convolutional codes are constructed by using negacyclic codes. These nonbinary quantum convolutional codes are different from quantum convolutional codes in the literature. Moreover, we construct a family of optimal quantum convolutional codes.

  18. SEQassembly: A Practical Tools Program for Coding Sequences Splicing

    NASA Astrophysics Data System (ADS)

    Lee, Hongbin; Yang, Hang; Fu, Lei; Qin, Long; Li, Huili; He, Feng; Wang, Bo; Wu, Xiaoming

    CDS (Coding Sequences) is a portion of mRNA sequences, which are composed by a number of exon sequence segments. The construction of CDS sequence is important for profound genetic analysis such as genotyping. A program in MATLAB environment is presented, which can process batch of samples sequences into code segments under the guide of reference exon models, and splice these code segments of same sample source into CDS according to the exon order in queue file. This program is useful in transcriptional polymorphism detection and gene function study.

  19. Problem-Based Test: An "In Vitro" Experiment to Analyze the Genetic Code

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon.…

  20. Problem-based test: An in vitro experiment to analyze the genetic code.

    PubMed

    Szeberényi, József

    2010-07-01

    Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon.

  1. Intragenic MBD5 familial deletion variant does not negatively impact MBD5 mRNA expression.

    PubMed

    Mullegama, Sureni V; Elsea, Sarah H

    2014-01-01

    2q23.1 deletion syndrome is characterized by intellectual disability, speech impairment, seizures, disturbed sleep pattern, behavioral problems, and hypotonia. Core features of this syndrome are due to haploinsufficiency of MBD5. Deletions that include coding and noncoding exons show reduced MBD5 mRNA expression. We report a patient with a neurological and behavioral phenotype similar to 2q23.1 deletion syndrome with an inherited intronic deletion in the 5-prime untranslated region of MBD5. Our data show that this patient has normal MBD5 mRNA expression; therefore, this deletion is likely not causative for 2q23.1 deletion syndrome. Overall, it is important to validate intronic deletions for pathogenicity.

  2. Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction.

    PubMed

    Weissberg, Itai; Wood, Lydia; Kamintsky, Lyn; Vazquez, Oscar; Milikovsky, Dan Z; Alexander, Allyson; Oppenheim, Hannah; Ardizzone, Carolyn; Becker, Albert; Frigerio, Federica; Vezzani, Annamaria; Buckwalter, Marion S; Huguenard, John R; Friedman, Alon; Kaufer, Daniela

    2015-06-01

    Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

  3. Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction.

    PubMed

    Weissberg, Itai; Wood, Lydia; Kamintsky, Lyn; Vazquez, Oscar; Milikovsky, Dan Z; Alexander, Allyson; Oppenheim, Hannah; Ardizzone, Carolyn; Becker, Albert; Frigerio, Federica; Vezzani, Annamaria; Buckwalter, Marion S; Huguenard, John R; Friedman, Alon; Kaufer, Daniela

    2015-06-01

    Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE. PMID:25836421

  4. Occurrence of alk(en)ylresorcinols in the fruits of two mango (Mangifera indica L.) cultivars during on-tree maturation and postharvest storage.

    PubMed

    Kienzle, Stefanie; Carle, Reinhold; Sruamsiri, Pittaya; Tosta, Carola; Neidhart, Sybille

    2014-01-01

    Regarding their relevance for the fungal resistance of mangoes in long supply chains, the alk(en)ylresorcinols (AR) were quantitated in peel and mesocarp throughout storage (27 days, 14 °C, ethylene absorption). The 12 'Chok Anan' and 11 'Nam Dokmai #4' lots picked between 83 and 115 days after full bloom (DAFB) had different harvest maturity indices. The development of dry matter and fruit growth indicated physiological maturity ∼100 DAFB. During storage, all fruits ripened slowly, mostly until over-ripeness and visible decay. The total AR contents always ranged at 73 ± 4.5 and 6.4 ± 0.7 mg hg(-1) of 'Chok Anan' and 'Nam Dokmai #4' peel dry weight, respectively, but only at 6.7 ± 0.7 and 0.9 ± 0.1 mg hg(-1) for the corresponding mesocarp (P ≤ 0.05). These narrow concentration ranges were contradictory to the decreasing fungal resistance. Accordingly, the alk(en)ylresorcinols have not been a deciding factor for the fungal resistance.

  5. Asymmetric quantum convolutional codes

    NASA Astrophysics Data System (ADS)

    La Guardia, Giuliano G.

    2016-01-01

    In this paper, we construct the first families of asymmetric quantum convolutional codes (AQCCs). These new AQCCs are constructed by means of the CSS-type construction applied to suitable families of classical convolutional codes, which are also constructed here. The new codes have non-catastrophic generator matrices, and they have great asymmetry. Since our constructions are performed algebraically, i.e. we develop general algebraic methods and properties to perform the constructions, it is possible to derive several families of such codes and not only codes with specific parameters. Additionally, several different types of such codes are obtained.

  6. Recent innovations in mRNA vaccines.

    PubMed

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  7. Decrease in class pi glutathione transferase mRNA levels by ultraviolet irradiation of cultured rat keratinocytes.

    PubMed

    Nakano, H; Kimura, J; Kumano, T; Hanada, K; Satoh, K; Hashimoto, I; Tsuchida, S

    1997-11-01

    The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST-P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST-P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10-25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST-P mRNA expression, with a 70% increase at 250 microM concentration. Alterations in possible trans-acting factors were examined to clarify the mechanism of repression by UV irradiation. c-Jun mRNA was induced 3.5-fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c-Fos mRNA was increased 10-fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c-Jun and c-Fos mRNA differed from that of GST-P mRNA. The level of mRNA for silencer factor-B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST-P gene did not suppress CAT activity. Although basal expression of the GST-P gene was mainly dependent on GPE I, altered expression of c-jun, c-fos and other genes coding for factors possibly trans-acting on GPE I did not appear to be responsible for the decreased GST-P mRNA levels.

