20 results on '"Annalisa Morano"'
Search Results
2. Reactive oxygen species regulate the levels of dual oxidase (Duox1-2) in human neuroblastoma cells.
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Simona Damiano, Roberta Fusco, Annalisa Morano, Mariarosaria De Mizio, Roberto Paternò, Antonella De Rosa, Rosa Spinelli, Stefano Amente, Rodolfo Frunzio, Paolo Mondola, Francoise Miot, Paolo Laccetti, Mariarosaria Santillo, and Enrico Vittorio Avvedimento
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Medicine ,Science - Abstract
Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox) (NOX2), or of the other membrane subunit of NADPH oxidase, p22(phox), blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.
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- 2012
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3. DNA damage, homology-directed repair, and DNA methylation.
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Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, Annalisa Morano, Bongyong Lee, Alba Di Pardo, Samantha Messina, Rodolfo Iuliano, Alfredo Fusco, Maria R Santillo, Mark T Muller, Lorenzo Chiariotti, Max E Gottesman, and Enrico V Avvedimento
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Genetics ,QH426-470 - Abstract
To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.
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- 2007
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4. Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia
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Maria Teresa Bochicchio, Luciana De Luca, Gabriella Bianchino, Giovanni Martinelli, Vittorio Simeon, Annalisa Morano, Elisabetta Signorino, Gianantonio Rosti, Giuseppe Pietrantuono, Pellegrino Musto, Luigi Del Vecchio, Claudia Venturi, Francesco La Rocca, Vitina Grieco, Ilaria Laurenzana, Stefania Trino, Alberto Fragasso, Antonella Caivano, Daniela Cilloni, Trino, Stefania, De Luca, Luciana, Simeon, Vittorio, Laurenzana, Ilaria, Morano, Annalisa, Caivano, Antonella, La Rocca, Francesco, Pietrantuono, Giuseppe, Bianchino, Gabriella, Grieco, Vitina, Signorino, Elisabetta, Fragasso, Alberto, Bochicchio, Maria Teresa, Venturi, Claudia, Rosti, Gianantonio, Martinelli, Giovanni, Del Vecchio, Luigi, Cilloni, Daniela, and Musto, Pellegrino
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0301 basic medicine ,Cytoplasm ,Cancer Research ,Bridging integrator 1 ,Fusion Proteins, bcr-abl ,Bone Marrow Cells ,Real-Time Polymerase Chain Reaction ,Endocytosis ,Receptor tyrosine kinase ,03 medical and health sciences ,Downregulation and upregulation ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,medicine ,Humans ,AXL ,BCR-ABL1 ,Chronic myeloid leukemia ,Rab interactor 1 ,Adaptor Proteins, Signal Transducing ,biology ,Gene Expression Regulation, Leukemic ,Gene Expression Profiling ,Tumor Suppressor Proteins ,Intracellular Signaling Peptides and Proteins ,Nuclear Proteins ,Receptor Protein-Tyrosine Kinases ,Myeloid leukemia ,Imatinib ,General Medicine ,Axl Receptor Tyrosine Kinase ,Up-Regulation ,Cell biology ,Gene Expression Regulation, Neoplastic ,HEK293 Cells ,030104 developmental biology ,Drug Resistance, Neoplasm ,Leukocytes, Mononuclear ,biology.protein ,Cancer research ,Rab ,K562 Cells ,Tyrosine kinase ,Signal Transduction ,medicine.drug ,K562 cells - Abstract
Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.
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- 2015
5. Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation
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Rodolfo Iuliano, Tiziana Angrisano, Bongyong Lee, Alfredo Fusco, Alba Di Pardo, Annalisa Morano, Antonio Porcellini, Enrico V. Avvedimento, Lorenzo Chiariotti, Mark T. Muller, Concetta Cuozzo, Samantha Messina, Maria R. Santillo, and Max E. Gottesman
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0301 basic medicine ,Cancer Research ,lcsh:QH426-470 ,DNA damage ,Computational biology ,Biology ,Homology directed repair ,03 medical and health sciences ,lcsh:Genetics ,030104 developmental biology ,DNA methylation ,Genetics ,DNA mismatch repair ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,Nucleotide excision repair - Abstract
[This corrects the article DOI: 10.1371/journal.pgen.0030110.].
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- 2017
6. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism
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Max E. Gottesman, Candida Zuchegna, Antonio Pezone, Annalisa Morano, Antonella Romano, Mark T. Muller, Rosaria Landi, Antonio Porcellini, Giusi Russo, Enrico V. Avvedimento, Russo, Giusi, Landi, Rosaria, Pezone, Antonio, Morano, Annalisa, Zuchegna, Candida, Romano, Antonella, Muller, Mark T, Gottesman, Max E, Porcellini, Antonio, and Avvedimento, VITTORIO ENRICO
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0301 basic medicine ,DNA Repair ,DNA repair ,DNA damage, epigenetics, gene expression ,Methylation ,Article ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Histone methylation ,Humans ,RNA-Directed DNA Methylation ,Alleles ,Epigenomics ,Multidisciplinary ,biology ,DNA Methylation ,Molecular biology ,Chromatin ,030104 developmental biology ,Histone ,030220 oncology & carcinogenesis ,DNA methylation ,biology.protein ,Illumina Methylation Assay ,DNA Damage - Abstract
We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.
