404 results on '"DE BORTOLI, M"'
Search Results
202. Feasibility and Outcome of Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant High-Dose Cyclophosphamide for Children and Adolescents with Hematologic Malignancies: An AIEOP-GITMO Retrospective Multicenter Study.
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Berger M, Lanino E, Cesaro S, Zecca M, Vassallo E, Faraci M, De Bortoli M, Barat V, Prete A, and Fagioli F
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- Adolescent, Adult, Allografts, Child, Child, Preschool, Disease-Free Survival, Female, Humans, Infant, Male, Retrospective Studies, Survival Rate, Time Factors, Cyclophosphamide administration & dosage, Hematologic Neoplasms mortality, Hematologic Neoplasms therapy, Hematopoietic Stem Cell Transplantation, Transplantation Conditioning
- Abstract
Post-transplant high-dose cyclophosphamide (PTCy) is a novel approach to prevent graft-versus-host disease (GVHD) and rejection in patients given haploidentical hematopoietic stem cell transplantation (HSCT). Thirty-three patients with high-risk hematologic malignancies and lacking a match-related or -unrelated donor were treated with PTCy haploidentical HSCT in 5 Italian AIEOP centers. Nineteen patients had a nonmyeloablative preparative regimen (57%), and 14 patients received a full myeloablative conditioning regimen (43%). No patients received serotherapy; GVHD prophylaxis was based on PTCy (50 mg/kg on days +3 and +4) combined with mycophenolate plus tacrolimus or cyclosporine A. Neutrophil and platelet engraftment was achieved on days +17 (range, 14 to 37) and +27 (range, 16 to 71). One patient had autologous reconstitution for anti-HLA antibodies. Acute GVHD grades II to IV and III to IV and chronic GVHD developed in 22% (95% CI, 11 to 42), 3% (95% CI, 0 to 21), and 4% (95% CI, 0 to 27) of cases, respectively. The 1-year overall survival rate was 72% (95% CI, 56 to 88), progression-free survival rate was 61% (95% CI, 43 to 80), cumulative incidence of relapse was 24% (95% CI, 13 to 44), and transplant-related mortality was 9% (95% CI, 3 to 26). The univariate analysis for risk of relapse incidence showed how 3 significant variables, mother as donor (P = .02), donor gender as female (P = .04), and patient gender as female (P = .02), were significantly associated with a lower risk of relapse. Disease progression was the main cause of death. PTCy is a safe procedure also for children and adolescents who have already received several lines of chemotherapy. Among the different diseases, a trend for better 1-year rates of overall survival was obtained for nonacute leukemia patients., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)
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- 2016
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203. Mutations in NEBL encoding the cardiac Z-disk protein nebulette are associated with various cardiomyopathies.
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Perrot A, Tomasov P, Villard E, Faludi R, Melacini P, Lossie J, Lohmann N, Richard P, De Bortoli M, Angelini A, Varga-Szemes A, Sperling SR, Simor T, Veselka J, Özcelik C, and Charron P
- Abstract
Introduction: Transgenic mice overexpressing mutated NEBL, encoding the cardiac-specific Z-disk protein nebulette, develop severe cardiac phenotypes. Since cardiomyopathies are commonly familial and because mutations in a single gene may result in variable phenotypes, we tested the hypothesis that NEBL mutations are associated with cardiomyopathy., Material and Methods: We analyzed 389 patients, including cohorts of patients with dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and left ventricular non-compaction cardiomyopathy (LVNC). The 28 coding exons of the NEBL gene were sequenced. Further bioinformatic analysis was used to distinguish variants., Results: In total, we identified six very rare heterozygous missense mutations in NEBL in 7 different patients (frequency 1.8%) in highly conserved codons. The mutations were not detectable in 320 Caucasian sex-matched unrelated individuals without cardiomyopathy and 192 Caucasian sex-matched blood donors without heart disease. Known cardiomyopathy genes were excluded in these patients. The mutations p.H171R and p.I652L were found in 2 HCM patients. Further, p.Q581R and p.S747L were detected in 2 DCM patients, while the mutation p.A175T was identified independently in two unrelated patients with DCM. One LVNC patient carried the mutation p.P916L. All HCM and DCM related mutations were located in the nebulin-like repeats, domains responsible for actin binding. Interestingly, the mutation associated with LVNC was located in the C-terminal serine-rich linker region., Conclusions: Our data suggest that NEBL mutations may cause various cardiomyopathies. We herein describe the first NEBL mutations in HCM and LVNC. Our findings underline the notion that the cardiomyopathies are true allelic diseases.