  8. Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation.

    PubMed

    Pang, Wei-Jun; Lin, Li-Gen; Xiong, Yan; Wei, Ning; Wang, Yu; Shen, Qing-Wu; Yang, Gong-She

    2013-11-01

    PU.1 is an Ets family transcription factor involved in the myelo-lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR-155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR-155 regulates adipogenesis not through PU.1, but via C/EBPβ which is another target of miR-155. We also checked the expression levels of PU.1 mRNA and antisense long non-coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral-mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes.

  9. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung

    PubMed Central

    1995-01-01

    Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing. PMID:7869037

  10. Cellulases and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2001-02-20

    The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

  11. Cellulases and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2001-01-01

    The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

  12. QR Code Mania!

    ERIC Educational Resources Information Center

    Shumack, Kellie A.; Reilly, Erin; Chamberlain, Nik

    2013-01-01

    space, has error-correction capacity, and can be read from any direction. These codes are used in manufacturing, shipping, and marketing, as well as in education. QR codes can be created to produce…

  13. Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes

    PubMed Central

    2015-01-01

    Background It is known that mRNA folding can affect and regulate various gene expression steps both in living organisms and in viruses. Previous studies have recognized functional RNA structures in the genome of the Dengue virus. However, these studies usually focused either on the viral untranslated regions or on very specific and limited regions at the beginning of the coding sequences, in a limited number of strains, and without considering evolutionary selection. Results Here we performed the first large scale comprehensive genomics analysis of selection for local mRNA folding strength in the Dengue virus coding sequences, based on a total of 1,670 genomes and 4 serotypes. Our analysis identified clusters of positions along the coding regions that may undergo a conserved evolutionary selection for strong or weak local folding maintained across different viral variants. Specifically, 53-66 clusters for strong folding and 49-73 clusters for weak folding (depending on serotype) aggregated of positions with a significant conservation of folding energy signals (related to partially overlapping local genomic regions) were recognized. In addition, up to 7% of these positions were found to be conserved in more than 90% of the viral genomes. Although some of the identified positions undergo frequent synonymous / non-synonymous substitutions, the selection for folding strength therein is preserved, and thus cannot be trivially explained based on sequence conservation alone. Conclusions The fact that many of the positions with significant folding related signals are conserved among different Dengue variants suggests that a better understanding of the mRNA structures in the corresponding regions may promote the development of prospective anti- Dengue vaccination strategies. The comparative genomics approach described here can be employed in the future for detecting functional regions in other pathogens with very high mutations rates. PMID:26449467

  14. EMF wire code research

    SciTech Connect

    Jones, T.

    1993-11-01

    This paper examines the results of previous wire code research to determines the relationship with childhood cancer, wire codes and electromagnetic fields. The paper suggests that, in the original Savitz study, biases toward producing a false positive association between high wire codes and childhood cancer were created by the selection procedure.

  15. Disappearance of stored polyadenylic acid and mRNA during early germination of radish (Raphanus sativus L.) embryo axes.

    PubMed

    Delseny, M; Aspart, L; Guitton, Y

    1977-01-01

    Polyadenylic acid [poly (A)] is detected, characterized and quantitated in dry radish embryo axis RNA using a (3)H poly (U) probe. The amount of poly (A) gradually decreases after the onset of soaking, and, after a few hours, recovers to the initial level. This variation is shown to result from the addition of two opposed phenomena: the decay of stored poly (A) and the accumulation of newly synthesized poly (A). Stored poly (A), as well as the "in vivo" protein synthesis coded for by preformed mRNA, decreases during early germination with a half-life of two hours. As a whole, these results demonstrate that at least a fraction of the stored mRNA is translated as soon as the seed is soaked and that its role is rapidly taken over by newly-made mRNA.

  16. Software Certification - Coding, Code, and Coders

    NASA Technical Reports Server (NTRS)

    Havelund, Klaus; Holzmann, Gerard J.

    2011-01-01

    We describe a certification approach for software development that has been adopted at our organization. JPL develops robotic spacecraft for the exploration of the solar system. The flight software that controls these spacecraft is considered to be mission critical. We argue that the goal of a software certification process cannot be the development of "perfect" software, i.e., software that can be formally proven to be correct under all imaginable and unimaginable circumstances. More realistically, the goal is to guarantee a software development process that is conducted by knowledgeable engineers, who follow generally accepted procedures to control known risks, while meeting agreed upon standards of workmanship. We target three specific issues that must be addressed in such a certification procedure: the coding process, the code that is developed, and the skills of the coders. The coding process is driven by standards (e.g., a coding standard) and tools. The code is mechanically checked against the standard with the help of state-of-the-art static source code analyzers. The coders, finally, are certified in on-site training courses that include formal exams.

  17. An expanding universe of mRNA modifications

    PubMed Central

    Jaffrey, Samie R.

    2015-01-01

    The fate of mRNA can be regulated by internal base modifications, with the currently known modified bases being N6-methyladenosine, 5-methylcytosine, and inosine. Three new studies show that yeast and human mRNA also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA. PMID:25372308

  18. Coding for Electronic Mail

    NASA Technical Reports Server (NTRS)

    Rice, R. F.; Lee, J. J.

    1986-01-01

    Scheme for coding facsimile messages promises to reduce data transmission requirements to one-tenth current level. Coding scheme paves way for true electronic mail in which handwritten, typed, or printed messages or diagrams sent virtually instantaneously - between buildings or between continents. Scheme, called Universal System for Efficient Electronic Mail (USEEM), uses unsupervised character recognition and adaptive noiseless coding of text. Image quality of resulting delivered messages improved over messages transmitted by conventional coding. Coding scheme compatible with direct-entry electronic mail as well as facsimile reproduction. Text transmitted in this scheme automatically translated to word-processor form.