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- 2016
7. Reactive oxygen species derived from NOX3 and NOX5 drive differentiation of human oligodendrocytes
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Roberta Accetta, Paolo Mondola, Simona Damiano, Roberto Paternò, Mariarosaria Santillo, Enrico V. Avvedimento, Annalisa Morano, Accetta, Roberta, Damiano, Simona, Morano, Annalisa, Mondola, Paolo, Paterno', Roberto, Avvedimento, VITTORIO ENRICO, and Santillo, Mariarosaria
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0301 basic medicine ,Cell signaling ,Multiple Sclerosis ,CREB ,lcsh:RC321-571 ,03 medical and health sciences ,Myelin ,Cellular and Molecular Neuroscience ,NOX5 ,0302 clinical medicine ,NOX3 ,Gene expression ,medicine ,Multiple sclerosi ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Protein kinase C ,Original Research ,biology ,Oligodendrocyte differentiation ,differentiation ,Molecular biology ,Oligodendrocyte ,Myelin basic protein ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,Reactive oxygen specie ,Reactive Oxygen Species ,030217 neurology & neurosurgery ,oligodendrocyte ,Neuroscience - Abstract
Reactive oxygen species (ROS) are signaling molecules that mediate stress response, apoptosis, DNA damage, gene expression and differentiation. We report here that differentiation of oligodendrocytes, the myelin forming cells in the CNS, is driven by ROS. To dissect the oligodendrocyte differentiation pathway, we used the cell line MO3-13, which display the molecular and cellular features of oligodendrocyte precursors. These cells exposed 1-4 days to low levels of H2O2 or to the protein kinase C (PKC) activator, phorbol-12-Myristate-13-Acetate (PMA) increased the expression of specific oligodendrocyte differentiation markers: the specific nuclear factor Olig-2, and Myelin Basic Protein (MBP), which was processed and accumulated selectively in membranes. The induction of differentiation genes was associated with the activation of ERK1-2 and phosphorylation of the nuclear cAMP responsive element binding protein 1 (CREB). PKC mediates ROS-induced differentiation because PKC depletion or bis-indolyl-maleimide (BIM), a PKC inhibitor, reversed the induction of differentiation markers by H2O2. H2O2 and PMA increased the expression of membrane-bound NADPH oxidases, NOX3 and NOX5. Selective depletion of these proteins inhibited differentiation induced by PMA. Furthermore, NOX5 silencing down regulated NOX3 mRNA levels, suggesting that ROS produced by NOX5 up-regulate NOX3 expression. These data unravel an elaborate network of ROS-generating enzymes (NOX5 to NOX3) activated by PKC and necessary for differentiation of oligodendrocytes. Furthermore, NOX3 and NOX5, as inducers of oligodendrocyte differentiation, represent novel targets for therapies of demyelinating diseases, including multiple sclerosis, associated with impairment of oligodendrocyte differentiation.
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- 2016
8. MiRNAs and piRNAs from bone marrow mesenchymal stem cell extracellular vesicles induce cell survival and inhibit cell differentiation of cord blood hematopoietic stem cells: a new insight in transplantation
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Marina Podestà, Antonella Caivano, Luciana De Luca, Daniela Cilloni, Giovanni Calice, Michele Santodirocco, Francesco Frassoni, Annalisa Morano, Ilaria Laurenzana, Stefania Trino, Stefania Raimondo, Pellegrino Musto, Luigi Del Vecchio, Vittorio Simeon, Lazzaro Di Mauro, Francesco La Rocca, De Luca, Luciana, Trino, Stefania, Laurenzana, Ilaria, Simeon, Vittorio, Calice, Giovanni, Raimondo, Stefania, Podestà, Marina, Santodirocco, Michele, Di Mauro, Lazzaro, La Rocca, Francesco, Caivano, Antonella, Morano, Annalisa, Frassoni, Francesco, Cilloni, Daniela, Del Vecchio, Luigi, and Musto, Pellegrino
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0301 basic medicine ,extracellular vesicles ,mesenchymal stem cells ,microRNAs ,piRNAs ,umbilical cord blood stem cells ,Cell Survival ,Cellular differentiation ,CD34 ,piRNA ,Biology ,Transfection ,CXCR4 ,Immunophenotyping ,03 medical and health sciences ,Mice ,medicine ,Animals ,Humans ,RNA, Small Interfering ,mesenchymal stem cell ,microRNA ,Mesenchymal stem cell ,Hematopoietic Stem Cell Transplantation ,Cell Differentiation ,Fetal Blood ,Hematopoietic Stem Cells ,Cell biology ,Haematopoiesis ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Cord blood ,Immunology ,Bone marrow ,extracellular vesicle ,Stem cell ,Research Paper - Abstract
Hematopoietic stem cells (HSC), including umbilical cord blood CD34+ stem cells (UCB-CD34+), are used for the treatment of several diseases. Although different studies suggest that bone marrow mesenchymal stem cells (BM-MSC) support hematopoiesis, the exact mechanism remains unclear. Recently, extracellular vesicles (EVs) have been described as a novel avenue of cell communication, which may mediate BM-MSC effect on HSC. In this work, we studied the interaction between UCB-CD34+ cells and BM-MSC derived EVs. First, by sequencing EV derived miRNAs and piRNAs we found that EVs contain RNAs able to influence UCB-CD34+ cell fate. Accordingly, a gene expression profile of UCB-CD34+ cells treated with EVs, identified about 100 down-regulated genes among those targeted by EV-derived miRNAs and piRNAs (e.g. miR-27b/MPL, miR-21/ANXA1, miR-181/EGR2), indicating that EV content was able to modify gene expression profile of receiving cells. Moreover, we demonstrated that UCB-CD34+ cells, exposed to EVs, significantly changed different biological functions, becoming more viable and less differentiated. UCB-CD34+ gene expression profile also identified 103 up-regulated genes, most of them codifying for chemokines, cytokines and their receptors, involved in chemotaxis of different BM cells, an essential function of hematopoietic reconstitution. Finally, the exposure of UCB-CD34+ cells to EVs caused an increased expression CXCR4, paralleled by an in vivo augmented migration from peripheral blood to BM niche in NSG mice. This study demonstrates the existence of a powerful cross talk between BM-MSC and UCB-CD34+ cells, mediated by EVs, providing new insight in the biology of cord blood transplantation.