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- 2016
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204. The Use of Splenectomy to Manage Platelet Transfusion Refractoriness due to Anti-Human Leukocyte Antibodies in Allogeneic Stem Cell Transplantation.
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Mauro M, Camoglio F, Piccoli P, De Bortoli M, Balter R, Pegoraro A, and Cesaro S
- Abstract
In patients undergoing hematopoietic stem cell transplantation (HSCT), refractoriness to platelet transfusion has been associated with graft failure, delayed engraftment, early mortality and decreased overall survival. Therapeutic strategies include plasma exchange, immunoglobulins, rituximab, and splenectomy. We describe here three patients with refractoriness to platelet transfusion due to anti-human leukocyte antibodies who were splenectomized before HSCT (two cases) and after HSCT (one case) due to the lack of efficacy of other therapies. Splenectomy was uneventful. All three patients achieved a full donor engraftment. We suggest that splenectomy is feasible and effective in HSCT patients to reduce the risk of graft failure or delayed engraftment.
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- 2016
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205. Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer.
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Miano V, Ferrero G, Reineri S, Caizzi L, Annaratone L, Ricci L, Cutrupi S, Castellano I, Cordero F, and De Bortoli M
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- Biomarkers, Tumor genetics, Cell Line, Tumor, Cell Proliferation genetics, Cell Survival genetics, Down-Regulation, Enzyme Activation, Epithelial-Mesenchymal Transition genetics, Estrogens metabolism, Female, HEK293 Cells, Humans, Ligands, MCF-7 Cells, RNA Interference, RNA, Small Interfering genetics, Breast Neoplasms genetics, Breast Neoplasms pathology, Estrogen Receptor alpha genetics, Gene Expression Regulation, Neoplastic, RNA, Long Noncoding genetics
- Abstract
Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.
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- 2016
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206. Homozygous Desmocollin-2 Mutations and Arrhythmogenic Cardiomyopathy.
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Lorenzon A, Pilichou K, Rigato I, Vazza G, De Bortoli M, Calore M, Occhi G, Carturan E, Lazzarini E, Cason M, Mazzotti E, Poloni G, Mostacciuolo ML, Daliento L, Thiene G, Corrado D, Basso C, Bauce B, and Rampazzo A
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- Adolescent, Adult, Aged, Aged, 80 and over, Child, Exons genetics, Female, Founder Effect, Homozygote, Humans, Italy, Male, Middle Aged, Pedigree, Young Adult, Arrhythmogenic Right Ventricular Dysplasia genetics, Desmocollins genetics, Mutation genetics
- Abstract
Dominant mutations in desmocollin-2 (DSC2) gene cause arrhythmogenic cardiomyopathy (ACM), a progressive heart muscle disease characterized by ventricular tachyarrhythmias, heart failure, and risk of juvenile sudden death. Recessive mutations are rare and are associated with a cardiac or cardiocutaneous phenotype. Here, we evaluated the impact of a homozygous founder DSC2 mutation on clinical expression of ACM. An exon-by-exon analysis of the DSC2 coding region was performed in 94 ACM index patients. The c.536A>G (p.D179G) mutation was identified in 5 patients (5.3%), 4 of which resulted to be homozygous carriers. The 5 subjects shared a conserved haplotype, strongly indicating a common founder. Genetic and clinical investigation of probands' families revealed that p.D179G homozygous carriers displayed severe forms of biventricular cardiomyopathy without hair or skin abnormalities. The only heterozygous proband, who carried an additional variant of unknown significance in αT-catenin gene, showed a mild form of ACM without left ventricular involvement. All heterozygous family members were clinically asymptomatic. In conclusion, this is the first homozygous founder mutation in DSC2 gene identified among Italian ACM probands. Our findings provide further evidence of the occurrence of recessive DSC2 mutations in patients with ACM predominantly presenting with biventricular forms of the disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)
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- 2015
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207. Immunophenotypic analysis of hematopoiesis in patients suffering from Shwachman-Bodian-Diamond Syndrome.