  19. mRNA Localization and Translational Control in Drosophila Oogenesis

    PubMed Central

    Lasko, Paul

    2012-01-01

    Localization of an mRNA species to a particular subcellular region can complement translational control mechanisms to produce a restricted spatial distribution of the protein it encodes. mRNA localization has been studied most in asymmetric cells such as budding yeast, early embryos, and neurons, but the process is likely to be more widespread. This article reviews the current state of knowledge about the mechanisms of mRNA localization and its functions in early embryonic development, focusing on Drosophila where the relevant knowledge is most advanced. Links between mRNA localization and translational control mechanisms also are examined. PMID:22865893

  20. XSOR codes users manual

    SciTech Connect

    Jow, Hong-Nian; Murfin, W.B.; Johnson, J.D.

    1993-11-01

    This report describes the source term estimation codes, XSORs. The codes are written for three pressurized water reactors (Surry, Sequoyah, and Zion) and two boiling water reactors (Peach Bottom and Grand Gulf). The ensemble of codes has been named ``XSOR``. The purpose of XSOR codes is to estimate the source terms which would be released to the atmosphere in severe accidents. A source term includes the release fractions of several radionuclide groups, the timing and duration of releases, the rates of energy release, and the elevation of releases. The codes have been developed by Sandia National Laboratories for the US Nuclear Regulatory Commission (NRC) in support of the NUREG-1150 program. The XSOR codes are fast running parametric codes and are used as surrogates for detailed mechanistic codes. The XSOR codes also provide the capability to explore the phenomena and their uncertainty which are not currently modeled by the mechanistic codes. The uncertainty distributions of input parameters may be used by an. XSOR code to estimate the uncertainty of source terms.

  1. DLLExternalCode

    SciTech Connect

    Greg Flach, Frank Smith

    2014-05-14

    DLLExternalCode is the a general dynamic-link library (DLL) interface for linking GoldSim (www.goldsim.com) with external codes. The overall concept is to use GoldSim as top level modeling software with interfaces to external codes for specific calculations. The DLLExternalCode DLL that performs the linking function is designed to take a list of code inputs from GoldSim, create an input file for the external application, run the external code, and return a list of outputs, read from files created by the external application, back to GoldSim. Instructions for creating the input file, running the external code, and reading the output are contained in an instructions file that is read and interpreted by the DLL.

  2. DLLExternalCode

    2014-05-14

    DLLExternalCode is the a general dynamic-link library (DLL) interface for linking GoldSim (www.goldsim.com) with external codes. The overall concept is to use GoldSim as top level modeling software with interfaces to external codes for specific calculations. The DLLExternalCode DLL that performs the linking function is designed to take a list of code inputs from GoldSim, create an input file for the external application, run the external code, and return a list of outputs, read frommore » files created by the external application, back to GoldSim. Instructions for creating the input file, running the external code, and reading the output are contained in an instructions file that is read and interpreted by the DLL.« less

  3. Parafermion stabilizer codes

    NASA Astrophysics Data System (ADS)

    Gungordu, Utkan; Nepal, Rabindra; Kovalev, Alexey

    2015-03-01

    We define and study parafermion stabilizer codes [Phys. Rev. A 90, 042326 (2014)] which can be viewed as generalizations of Kitaev's one dimensional model of unpaired Majorana fermions. Parafermion stabilizer codes can protect against low-weight errors acting on a small subset of parafermion modes in analogy to qudit stabilizer codes. Examples of several smallest parafermion stabilizer codes are given. Our results show that parafermions can achieve a better encoding rate than Majorana fermions. A locality preserving embedding of qudit operators into parafermion operators is established which allows one to map known qudit stabilizer codes to parafermion codes. We also present a local 2D parafermion construction that combines topological protection of Kitaev's toric code with additional protection relying on parity conservation. This work was supported in part by the NSF under Grants No. Phy-1415600 and No. NSF-EPSCoR 1004094.

  4. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    PubMed

    Iborra, A; Sentandreu, R; Gozalbo, D

    1996-09-01

    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  5. Bio—Cryptography: A Possible Coding Role for RNA Redundancy

    NASA Astrophysics Data System (ADS)

    Regoli, M.

    2009-03-01

    The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. The RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions," are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behavior in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

  6. Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export

    PubMed Central

    Lei, Haixin; Zhai, Bo; Yin, Shanye; Gygi, Steve; Reed, Robin

    2013-01-01

    We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery. PMID:23275560

  7. Graph based fusion of miRNA and mRNA expression data improves clinical outcome prediction in prostate cancer

    PubMed Central

    2011-01-01

    Background One of the main goals in cancer studies including high-throughput microRNA (miRNA) and mRNA data is to find and assess prognostic signatures capable of predicting clinical outcome. Both mRNA and miRNA expression changes in cancer diseases are described to reflect clinical characteristics like staging and prognosis. Furthermore, miRNA abundance can directly affect target transcripts and translation in tumor cells. Prediction models are trained to identify either mRNA or miRNA signatures for patient stratification. With the increasing number of microarray studies collecting mRNA and miRNA from the same patient cohort there is a need for statistical methods to integrate or fuse both kinds of data into one prediction model in order to find a combined signature that improves the prediction. Results Here, we propose a new method to fuse miRNA and mRNA data into one prediction model. Since miRNAs are known regulators of mRNAs we used the correlations between them as well as the target prediction information to build a bipartite graph representing the relations between miRNAs and mRNAs. This graph was used to guide the feature selection in order to improve the prediction. The method is illustrated on a prostate cancer data set comprising 98 patient samples with miRNA and mRNA expression data. The biochemical relapse was used as clinical endpoint. It could be shown that the bipartite graph in combination with both data sets could improve prediction performance as well as the stability of the feature selection. Conclusions Fusion of mRNA and miRNA expression data into one prediction model improves clinical outcome prediction in terms of prediction error and stable feature selection. The R source code of the proposed method is available in the supplement. PMID:22188670

  8. Codon influence on protein expression in E. coli correlates with mRNA levels

    PubMed Central

    Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

    2016-01-01

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  9. Beta Thalassemia: mutations which affect processing of the beta-Globin mRNA precursor.