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- 2016
9. Gene expression profiling of normal thyroid tissue from patients with thyroid carcinoma
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Vittorio Simeon, Carmela Mazzoccoli, Giovanna Di Meo, Roberto Ria, Pellegrino Musto, Angela Gurrado, Gaetano Lastilla, Angelo Vacca, Annalisa Morano, Antonia Reale, Stefania Trino, Alessandro Pasculli, Aurelia Lamanuzzi, Mario Testini, Assunta Melaccio, Franco Dammacco, Ilaria Saltarella, Ria, Roberto, Simeon, Vittorio, Melaccio, Assunta, Di Meo, Giovanna, Trino, Stefania, Mazzoccoli, Carmela, Saltarella, Ilaria, Lamanuzzi, Aurelia, Morano, Annalisa, Gurrado, Angela, Pasculli, Alessandro, Lastilla, Gaetano, Musto, Pellegrino, Reale, Antonia, Dammacco, Franco, Vacca, Angelo, and Testini, Mario
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0301 basic medicine ,Adult ,Male ,Pathology ,medicine.medical_specialty ,endocrine system ,endocrine system diseases ,oncogenes ,Thyroid Gland ,medicine.disease_cause ,Thyroid carcinoma ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,oncogene ,medicine ,Biomarkers, Tumor ,thyroid cancer ,Humans ,Thyroid Neoplasms ,Thyroid cancer ,Aged ,business.industry ,hypoxia ,Gene Expression Profiling ,Thyroid adenoma ,Thyroid ,Cancer ,Middle Aged ,medicine.disease ,microenvironment ,3. Good health ,Gene expression profiling ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,gene expression profile ,Female ,Carcinogenesis ,business ,Research Paper - Abstract
Gene expression profiling (GEP) of normal thyroid tissue from 43 patients with thyroid carcinoma, 6 with thyroid adenoma, 42 with multinodular goiter, and 6 with Graves-Basedow disease was carried out with the aim of achieving a better understanding of the genetic mechanisms underlying the role of normal cells surrounding the tumor in the thyroid cancer progression. Unsupervised and supervised analyses were performed to compare samples from neoplastic and non-neoplastic diseases. GEP and subsequent RT-PCR analysis identified 28 differentially expressed genes. Functional assessment revealed that they are involved in tumorigenesis and cancer progression. The distinct GEP is likely to reflect the onset and/or progression of thyroid cancer, its molecular classification, and the identification of new potential prognostic factors, thus allowing to pinpoint selective gene targets with the aim of realizing more precise preoperative diagnostic procedures and novel therapeutic approaches. STATEMENT OF SIGNIFICANCE This study is focused on the gene expression profiling analysis followed by RT-PCR of normal thyroid tissues from patients with neoplastic and non-neoplastic thyroid diseases. Twenty-eight genes were found to be differentially expressed in normal cells surrounding the tumor in the thyroid cancer. The genes dysregulated in normal tissue samples from patients with thyroid tumors may represent new molecular markers, useful for their diagnostic, prognostic and possibly therapeutic implications.
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- 2016
10. LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription
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Alessandra Bertoni, Luigi Lania, Barbara Majello, Annalisa Morano, Stefano Amente, Enrico V. Avvedimento, Amente, Stefano, Bertoni, A., Morano, A., Lania, Luigi, Avvedimento, VITTORIO ENRICO, and Majello, Barbara
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Cancer Research ,Histone H3 Lysine 4 ,animal structures ,Transcription, Genetic ,LSD1 ,Myc ,histone ,Biology ,Methylation ,Cell Line ,E-Box Elements ,Histones ,Proto-Oncogene Proteins c-myc ,Histone H3 ,Genetics ,Animals ,Histone code ,oxidative burst ,Molecular Biology ,Transcription factor ,Histone Demethylases ,Lysine ,DNA ,Molecular biology ,Chromatin ,Rats ,DNA Repair Enzymes ,DNA demethylation ,Histone ,Transcription preinitiation complex ,biology.protein ,Mitogens ,transcription ,Oxidation-Reduction - Abstract
Myc is a transcription factor that significantly contributes to cancer progression by modulating the expression of important genes through binding to a DNA sequence, CACGTG, called E-box. We find that on Myc binding to chromatin, the lysine-demethylating enzyme, LSD1, triggers a transient demethylation of lysine 4 in the histone H3. In addition, we demonstrate that Myc binds and recruits LSD1 to the E-box chromatin and the formation of this complex is stimulated by cAMP-PKA. Demethylation by LSD1 produces H(2)O(2), which locally oxidizes guanine and induces the recruitment of 8-oxoguanine-DNA glycosylase (OGG1) and of the nuclease Ape1 on the E-box chromatin. Inhibition of oxidation or silencing of LSD1, OGG1 or Ape1 significantly reduce transcription and inhibit mRNA accumulation of Myc-target genes. Collectively, these data highlight the role of transient LSD1-mediated demethylation of H3K4 leading to local DNA oxidation as driving force in the assembly of the Myc-induced transcription initiation complex.