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Mercuri A, Cannata E, Perbellini O, Cugno C, Balter R, Zaccaron A, Tridello G, Pizzolo G, De Bortoli M, Krampera M, Cipolli M, and Cesaro S
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- Adolescent, Adult, Antigens, CD metabolism, Bone Marrow pathology, Bone Marrow Cells metabolism, Bone Marrow Cells pathology, Bone Marrow Diseases genetics, Case-Control Studies, Cell Differentiation, Cell Lineage, Child, Child, Preschool, Exocrine Pancreatic Insufficiency genetics, Female, Flow Cytometry, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Infant, Karyotype, Lipomatosis genetics, Male, Mutation, Shwachman-Diamond Syndrome, Young Adult, Bone Marrow Diseases diagnosis, Bone Marrow Diseases metabolism, Exocrine Pancreatic Insufficiency diagnosis, Exocrine Pancreatic Insufficiency metabolism, Hematopoiesis genetics, Immunophenotyping, Lipomatosis diagnosis, Lipomatosis metabolism
- Abstract
Objectives: Shwachman-Diamond syndrome is a rare disorder characterized by exocrine pancreatic insufficiency, skeletal abnormalities, and bone marrow failure, with high risk of leukemic evolution. The aim of the study was the immunophenotypic characterization of bone marrow cells from patients with Shwachman-Diamond syndrome to assess the maturation pathway of blood progenitor cells and to identify the presence of recurrent abnormalities., Methods: Bone marrow samples from nineteen patients and eleven controls were analyzed by multiparameter flow cytometry., Results: We found a low frequency of CD34+ cells (P = 0.0179) and myeloid progenitors (P = 0.025), in the bone marrow of patients with Shwachman-Diamond syndrome as compared to the controls. A significant reduction in the percentage of granulocytes (P = 0.002) and an increase of monocytes (P < 0.001) were also evident in the bone marrow of patients., Conclusions: On the basis of these observations, future prospective assessments may be useful to verify the contribution of bone marrow immunophenotype in the early identification of the evolution toward aplasia or myelodysplasia., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
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- 2015
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208. 4-IPP, a selective MIF inhibitor, causes mitotic catastrophe in thyroid carcinomas.
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Varinelli L, Caccia D, Volpi CC, Caccia C, De Bortoli M, Taverna E, Gualeni AV, Leoni V, Gloghini A, Manenti G, and Bongarzone I
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- Apoptosis, Biomarkers, Tumor metabolism, Blotting, Western, Carcinoma drug therapy, Carcinoma metabolism, Carcinoma, Papillary, Cell Cycle, Cell Proliferation, Chromatography, Liquid methods, Fluorescent Antibody Technique, Humans, Immunoenzyme Techniques, Intramolecular Oxidoreductases metabolism, Macrophage Migration-Inhibitory Factors metabolism, Receptors, Immunologic metabolism, Tandem Mass Spectrometry methods, Thyroid Cancer, Papillary, Thyroid Neoplasms drug therapy, Thyroid Neoplasms metabolism, Tumor Cells, Cultured, Carcinoma pathology, Indoles pharmacology, Intramolecular Oxidoreductases antagonists & inhibitors, Macrophage Migration-Inhibitory Factors antagonists & inhibitors, Metabolome, Mitosis drug effects, Proteome analysis, Thyroid Neoplasms pathology
- Abstract
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is over-expressed in several human neoplastic cells. When MIF binds its receptor (CD74) and co-receptor (CD44), it initiates signaling cascades that orchestrate cell proliferation and survival, and it can directly modulate the activity of AMPK. These activities indicate that MIF potentially regulates cell survival and metabolism. We found that MIF was primarily co-expressed with CD74 in 16 out of 23 papillary thyroid carcinoma (PTC) and in all the 27 available anaplastic thyroid carcinoma (ATC) biopsy samples. MIF and CD74 were co-expressed in TPC-1 and HTC-C3 cell lines. The selective MIF inhibitor, 4-iodo-6-phenylpyrimidine (4-IPP), blocked MIF/CD74 internalization, activated JNK, and dose-dependently inhibited proliferation inducing apoptosis and mitotic cell death. In two CD74-negative cell lines, NIM-1 and K1, 4-IPP treatment partially reduced proliferation. Coordinated MIF and CD74 expression appeared to confer in tumor cells the plasticity necessary to escape cell cycle regulation, metabolic changes, and stress conditions. MIF/CD74 signaling removal made cells susceptible to apoptosis and mitotic cell death. This finding suggests a possible avenue for targeting DNA endoreduplication, thus preventing the proliferation of therapy-resistant cell subpopulations. This study highlights MIF/CD74 axis as an important player in the biology of aggressive thyroid neoplasms., (© 2015 Society for Endocrinology.)
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- 2015
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209. Arrhythmogenic cardiomyopathy: a disease of intercalated discs.