    PubMed

    Kantor, J A; Turner, P H; Nienhuis, A W

    1980-08-01

    To define the molecular lesion which causes decreased beta-globin synthesis in beta+ thalessemia, four patients of diverse ethnic origin were studied. Each had a 2--3 fold higher concentration of beta-globin mRNA precursor than that found in control bone marrow cells from patients with sickle cell anemia. Globin RNA metabolism was analyzed in two of these patients. Transcription of the beta-globin gene appeared to be normal, since analysis of nuclear RNA indicated that beta-globin mRNA synthesis exceeded that of alpha in a 2 hr pulse but the cytoplasm contained a relative deficiency of labeled beta-globin mRNA. An abnormal RNA species approximately 650 nucleotides in length, which contained sequences transcribed from both the large intron and coding portions of the beta-globin gene, was found in one patient's bone marrow cells. The second patient's cells contained a significant amount of a 1320 nucleotide RNA species, not initially evident in normal cells, from which part but not all of the large intervening sequence had been removed. Our data thus indicate that mutations which affect RNA processing cause beta thalessemia.

  10. Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

    PubMed

    Zhang, Jichuan; Fei, Jingyi; Leslie, Benjamin J; Han, Kyu Young; Kuhlman, Thomas E; Ha, Taekjip

    2015-11-27

    Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

  11. Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues.

    PubMed Central

    Tuck, M T

    1992-01-01

    Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity. PMID:1445268

  12. Structural Distinction of Diacyl-, Alkylacyl, and Alk-1-Enylacyl Glycerophosphocholines as [M - 15]- Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

    NASA Astrophysics Data System (ADS)

    Hsu, Fong-Fu; Lodhi, Irfan J.; Turk, John; Semenkovich, Clay F.

    2014-08-01

    We describe a linear ion-trap (LIT) multiple-stage (MSn) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M - 15]- ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS4 mass spectra of the [M - 15 - R2'CH = CO]- ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

  13. Structure of the E. coli DNA Glycosylase AlkA Bound to the Ends of Duplex DNA: A System for the Structure Determination of Lesion-Containing DNA

    SciTech Connect

    Bowman, B.R.; Lee, S.; Wang, S.; Verdine, G.L.

    2008-10-24

    The constant attack on DNA by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. Visualizing the effects of these lesions on the structure of duplex DNA is key to understanding their biologic consequences. The most definitive method of obtaining such structures, X-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of DNA. Here, we present a crystallization system that uses a protein, the DNA glycosylase AlkA, as a scaffold to mediate the crystallization of lesion-containing duplex DNA. We demonstrate the use of this system to facilitate the rapid structure determination of DNA containing the lesion 8-oxoguanine in several different sequence contexts, and also deoxyinosine and 1,N{sup 6}-ethenoadenine, each stabilized as the corresponding 2{prime}-flouro analog. The structures of 8-oxoguanine provide a correct atomic-level view of this important endogenous lesion in DNA.

  14. Functional Integration of mRNA Translational Control Programs.

    PubMed

    MacNicol, Melanie C; Cragle, Chad E; Arumugam, Karthik; Fosso, Bruno; Pesole, Graziano; MacNicol, Angus M

    2015-01-01

    Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease. PMID:26197342

  15. Sodium Channel Inhibitors Reduce DMPK mRNA and Protein.

    PubMed

    Witherspoon, Luke; O'Reilly, Sean; Hadwen, Jeremiah; Tasnim, Nafisa; MacKenzie, Alex; Farooq, Faraz

    2015-08-01

    Myotonic dystrophy type 1 (DM1) is caused by an expanded trinucleotide (CTG)n tract in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This results in the aggregation of an expanded mRNA forming toxic intranuclear foci which sequester splicing factors. We believe down-regulation of DMPK mRNA represents a potential, and as yet unexplored, DM1 therapeutic avenue. Consequently, a computational screen for agents which down-regulate DMPK mRNA was undertaken, unexpectedly identifying the sodium channel blockers mexiletine, prilocaine, procainamide, and sparteine as effective suppressors of DMPK mRNA. Analysis of DMPK mRNA in C2C12 myoblasts following treatment with these agents revealed a reduction in the mRNA levels. In vivo analysis of CD1 mice also showed DMPK mRNA and protein down-regulation. The role of DMPK mRNA suppression in the documented efficacy of this class of compounds in DM1 is worthy of further investigation. PMID:26011798

  16. Functional Integration of mRNA Translational Control Programs.

    PubMed

    MacNicol, Melanie C; Cragle, Chad E; Arumugam, Karthik; Fosso, Bruno; Pesole, Graziano; MacNicol, Angus M

    2015-01-01

    Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  17. Sarcomatoid variant of ALK- anaplastic large cell lymphoma involving multiple lymph nodes and both lungs with production of proinflammatory cytokines: report of a case and review of literature

    PubMed Central

    Yu, Lu; Yan, Lin Li; Yang, Shou Jing

    2014-01-01

    Sarcomatoid variant of anaplastic large cell lymphoma (ALCL) is one of the rarest histologic variants of ALCL that consists of large, bizarre, often spindle-shaped, neoplastic cells resembling a soft tissue sarcoma. We report here such a case of ALCL with both pulmonary and multiple nodal involvement in a 47-year-old woman who initially presented with fever, cough, sputum, itching skin, and weight loss. The initial transbronchial lung biopsy showed discohesive pleomorphic malignant cells in a strong inflammatory milieu reminiscent of inflammatory malignant fibrous histiocytoma (MFH). Subsequent cervical lymph node biopsy revealed a spindle cell sarcoma predominantly composed of plump spindle and oval neoplastic cells in interweaving fascicles, with sparse inflammatory infiltrates, resembling pleomorphic-storiform type of MFH. However, these tumor cells in the lung and node lesions revealed essentially similar immunohistochemical features that were positive for CD30, EMA, TIA-1, granzyme B, and fascin, but negative for anaplastic lymphoma kinase (ALK), and T- or B-lineage-specific marker. The spindled cells stains diffuse strong positive for smooth muscle actin (SMA), along with vimentin. Further studies showed that the tumor produced large quantities of the proinflammatory cytokines interleukin-2 (IL-2), IL-6, and IL-8, which we believe may contribute to the pathogenesis of sarcomatoid transformation of this tumor, and was associated with the patient’s inflammatory symptoms. To the best of our knowledge, this is the first reported case of sarcomatoid variant of ALK-negative ALCL with null cell phenotype and in situ production of proinflammatory cytokines presenting as multiple nodes and pulmonary involvement. PMID:25197351

  18. Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB

    PubMed Central

    Cooper, Harriet L. R.; Mishra, Girish; Huang, Xiongyi; Pender-Cudlip, Marilla; Austin, Rachel N.; Shanklin, John; Groves, John T.