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- 2010
11. Differential Phosphorylation of c-Jun and JunD in Response to the Epidermal Growth Factor Is Determined by the Structure of MAPK Targeting Sequences
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Annalisa Morano, Giovanna Fasanella, Anna M. Musti, Marcello Maggiolini, Adriana Gallo, Maria Vinciguerra, Adele Vivacqua, and Concetta Cuozzo
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MAPK/ERK pathway ,Molecular Sequence Data ,Biochemistry ,Cell Line ,Mice ,Structure-Activity Relationship ,Protein structure ,Epidermal growth factor ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Peptide sequence ,Binding Sites ,Epidermal Growth Factor ,integumentary system ,Chemistry ,c-jun ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Cell culture ,Mitogen-Activated Protein Kinases ,Signal transduction ,Sequence Analysis ,Signal Transduction - Abstract
MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.
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- 2004
12. Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes
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Annalisa Morano, Valentina Ucci, V. Enrico Avvedimento, Roberto Paternò, Paolo Mondola, Mariarosaria Santillo, Simona Damiano, Roberta Accetta, Damiano, S, Morano, A, Ucci, V, Accetta, R, Mondola, Paolo, Paterno', Roberto, Avvedimento, VITTORIO ENRICO, and Santillo, Mariarosaria
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Cell signaling ,Blotting, Western ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,chemistry.chemical_compound ,Epidermal growth factor ,Humans ,Epidermal growth factor receptor ,chemistry.chemical_classification ,Reactive oxygen species ,Epidermal Growth Factor ,Mucins ,NADPH Oxidases ,Tyrosine phosphorylation ,Dual oxidase 2 ,Cell Biology ,Dual Oxidases ,Cell biology ,Enterocytes ,chemistry ,biology.protein ,Phosphorylation ,RNA Interference ,Caco-2 Cells ,Reactive Oxygen Species - Abstract
Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2-protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2-protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.
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- 2014
13. Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene
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Silvia Bartollino, Federica Babbio, Annalisa Morano, Lorenzo Chiariotti, Max E. Gottesman, Tiziana Angrisano, Enrico V. Avvedimento, Candida Zuchegna, Ian Marc Bonapace, Brittany Allen, Giusi Russo, Antonio Pezone, Antonio Porcellini, Mark T. Muller, Rosaria Landi, Morano, Annalisa, Angrisano, Tiziana, Russo, Giusi, Landi, Rosaria, Pezone, Antonio, S., Bartollino, Zuchegna, Candida, F., Babbio, I. M., Bonapace, B., Allen, M. T., Muller, Chiariotti, Lorenzo, M. E., Gottesman, Porcellini, Antonio, and Avvedimento, VITTORIO ENRICO
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Transcription, Genetic ,DNA repair ,Ubiquitin-Protein Ligases ,Green Fluorescent Proteins ,Cell Cycle Proteins ,Gene Regulation, Chromatin and Epigenetics ,Biology ,CCAAT-Enhancer-Binding Proteins ,DNA (Cytosine-5-)-Methyltransferase ,DNA Breaks, Double-Stranded ,HeLa Cells ,Humans ,Nuclear Proteins ,DNA Methylation ,Recombinational DNA Repair ,Genetics ,DNA Methyltransferase 3A ,Double-Stranded ,Epigenetics of physical exercise ,Genetic ,Histone methylation ,DNA (Cytosine-5-)-Methyltransferases ,RNA-Directed DNA Methylation ,Epigenomics ,"epigenetics" ,DNA Breaks ,Methylation ,Molecular biology ,DNA methylation ,gene expression ,DNA damage ,Illumina Methylation Assay ,gene regulation ,Transcription - Abstract
We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.
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- 2014
14. Identification of EpHA3 As a New Potential Molecular Target in Multiple Myeloma
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Ilaria Laurenzana, Daniela Cilloni, Luciana De Luca, Alessandra Favole, Angelo Vacca, Simona Berardi, Oreste Villani, Enrico Bracco, Francesco La Rocca, Pellegrino Musto, Antonio Basile, Sonia Carturan, Vittorio Simeon, Antonella Caivano, Annalisa Morano, Giuseppe Pietrantuono, and Stefania Trino
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Tube formation ,Tumor microenvironment ,Angiogenesis ,medicine.drug_class ,Immunology ,Erythropoietin-producing hepatocellular (Eph) receptor ,Cell Biology ,Hematology ,Biology ,Plasma cell ,Monoclonal antibody ,medicine.disease ,Biochemistry ,Molecular biology ,medicine.anatomical_structure ,medicine ,Viability assay ,Monoclonal gammopathy of undetermined significance - Abstract
Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.