    2012-01-01

    A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analog, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ~20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicates that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step. PMID:23157204

  19. Remission of ALK-negative primary pulmonary inflammatory myofibroblastic tumor on treatment with clarithromycin: A case report and review of the literature

    PubMed Central

    WATANABE, HIDEHIRO; URUMA, TOMONORI; TAZAKI, GEN; TAJIRI, TAKUMA; KIKUCHI, RYOTA; ITOH, MASAYUKI; AOSHIBA, KAZUTETSU; NAKAMURA, HIROYUKI

    2016-01-01

    Inflammatory myofibroblastic tumors (IMTs) belong to an intermediate group of soft-tissue tumors, they are relatively rare but exhibit a wide range of pathologies, from benign to malignant. At present, no standard treatment has been established, however, it is known to be important to determine the grade of malignancy of the tumor, prior to treatment. The present study reports a 73-year-old female patient with no clinical manifestations, who, when examined radiographically at a health check exhibited bilateral thoracic infiltrative shadows and nodular shadows by chest CT. A metastatic tumor or an organizing pneumonia were suspected. Blood examination showed no abnormal findings, and a pathological diagnosis of IMT was given from the histological findings of the tissue extracted by video-assisted thoracic surgery. Histological analysis established the lack of expression of anaplastic lymphoma kinase (ALK1) and immunoglobulin subtype G4 (IgG4). Alteration of the radiological shadows was observed over several weeks, and after concluding that chronic inflammation was worsening the patient's condition, clarithromycin was administered as a long-term macrolide therapy. The IMT decreased in size, and eight months later it had almost resolved. The patient was last reported to be maintaining a stable condition with no relapse. Some IMT cases have malignant pathology, and should be carefully followed-up. However, in the present case, where the IMT is both ALK1-negative and IgG4-negative, its biological immune responsiveness appears to differ from positive cases, and an inflammatory response was predominant. Clarithromycin, has immunomodulatory and anti-inflammatory effects and appeared to be effective in treating the IMT of the patient in the present study. PMID:26998073

  20. Industrial Code Development

    NASA Technical Reports Server (NTRS)

    Shapiro, Wilbur

    1991-01-01

    The industrial codes will consist of modules of 2-D and simplified 2-D or 1-D codes, intended for expeditious parametric studies, analysis, and design of a wide variety of seals. Integration into a unified system is accomplished by the industrial Knowledge Based System (KBS), which will also provide user friendly interaction, contact sensitive and hypertext help, design guidance, and an expandable database. The types of analysis to be included with the industrial codes are interfacial performance (leakage, load, stiffness, friction losses, etc.), thermoelastic distortions, and dynamic response to rotor excursions. The first three codes to be completed and which are presently being incorporated into the KBS are the incompressible cylindrical code, ICYL, and the compressible cylindrical code, GCYL.

  1. Updating the Read Codes

    PubMed Central

    Robinson, David; Comp, Dip; Schulz, Erich; Brown, Philip; Price, Colin

    1997-01-01

    Abstract The Read Codes are a hierarchically-arranged controlled clinical vocabulary introduced in the early 1980s and now consisting of three maintained versions of differing complexity. The code sets are dynamic, and are updated quarterly in response to requests from users including clinicians in both primary and secondary care, software suppliers, and advice from a network of specialist healthcare professionals. The codes' continual evolution of content, both across and within versions, highlights tensions between different users and uses of coded clinical data. Internal processes, external interactions and new structural features implemented by the NHS Centre for Coding and Classification (NHSCCC) for user interactive maintenance of the Read Codes are described, and over 2000 items of user feedback episodes received over a 15-month period are analysed. PMID:9391934

  2. Mechanical code comparator

    DOEpatents

    Peter, Frank J.; Dalton, Larry J.; Plummer, David W.

    2002-01-01

    A new class of mechanical code comparators is described which have broad potential for application in safety, surety, and security applications. These devices can be implemented as micro-scale electromechanical systems that isolate a secure or otherwise controlled device until an access code is entered. This access code is converted into a series of mechanical inputs to the mechanical code comparator, which compares the access code to a pre-input combination, entered previously into the mechanical code comparator by an operator at the system security control point. These devices provide extremely high levels of robust security. Being totally mechanical in operation, an access control system properly based on such devices cannot be circumvented by software attack alone.

  3. Generating code adapted for interlinking legacy scalar code and extended vector code

    DOEpatents

    Gschwind, Michael K

    2013-06-04

    Mechanisms for intermixing code are provided. Source code is received for compilation using an extended Application Binary Interface (ABI) that extends a legacy ABI and uses a different register configuration than the legacy ABI. First compiled code is generated based on the source code, the first compiled code comprising code for accommodating the difference in register configurations used by the extended ABI and the legacy ABI. The first compiled code and second compiled code are intermixed to generate intermixed code, the second compiled code being compiled code that uses the legacy ABI. The intermixed code comprises at least one call instruction that is one of a call from the first compiled code to the second compiled code or a call from the second compiled code to the first compiled code. The code for accommodating the difference in register configurations is associated with the at least one call instruction.