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- 2012
15. Absence of Spred, a Negative Regulator of Tyrosine Kinase Activity, in Acute Myeloid Leukemia Patients
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Francesco Frassoni, Luciana De Luca, Alessandra Favole, Giuseppe Saglio, Valentina Campia, Enrico Bracco, Antonella Caivano, Valentina Rosso, Cristina Panuzzo, Daniela Cilloni, Chiara Calabrese, Elisabetta Signorino, Sonia Carturan, Pimjai Niparuk, and Annalisa Morano
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MAPK/ERK pathway ,medicine.diagnostic_test ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Receptor tyrosine kinase ,medicine.anatomical_structure ,Western blot ,Fibroblast growth factor receptor ,microRNA ,medicine ,biology.protein ,Cancer research ,Bone marrow ,Tyrosine kinase - Abstract
Abstract 4616 Background: The Sprouty family proteins, including Spred, were originally identified in Drosophila melanogaster as an antagonist of Breathless, the ortholog of Fibroblast Growth Factor Receptor (FGFR) in mammals. These proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. Their main function is to deregulate the RAS/MAPK and RAS/RAF/ERK signal pathways by physically interacting. The role of Spread proteins in haematological malignancies is still not been clarified. Aim: The aim of this study was to investigate a possible involvement of Spred in maintaining the aberrant TK signalling in patients affected by acute myeloid leukaemia (AML). Methods: Bone marrow (BM) cells were collected from 82 AML patients at diagnosis and 10 BM samples from healthy donors as control. In addition 20 patients were analyzed during follow-up at the time haematological remission. All the patients included had been characterized at the cytogenetic level by conventional karyotyping, and screened by reverse transcriptase-PCR for the presence of the most frequent fusion transcripts or mutations. We analysed Spread mRNA expression by RQ-PCR and the protein by Western blot and immunofluorescence assay. In addition gain of function experiments were performed by transfecting with Spred coding sequence different leukemic cells lines lacking Spred activity. Results: We found that Spread mRNA levels were significantly decreased in AML compared to healthy subjects (2−DDct = 0,1 in AML compared to 0,6 in controls) (p Conclusions: A decreased expression and activity of Spred, a negative regulator of TK activity through Ras pathway, is implicated in sustaining the oncogenetic signalling and abnormal proliferation induced by tyrosine kinase proteins in acute myeloid leukemia patients. Disclosures: No relevant conflicts of interest to declare.
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- 2012
16. Reactive oxygen species regulate the levels of dual oxidase (duox1-2) in human neuroblastoma cells
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Mariarosaria De Mizio, Annalisa Morano, Roberta Fusco, Roberto Paternò, Françoise Miot, Mariarosaria Santillo, Paolo Laccetti, Antonella De Rosa, Enrico V. Avvedimento, Simona Damiano, Rodolfo Frunzio, Paolo Mondola, Stefano Amente, Rosa Spinelli, Damiano, S., Fusco, R., Morano, A., de Mizio, M., Paterno, R., de Rosa, A., Spinelli, R., Amente, S., Frunzio, R., Mondola, P., Miot, F., Laccetti, P., Santillo, M., Avvedimento, E. V., Damiano, S, Fusco, R, Morano, A, De Mizio, M, Paterno', Roberto, De Rosa, A, Spinelli, R, Amente, Stefano, Frunzio, Rodolfo, Mondola, Paolo, Miot, F, Laccetti, P, Santillo, Mariarosaria, and Avvedimento, VITTORIO ENRICO
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Science ,NADPH Oxidase ,Signaling Pathways ,Reactive Oxygen Species -- metabolism ,Neuroblastoma ,Cell Line, Tumor ,Tumor Cells, Cultured ,Animals ,Humans ,RNA, Messenger ,Biology ,chemistry.chemical_classification ,Platelet-Derived Growth Factor ,Reactive oxygen species ,Oxidase test ,Multidisciplinary ,NADPH oxidase ,biology ,Animal ,Platelet-Derived Growth Factor -- pharmacology ,NADPH Oxidases ,Dual Oxidases ,RNA, Messenger -- metabolism ,Sciences biomédicales ,Rats ,Membrane protein ,chemistry ,Biochemistry ,Cell culture ,Neuroblastoma -- metabolism ,biology.protein ,Medicine ,Rat ,P22phox ,Molecular Neuroscience ,Reactive Oxygen Species ,Dual Oxidase ,Reactive Oxygen Specie ,NADPH Oxidase -- metabolism ,Platelet-derived growth factor receptor ,Research Article ,Neuroscience ,Human - Abstract
Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox) (NOX2), or of the other membrane subunit of NADPH oxidase, p22(phox), blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published
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- 2012
17. High Serum Levels of Extracellular Vesicles which Express Specific Markers of Malignancy Are Released in Patients with Various Types of Hematological Neoplastic Disorders
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Pellegrino Musto, Annalisa Morano, Luigi Del Vecchio, Vittorio Simeon, Fiorella D'Auria, Francesco La Rocca, Stefania Trino, Luciana De Luca, Antonella Caivano, and Ilaria Laurenzana
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CD20 ,education.field_of_study ,Myeloid ,biology ,business.industry ,Chronic lymphocytic leukemia ,Immunology ,Population ,Macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,CD19 ,medicine.anatomical_structure ,hemic and lymphatic diseases ,medicine ,biology.protein ,education ,business ,Myelofibrosis ,Chronic myelogenous leukemia - Abstract
INTRODUCTION: Extracellular vesicles (EVs), including exosomes, shedding vesicles (microvesicles: MVs) and apoptotic bodies, are membrane vesicles of 40-3,000 nm that are released from many cell types, such as red blood cells, platelets, lymphocytes, dendritic cells, endothelial cells and tumour cells. They have a key role not only in the regulation of normal physiological processes, but also in the pathology underlying several diseases. Recently, it was discovered that EVs circulate in bodily fluids of cancer patients and could serve as potential diagnostic biomarkers. However, the presence and role of peripheral circulating EVs in hematological malignancies (HMs) remain unclear. The aim of this study was to investigate EVs in peripheral blood of HM patients, defining the EVs count, size and phenotype. METHODS: Peripheral blood samples were obtained prior to treatment from 87 subjects: 6 Chronic Lymphocytic Leukemia (CLL), 10 Non-Hodgkin's Lymphoma (NHL; 5 follicular, 5 diffuse large B-cell), 6 Waldenstrom's Macroglobulinemia (WM), 6 Hodgkin's Lymphoma (HL), 6 Multiple Myeloma (MM), 5 Acute Myeloid Lukemia (AML), 19 Myeloproliferative Neoplasms [(MPNs; 5 Essential Thrombocythemia (ET), 5 Polycythemia Vera (PV), 5 Primary Myelofibrosis (PMF), 4 Chronic Myelogenous Leukemia (CML)], 5 Myelodysplastic Syndrome (MDS) and 28 healthy controls. EVs were isolated from serum of peripheral blood by ultracentrifugation steps. For calculation of counts, TruCOUNT beads were added immediately prior to analysis by flow cytometry (FACS). For size assessment, Megamix beads with specific size (0.3,0.9, 3.0 micrometer) were used. For phenotype characterization, a panel of specific antibodies (anti CD59, CD61, CD62E, CD19, CD20, CD38, CD138, CD13, CD30) were employed in a FACS analysis. RESULTS: MVs count was significantly higher in patients with CLL, WM, HL, MM, AML and CML (median values 890, 3227, 1218, 914, 656 and 1275 MVs/microliter of serum, respectively) compared to healthy controls (median value 270 MVs/ microliter of serum, ***P In CLL and MDS, through the number of cases was small, MVs counts directly correlated with Rai stage and R-IPSS risk, respectively. As regards vesicles volume, all HMs generated a distinct population of MVs with smaller size when compared with controls. As expected, MVs were consistently Annexin V positive and expressed a common membrane protein, CD59, an ubiquitous complement regulator. Part of MVs originated from platelets (median value 400 CD61 positive/ microliter of serum) and endothelial cells (median value 60 CD62E positive/ microliter of serum). Interestingly, MV phenotype was disease specific: a relevant amount of MVs expressed surface specific proteins related to malignancy. In particular, we found MVs positive for CD19 and/or CD20 in CLL, WM and NHL, CD38 and/or CD138 in MM, CD13 in AML, MPNs and MDS, CD30 in HL. These markers were not significantly expressed on the surface of healthy subject MVs. CONCLUSIONS: In this study, for the first time, serum MVs in a panel of HMs were analysed. We found that patients with various types of HMs release elevated peripheral blood MV levels selectively displaying markers of the underlying malignancy. MVs could be therefore useful as potential novel biomarkers in HMs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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- 2014
18. Exposure of Cord Blood Hematopoietic Stem Cells to Bone Marrow Mesenchimal Cells-Derived Microvesicles Induces Cell Survival and Inhibition of Differentiation
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Ilaria Laurenzana, Annalisa Morano, Lazzaro Di Mauro, Vittorio Simeon, Francesco Frassoni, Michele Santodirocco, Pellegrino Musto, Stefania Trino, Francesco La Rocca, Marina Podestà, Antonella Caivano, Luigi Del Vecchio, Daniela Cilloni, Luciana De Luca, and Stefania Raimondo
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Cellular differentiation ,Immunology ,CD34 ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Exosome ,Molecular biology ,Transplantation ,Haematopoiesis ,Cord blood ,CD90 ,Stem cell - Abstract
Introduction Hematopoietic stem cells (HSC) are used in transplantation for the treatment of several hematological or non-hematological diseases. HSC fate is bound to different factors and, among these, the cross-talk with the bone marrow microenvironment represents an important regulatory mechanism of aging, self-renewal, stemness and/or differentiation. Cells communicate by both direct interaction and through the secretion of soluble factor and extracellular vesicles (EVs). EVs are cell-derived vesicles enclosed by a lipid bilayer and enriched in phosphatidylserine, cytoplasmic protein, mRNA, miRNA and DNA. Recent studies suggest that the release of EVs is a highly regulated and important process for surface membrane traffic and horizontal transfer of RNAs, protein and DNA, present in different kind of cells, including tumor cells. Bone marrow mesenchimal stem cells (BM-MSC) are a component of hematopoietic microenvironment and support hematopoiesis by the constitutive ability to secrete soluble factor and EVs. On these basis, we decided to study the interaction between the umbilical cord blood (UCBs) stem cells and the BM-MSCs mediated by EVs. The overarching goal of this project was to find out if and how the EVs can influence the fate of UCBs in transplantation. Methods Primary BM-MSC were cultured in DMEM containing 10% of fetal bovine serum (FBS). EVs were isolated from supernatant of BM-MSC by ultracentrifugation at 100.000 x g for 70 min at 4 ºC. After characterization by FACS analysis, EVs were stocked at -80 ºC