  4. Probing dimensionality beyond the linear sequence of mRNA.

    PubMed

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions. PMID:26650615

  5. Phonological coding during reading

    PubMed Central

    Leinenger, Mallorie

    2014-01-01

    The exact role that phonological coding (the recoding of written, orthographic information into a sound based code) plays during silent reading has been extensively studied for more than a century. Despite the large body of research surrounding the topic, varying theories as to the time course and function of this recoding still exist. The present review synthesizes this body of research, addressing the topics of time course and function in tandem. The varying theories surrounding the function of phonological coding (e.g., that phonological codes aid lexical access, that phonological codes aid comprehension and bolster short-term memory, or that phonological codes are largely epiphenomenal in skilled readers) are first outlined, and the time courses that each maps onto (e.g., that phonological codes come online early (pre-lexical) or that phonological codes come online late (post-lexical)) are discussed. Next the research relevant to each of these proposed functions is reviewed, discussing the varying methodologies that have been used to investigate phonological coding (e.g., response time methods, reading while eyetracking or recording EEG and MEG, concurrent articulation) and highlighting the advantages and limitations of each with respect to the study of phonological coding. In response to the view that phonological coding is largely epiphenomenal in skilled readers, research on the use of phonological codes in prelingually, profoundly deaf readers is reviewed. Finally, implications for current models of word identification (activation-verification model (Van Order, 1987), dual-route model (e.g., Coltheart, Rastle, Perry, Langdon, & Ziegler, 2001), parallel distributed processing model (Seidenberg & McClelland, 1989)) are discussed. PMID:25150679

  6. Industrial Computer Codes

    NASA Technical Reports Server (NTRS)

    Shapiro, Wilbur

    1996-01-01

    This is an overview of new and updated industrial codes for seal design and testing. GCYLT (gas cylindrical seals -- turbulent), SPIRALI (spiral-groove seals -- incompressible), KTK (knife to knife) Labyrinth Seal Code, and DYSEAL (dynamic seal analysis) are covered. CGYLT uses G-factors for Poiseuille and Couette turbulence coefficients. SPIRALI is updated to include turbulence and inertia, but maintains the narrow groove theory. KTK labyrinth seal code handles straight or stepped seals. And DYSEAL provides dynamics for the seal geometry.

  7. Doubled Color Codes

    NASA Astrophysics Data System (ADS)

    Bravyi, Sergey

    Combining protection from noise and computational universality is one of the biggest challenges in the fault-tolerant quantum computing. Topological stabilizer codes such as the 2D surface code can tolerate a high level of noise but implementing logical gates, especially non-Clifford ones, requires a prohibitively large overhead due to the need of state distillation. In this talk I will describe a new family of 2D quantum error correcting codes that enable a transversal implementation of all logical gates required for the universal quantum computing. Transversal logical gates (TLG) are encoded operations that can be realized by applying some single-qubit rotation to each physical qubit. TLG are highly desirable since they introduce no overhead and do not spread errors. It has been known before that a quantum code can have only a finite number of TLGs which rules out computational universality. Our scheme circumvents this no-go result by combining TLGs of two different quantum codes using the gauge-fixing method pioneered by Paetznick and Reichardt. The first code, closely related to the 2D color code, enables a transversal implementation of all single-qubit Clifford gates such as the Hadamard gate and the π / 2 phase shift. The second code that we call a doubled color code provides a transversal T-gate, where T is the π / 4 phase shift. The Clifford+T gate set is known to be computationally universal. The two codes can be laid out on the honeycomb lattice with two qubits per site such that the code conversion requires parity measurements for six-qubit Pauli operators supported on faces of the lattice. I will also describe numerical simulations of logical Clifford+T circuits encoded by the distance-3 doubled color code. Based on a joint work with Andrew Cross.

  8. GeneTack database: genes with frameshifts in prokaryotic genomes and eukaryotic mRNA sequences.

    PubMed

    Antonov, Ivan; Baranov, Pavel; Borodovsky, Mark

    2013-01-01

    Database annotations of prokaryotic genomes and eukaryotic mRNA sequences pay relatively low attention to frame transitions that disrupt protein-coding genes. Frame transitions (frameshifts) could be caused by sequencing errors or indel mutations inside protein-coding regions. Other observed frameshifts are related to recoding events (that evolved to control expression of some genes). Earlier, we have developed an algorithm and software program GeneTack for ab initio frameshift finding in intronless genes. Here, we describe a database (freely available at http://topaz.gatech.edu/GeneTack/db.html) containing genes with frameshifts (fs-genes) predicted by GeneTack. The database includes 206 991 fs-genes from 1106 complete prokaryotic genomes and 45 295 frameshifts predicted in mRNA sequences from 100 eukaryotic genomes. The whole set of fs-genes was grouped into clusters based on sequence similarity between fs-proteins (conceptually translated fs-genes), conservation of the frameshift position and frameshift direction (-1, +1). The fs-genes can be retrieved by similarity search to a given query sequence via a web interface, by fs-gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, etc. The largest clusters contain fs-genes with programed frameshifts (related to recoding events).

  9. FAA Smoke Transport Code

    SciTech Connect

    Domino, Stefan; Luketa-Hanlin, Anay; Gallegos, Carlos

    2006-10-27

    FAA Smoke Transport Code, a physics-based Computational Fluid Dynamics tool, which couples heat, mass, and momentum transfer, has been developed to provide information on smoke transport in cargo compartments with various geometries and flight conditions. The software package contains a graphical user interface for specification of geometry and boundary conditions, analysis module for solving the governing equations, and a post-processing tool. The current code was produced by making substantial improvements and additions to a code obtained from a university. The original code was able to compute steady, uniform, isothermal turbulent pressurization. In addition, a preprocessor and postprocessor were added to arrive at the current software package.

  10. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  11. Tokamak Systems Code

    SciTech Connect

    Reid, R.L.; Barrett, R.J.; Brown, T.G.; Gorker, G.E.; Hooper, R.J.; Kalsi, S.S.; Metzler, D.H.; Peng, Y.K.M.; Roth, K.E.; Spampinato, P.T.