and partially used for RNA extraction using TRIZOL reagent. EVs smallRNA was sequenced with HiScan SQ Illumina. UCBs CD34+ cells were isolated by Miltenyi Biotec separation kit and were cultured in DMEM containing 10% of FBS with or without BM-MSC EVs. After 24h of co-culture, viability by trypan blue count, apoptosis by Propide Iodure and Annexin V test and cell differentiation by FACS analysis of different markers (CD45, CD34, CD33, CD19 and CD38) were evaluated. RNA was extracted from the same samples and a gene expression profile experiment was performed using Illumina array. All genomic data were analyzed by Ingenuity Pathways Analysis software (IPA). Results EVs isolated from BM-MSC were positive for MSC markers like CD29 (61%), CD90 (72%), CD73 (63%), CD105 (35%), CD146 (32%), CD44 (70%) and for the exosome antigen CD81 (40%); they were negative, instead, for the hematopoietic marker CD45. The sequencing of smallRNA-EVs identified 87 miRNAs in BM-MSC EVs. Analysis of gene expression profile of UCBs CD34+ cells treated with EVs revealed 103 up-regulated and 100 down-regulated genes (with padj < 0.05 and logFC=0.7), when compared to UCBs CD34+ cells not treated with EVs. Interestingly, analyzing together the sequencing of EVs and the gene expression data of UCBs CD34+ cells treated with EVs, we found a direct correlation between EVs miRNAs and down-regulated genes, identifying at least one target gene for each EVs miRNA (e.g., miR-3168/LYZ, miR-27b-3p/ZFP36, miR21-5p/ANXA1). These results indicate that the EVs RNA content modifies the genes profile of receiving cells. Gene ontology analysis, using IPA, of the miRNA targeted genes identified different down-regulated biological function, like cell death and cellular development (e.g., CDKN1B, CEBPA, ANXA1, MPL). To confirm these data, we evaluated viability and apoptosis of UCBs treated with EVs respect to the UCBs control culture, and we observed an increase of 50% of cell viability and a reduction of 42% of apoptosis (p Furthermore, using IPA, we found that some up-regulated genes (e.g., IL6, CSF2, CCL3) are under the control of miRNA targeted genes (e.g., ZFP36/miR-27b-3p). The analysis of these genes identified some biological function up-regulated in cell after EVs treatment, the most important one being chemotaxis of the cells. Conclusion This study indicates, for the first time, the existence of a cross-talk between MSC and UCBs mediated by EVs. Moreover, it identifies a gene profile modification of UCBs cells after miRNA EVs treatment, providing new insights for cord blood transplantation. Disclosures No relevant conflicts of interest to declare.
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- 2014
19. EphA3 As a Molecular Target In Multiple Myeloma: Opportunity For a Novel Therapeutic Approach With a Specific Monoclonal Antibody
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Luciana De Luca, Pellegrino Musto, Stefania Trino, Ilaria Laurenzana, Daniela Cilloni, Francesco Frassoni, Annalisa Morano, Enrico Bracco, Emma Di Carlo, Alessandra Favole, Antonella Caivano, Francesco La Rocca, Vittorio Simeon, Simona Berardi, Irma Airoldi, Valentina Gaidano, Oreste Villani, Martin Lackmann, Antonio Basile, Angelo Vacca, and Giuseppe Pietrantuono
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Pathology ,medicine.medical_specialty ,Tumor microenvironment ,Angiogenesis ,medicine.drug_class ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Monoclonal antibody ,Biochemistry ,In vitro ,medicine.anatomical_structure ,In vivo ,medicine ,biology.protein ,Cancer research ,Bone marrow ,Antibody ,Monoclonal gammopathy of undetermined significance - Abstract
Introduction Multiple myeloma (MM) remains an incurable malignancy despite important recent advances in treatments. Neo-vascularization entails a crucial aspect of interactions between neoplastic plasma cells (PCs) and their microenvironment. Without it, MM would be unable to grow and progress, and would probably regress to a low-mass steady-state comparable to monoclonal gammopathy of undetermined significance (MGUS). To overcome drug resistance and improve clinical response to novel therapeutic approaches halting both PC growth and the increased bone marrow (BM) microvascular density are needed. In this setting, monoclonal antibodies against MM-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate membrane target structures expressed across all MM cells. The Eph receptors, a large family of receptor tyrosine kinases, have been implicated in many processes involved in malignancy, including alteration of the tumour microenvironment, and in angiogenesis, in both of which EphA3 likely plays an active role. Interestingly, the over-expression of EphA3 is sufficient to confer tumorigenic potential, although probably further mechanisms can occur to abnormally activate the receptor. A first-in-class engineered IgG1 antibody targeting the EphA3 was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EphA3 over-expressing hematological myeloid malignancies refractory to conventional treatment. Methods We investigated the EphA3 role in MM patients in order to define whether it may represent a potential new molecular target for a novel therapeutic approach with a specific anti EphA3 monoclonal antibody. The EphA3 expression was studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with MGUS (MGECs), of control subjects (normal ECs). Moreover, the effects of anti EphA3 antibody in MM were studied in vitro and in vivo in a MM xenograft mouse model. After written informed consent, BM