    1985-03-01

    The FEDC Tokamak Systems Code calculates tokamak performance, cost, and configuration as a function of plasma engineering parameters. This version of the code models experimental tokamaks. It does not currently consider tokamak configurations that generate electrical power or incorporate breeding blankets. The code has a modular (or subroutine) structure to allow independent modeling for each major tokamak component or system. A primary benefit of modularization is that a component module may be updated without disturbing the remainder of the systems code as long as the imput to or output from the module remains unchanged.

  12. MORSE Monte Carlo code

    SciTech Connect

    Cramer, S.N.

    1984-01-01

    The MORSE code is a large general-use multigroup Monte Carlo code system. Although no claims can be made regarding its superiority in either theoretical details or Monte Carlo techniques, MORSE has been, since its inception at ORNL in the late 1960s, the most widely used Monte Carlo radiation transport code. The principal reason for this popularity is that MORSE is relatively easy to use, independent of any installation or distribution center, and it can be easily customized to fit almost any specific need. Features of the MORSE code are described.

  13. An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    PubMed Central

    Toribio, René; Díaz-López, Irene; Boskovic, Jasminka; Ventoso, Iván

    2016-01-01

    During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680–914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. PMID:26984530

  14. Optimization of mRNA design for protein expression in the crustacean Daphnia magna.

    PubMed

    Törner, Kerstin; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

    2014-08-01

    The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3' UTR failed to induce fluorescence. To assess reporter expression, the length of the 3' UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3' end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3' UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3' UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3' UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

  15. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

    PubMed Central

    Garrett, J E; Collard, M W; Douglass, J O

    1989-01-01

    The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed. Images PMID:2573832

  16. An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

    PubMed Central

    Moormann, R J; van der Velden, H M; Dodemont, H J; Andreoli, P M; Bloemendal, H; Schoenmakers, J G

    1981-01-01

    Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs. Images PMID:6171772

  17. Research on universal combinatorial coding.

    PubMed

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value.

  18. Research on universal combinatorial coding.

    PubMed

    Lu, Jun; Zhang, Zhuo; Mo, Juan

    2014-01-01

    The conception of universal combinatorial coding is proposed. Relations exist more or less in many coding methods. It means that a kind of universal coding method is objectively existent. It can be a bridge connecting many coding methods. Universal combinatorial coding is lossless and it is based on the combinatorics theory. The combinational and exhaustive property make it closely related with the existing code methods. Universal combinatorial coding does not depend on the probability statistic characteristic of information source, and it has the characteristics across three coding branches. It has analyzed the relationship between the universal combinatorial coding and the variety of coding method and has researched many applications technologies of this coding method. In addition, the efficiency of universal combinatorial coding is analyzed theoretically. The multicharacteristic and multiapplication of universal combinatorial coding are unique in the existing coding methods. Universal combinatorial coding has theoretical research and practical application value. PMID:24772019

  19. Myelin basic protein synthesis is regulated by small non-coding RNA 715.

    PubMed

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-09-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein.

  20. Fast Coding Unit Encoding Mechanism for Low Complexity Video Coding

    PubMed Central

    Wu, Yueying; Jia, Kebin; Gao, Guandong

    2016-01-01

    In high efficiency video coding (HEVC), coding tree contributes to excellent compression performance. However, coding tree brings extremely high computational complexity. Innovative works for improving coding tree to further reduce encoding time are stated in this paper. A novel low complexity coding tree mechanism is proposed for HEVC fast coding unit (CU) encoding. Firstly, this paper makes an in-depth study of the relationship among CU distribution, quantization parameter (QP) and content change (CC). Secondly, a CU coding tree probability model is proposed for modeling and predicting CU distribution. Eventually, a CU coding tree probability update is proposed, aiming to address probabilistic model distortion problems caused by CC. Experimental results show that the proposed low complexity CU coding tree mechanism significantly reduces encoding time by 27% for lossy coding and 42% for visually lossless coding and lossless coding. The proposed low complexity CU coding tree mechanism devotes to improving coding performance under various application conditions. PMID:26999741

  1. Code of Ethics

    ERIC Educational Resources Information Center

    Division for Early Childhood, Council for Exceptional Children, 2009

    2009-01-01

    The Code of Ethics of the Division for Early Childhood (DEC) of the Council for Exceptional Children is a public statement of principles and practice guidelines supported by the mission of DEC. The foundation of this Code is based on sound ethical reasoning related to professional practice with young children with disabilities and their families…

  2. Lichenase and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2000-08-15

    The present invention provides a fungal lichenase, i.e., an endo-1,3-1,4-.beta.-D-glucanohydrolase, its coding sequence, recombinant DNA molecules comprising the lichenase coding sequences, recombinant host cells and methods for producing same. The present lichenase is from Orpinomyces PC-2.

  3. Legacy Code Modernization

    NASA Technical Reports Server (NTRS)

    Hribar, Michelle R.; Frumkin, Michael; Jin, Haoqiang; Waheed, Abdul; Yan, Jerry; Saini, Subhash (Technical Monitor)

    1998-01-01

    Over the past decade, high performance computing has evolved rapidly; systems based on commodity microprocessors have been introduced in quick succession from at least seven vendors/families. Porting codes to every new architecture is a difficult problem; in particular, here at NASA, there are many large CFD applications that are very costly to port to new machines by hand. The LCM ("Legacy Code Modernization") Project is the development of an integrated parallelization environment (IPE) which performs the automated mapping of legacy CFD (Fortran) applications to state-of-the-art high performance computers. While most projects to port codes focus on the parallelization of the code, we consider porting to be an iterative process consisting of several steps: 1) code cleanup, 2) serial optimization,3) parallelization, 4) performance monitoring and visualization, 5) intelligent tools for automated tuning using performance prediction and 6) machine specific optimization. The approach for building this parallelization environment is to build the components for each of the steps simultaneously and then integrate them together. The demonstration will exhibit our latest research in building this environment: 1. Parallelizing tools and compiler evaluation. 2. Code cleanup and serial optimization using automated scripts 3. Development of a code generator for performance prediction 4. Automated partitioning 5. Automated insertion of directives. These demonstrations will exhibit the effectiveness of an automated approach for all the steps involved with porting and tuning a legacy code application for a new architecture.

  4. Synthesizing Certified Code

    NASA Technical Reports Server (NTRS)

    Whalen, Michael; Schumann, Johann; Fischer, Bernd

    2002-01-01

    Code certification is a lightweight approach to demonstrate software quality on a formal level. Its basic idea is to require producers to provide formal proofs that their code satisfies certain quality properties. These proofs serve as certificates which can be checked independently. Since code certification uses the same underlying technology as program verification, it also requires many detailed annotations (e.g., loop invariants) to make the proofs possible. However, manually adding theses annotations to the code is time-consuming and error-prone. We address this problem by combining code certification with automatic program synthesis. We propose an approach to generate simultaneously, from a high-level specification, code and all annotations required to certify generated code. Here, we describe a certification extension of AUTOBAYES, a synthesis tool which automatically generates complex data analysis programs from compact specifications. AUTOBAYES contains sufficient high-level domain knowledge to generate detailed annotations. This allows us to use a general-purpose verification condition generator to produce a set of proof obligations in first-order logic. The obligations are then discharged using the automated theorem E-SETHEO. We demonstrate our approach by certifying operator safety for a generated iterative data classification program without manual annotation of the code.

  5. Effects of DNA replication on mRNA noise

    PubMed Central

    Peterson, Joseph R.; Cole, John A.; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A.

    2015-01-01

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript’s half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  6. Combustion chamber analysis code

    NASA Astrophysics Data System (ADS)

    Przekwas, A. J.; Lai, Y. G.; Krishnan, A.; Avva, R. K.; Giridharan, M. G.

    1993-05-01

    A three-dimensional, time dependent, Favre averaged, finite volume Navier-Stokes code has been developed to model compressible and incompressible flows (with and without chemical reactions) in liquid rocket engines. The code has a non-staggered formulation with generalized body-fitted-coordinates (BFC) capability. Higher order differencing methodologies such as MUSCL and Osher-Chakravarthy schemes are available. Turbulent flows can be modeled using any of the five turbulent models present in the code. A two-phase, two-liquid, Lagrangian spray model has been incorporated into the code. Chemical equilibrium and finite rate reaction models are available to model chemically reacting flows. The discrete ordinate method is used to model effects of thermal radiation. The code has been validated extensively against benchmark experimental data and has been applied to model flows in several propulsion system components of the SSME and the STME.

  7. Combustion chamber analysis code

    NASA Technical Reports Server (NTRS)

    Przekwas, A. J.; Lai, Y. G.; Krishnan, A.; Avva, R. K.; Giridharan, M. G.

    1993-01-01

    A three-dimensional, time dependent, Favre averaged, finite volume Navier-Stokes code has been developed to model compressible and incompressible flows (with and without chemical reactions) in liquid rocket engines. The code has a non-staggered formulation with generalized body-fitted-coordinates (BFC) capability. Higher order differencing methodologies such as MUSCL and Osher-Chakravarthy schemes are available. Turbulent flows can be modeled using any of the five turbulent models present in the code. A two-phase, two-liquid, Lagrangian spray model has been incorporated into the code. Chemical equilibrium and finite rate reaction models are available to model chemically reacting flows. The discrete ordinate method is used to model effects of thermal radiation. The code has been validated extensively against benchmark experimental data and has been applied to model flows in several propulsion system components of the SSME and the STME.

  8. Energy Conservation Code Decoded

    SciTech Connect

    Cole, Pam C.; Taylor, Zachary T.

    2006-09-01

    Designing an energy-efficient, affordable, and comfortable home is a lot easier thanks to a slime, easier to read booklet, the 2006 International Energy Conservation Code (IECC), published in March 2006. States, counties, and cities have begun reviewing the new code as a potential upgrade to their existing codes. Maintained under the public consensus process of the International Code Council, the IECC is designed to do just what its title says: promote the design and construction of energy-efficient homes and commercial buildings. Homes in this case means traditional single-family homes, duplexes, condominiums, and apartment buildings having three or fewer stories. The U.S. Department of Energy, which played a key role in proposing the changes that resulted in the new code, is offering a free training course that covers the residential provisions of the 2006 IECC.

  9. Evolving genetic code

    PubMed Central

    OHAMA, Takeshi; INAGAKI, Yuji; BESSHO, Yoshitaka; OSAWA, Syozo

    2008-01-01

    In 1985, we reported that a bacterium, Mycoplasma capricolum, used a deviant genetic code, namely UGA, a “universal” stop codon, was read as tryptophan. This finding, together with the deviant nuclear genetic codes in not a few organisms and a number of mitochondria, shows that the genetic code is not universal, and is in a state of evolution. To account for the changes in codon meanings, we proposed the codon capture theory stating that all the code changes are non-disruptive without accompanied changes of amino acid sequences of proteins. Supporting evidence for the theory is presented in this review. A possible evolutionary process from the ancient to the present-day genetic code is also discussed. PMID:18941287

  10. Identification of new M23A mRNA of mouse aquaporin-4 expressed in brain, liver, and kidney.

    PubMed

    Alikina, T Yu; Illarionova, N B; Zelenin, S M; Bondar, A A

    2012-05-01

    Aquaporins (AQPs) belong to a transmembrane protein family of water channels that are permeable to water by the osmotic gradient. There are two isoforms of mouse AQP4 - M1 and M23. Their balance in the cell determines water permeability of the plasma membrane. These two isoforms are encoded by three mRNAs: M1 isoform is encoded by M1 mRNA and M23 isoform is encoded by M23 and M23X mRNAs. Here we found a new fourth mRNA of mouse AQP4 - M23A mRNA. The start of transcription is different for M23A mRNA