aspirates were collected from 26 MM and 6MGUS patients. Normal ECs were derived from 5 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed both mRNA and protein levels of EphA3 in normal ECs, MGECs and MMECs and in MM cell lines by absolute RT-PCR and by WB coupled to immunofluorescence and FACS analysis respectively. Immunoistochemistry was also performed on MM BM biopsies. The biological effects of EphA3 targeting were studied in vitro silencing (siRNA) the EphA3 mRNA in MMECs and using the anti EphA3 antibody testing them in series of in vitro functional assays including viability, apoptosis, adhesion, migration, wound healing and angiogenesis tests. We further examined the inhibitory capacity of anti-EphA3 Ab on tumor growth in SCID mice bearing MM tumor cell xenografts. Finally, we assessed morphology, vessel density, and apoptosis of excised xenotransplanted tumors. Results Briefly, our data showed that EphA3 mRNA and protein levels are progressively increased from ECs to MGECs, reaching the highest values in MMECs. EphA3 stained intensely and diffusely MM microvessels and PC in MM BM biopsies. The EphA3siRNA MMECs revealed a protein level reduction of approximately 80% when compared to the control. We not detected viability or apoptotic defects, whereas in vitro adhesion, migration and angiogenesis inhibition was evident when compared to the not silenced counterpart. The anti EphA3 antibody inhibited MMECs migration and reduced in vitro MM angiogenesis. In particular, tumour masses developed in xenograft mice treated with anti-EphA3 Abs were smaller in size and showed foci of ischemic-hemorrhagic necrosis, in association with a significant (P < 0.05) reduction in the number of intact tumor microvessels. The proliferative activity was not significantly different from that observed in tumors from untreated or control isotype treated mice, while the apoptotic index was significantly (P < 0.05) increased in comparison with tumors from both groups of mice. Conclusions In this study we have characterized the role of the EphA3in MM patients, providing in vitro and in vivo experimental evidences that support the possibility of using EphA3 as a new molecular target for MM. Disclosures: No relevant conflicts of interest to declare.
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- 2013
20. DNA Damage, Homology-Directed Repair, and DNA Methylation
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Samantha Messina, Mark T. Muller, Antonio Porcellini, Concetta Cuozzo, Enrico V. Avvedimento, Mariarosaria Santillo, Alfredo Fusco, Bongyong Lee, Annalisa Morano, Rodolfo Iuliano, Alba Di Pardo, Tiziana Angrisano, Max E. Gottesman, Lorenzo Chiariotti, C, Cuozzo, A, Porcellini, T, Angrisano, A, Morano, B, Lee, Ad, Pardo, Messina, S., R, Iuliano, A, Fusco, Mr, Santillo, Mt, Muller, L, Chiariotti, Me, Gottesman, Ev, Avvedimento, C., Cuozzo, Porcellini, A, Angrisano, Tiziana, Morano, A, Lee, B, Pardo, Ad, Messina, S, Iuliano, R, Fusco, Alfredo, Santillo, Mr, Muller, Mt, Chiariotti, Lorenzo, Gottesman, Me, Avvedimento, VITTORIO ENRICO, Cuozzo, C, Porcellini, Antonio, Di Pardo, A, and Avvedimento, Ev
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Cancer Research ,DNA Repair ,Gene Expression ,Loss of Heterozygosity ,Mice ,Histone methylation ,DNA Breaks, Double-Stranded ,DNA (Cytosine-5-)-Methyltransferases ,RNA-Directed DNA Methylation ,Genetics (clinical) ,Epigenomics ,Mammals ,Recombination, Genetic ,Homo (human) ,Methylation ,Mus (mouse) ,Chromatin ,Recombinant Proteins ,In Vitro ,DNA methylation ,Research Article ,DNA (Cytosine-5-)-Methyltransferase 1 ,lcsh:QH426-470 ,DNA damage ,DNA repair ,Green Fluorescent Proteins ,Biology ,Transfection ,Cell Line ,Genetics ,Animals ,Humans ,Gene Silencing ,RNA, Messenger ,Thyroid Neoplasms ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,DNA Primers ,Base Sequence ,Models, Genetic ,Correction ,Cell Biology ,DNA Methylation ,Molecular biology ,lcsh:Genetics ,repair ,CpG Islands ,methylation ,In vitro recombination ,DNA Damage ,HeLa Cells - Abstract
To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments., Author Summary Genomic DNA can be modified by cytosine methylation. This epigenetic modification is layered on the primary genetic information and can silence the affected gene. Epigenetic modification has been implicated in cancer and aging. To date, the primary cause and the mechanism leading to DNA methylation are not known. By using a sophisticated genetic system, we have induced a single break in the double helix of the genomes of mouse or human cells. This rupture was repaired by a very precise mechanism: the damaged chromosome pairs and retrieves genetic information from an undamaged and homologous DNA partner. This homology-directed repair was marked in half of the repaired molecules by de novo methylation of cytosines flanking the cut. As a direct consequence, the gene in these repaired molecules was silenced. In the remaining molecules, the recombinant DNA was undermethylated and expressed the reconstituted gene. Since homology-directed repair may duplicate or delete genetic information, epigenetic modification of repaired DNA represents a powerful evolutionary force. If the expression of the repaired gene is harmful, only cells inheriting the silenced copy will survive. Conversely, if the function of the repaired gene is beneficial, cells inheriting the under-methylated copy will have a selective advantage.
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- 2007
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