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Calore M, Lorenzon A, De Bortoli M, Poloni G, and Rampazzo A
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- Animals, Arrhythmias, Cardiac genetics, Cardiomyopathies genetics, Disease Models, Animal, Humans, Models, Biological, Arrhythmias, Cardiac complications, Arrhythmias, Cardiac pathology, Cardiomyopathies complications, Cardiomyopathies pathology, Intercellular Junctions pathology
- Abstract
Arrhythmogenic cardiomyopathy (ACM) is an acquired progressive disease having an age-related penetrance and showing clinical manifestations usually during adolescence and young adulthood. It is characterized clinically by a high incidence of severe ventricular tachyarrhythmias and sudden cardiac death and pathologically by degeneration of ventricular cardiomyocytes with replacement by fibro-fatty tissue. Whereas, in the past, the disease was considered to involve only the right ventricle, more recent clinical studies have established that the left ventricle is frequently involved. ACM is an inherited disease in up to 50% of cases, with predominantly an autosomal dominant pattern of transmission, although recessive inheritance has also been described. Since most of the pathogenic mutations have been identified in genes encoding desmosomal proteins, ACM is currently defined as a disease of desmosomes. However, on the basis of the most recent description of the intercalated disc organization and of the identification of a novel ACM gene encoding for an area composita protein, ACM can be considered as a disease of the intercalated disc, rather than only as a desmosomal disease. Despite increasing knowledge of the genetic basis of ACM, we are just beginning to understand early molecular events leading to cardiomyocyte degeneration, fibrosis and fibro-fatty substitution. This review summarizes recent advances in our comprehension of the link between the molecular genetics and pathogenesis of ACM and of the novel role of cardiac intercalated discs.
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- 2015
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210. Molecular characterization of LASP-1 expression reveals vimentin as its new partner in human hepatocellular carcinoma cells.
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Salvi A, Bongarzone I, Ferrari L, Abeni E, Arici B, De Bortoli M, Scuri S, Bonini D, Grossi I, Benetti A, Baiocchi G, Portolani N, and De Petro G
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- Adult, Aged, Aged, 80 and over, Female, Fluorescent Antibody Technique, Gene Expression Profiling, Humans, Male, Middle Aged, RNA, Messenger genetics, Sex Factors, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tumor Cells, Cultured, Adaptor Proteins, Signal Transducing genetics, Carcinoma, Hepatocellular genetics, Cytoskeletal Proteins genetics, Gene Expression Regulation, Neoplastic physiology, LIM Domain Proteins genetics, Liver Neoplasms genetics, Vimentin metabolism
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Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We have previously reported that LASP-1 is a downstream protein of the urokinase type plasminogen activator (uPA). Here we investigated the role of LASP-1 in HCC by a molecular and biological characterization of LASP-1 expression in human HCC specimens and in cultured HCC cells. We determined the LASP-1 mRNA expression levels in 55 HCC cases with different hepatic background disease. We identified 3 groups of patients with high, equal or low LASP-1 mRNA levels in HCC tissues compared to the peritumoral (PT) tissues. In particular we found that i) the HCCs displayed a higher LASP-1 mRNA level in HCC compared to PT tissues; ii) the expression levels of LASP-1 mRNA in female HCCs were significantly higher compared to male HCCs; iii) the cirrhotic HCCs displayed a higher LASP-1 mRNA. Further, the biological characterization of the ectopic LASP-1 overexpression in HCC cells, using MALDI-TOF mass spectrometer on the LASP-1 co-immunoprecipitated fractions, displayed vimentin as a novel putative partner of LASP-1. Our results suggest that LASP-1 mRNA overexpression may be mainly implicated in female HCCs and cirrhotic HCCs; and that LASP1 may play its role with vimentin in HCC cells.
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- 2015
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211. A founder MYBPC3 mutation results in HCM with a high risk of sudden death after the fourth decade of life.
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Calore C, De Bortoli M, Romualdi C, Lorenzon A, Angelini A, Basso C, Thiene G, Iliceto S, Rampazzo A, and Melacini P
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- Adolescent, Adult, Age Factors, Aged, Cardiomyopathy, Hypertrophic diagnosis, Cardiomyopathy, Hypertrophic mortality, Cardiomyopathy, Hypertrophic therapy, Child, Child, Preschool, Echocardiography, Female, Follow-Up Studies, Genotype, Humans, Infant, Kaplan-Meier Estimate, Male, Middle Aged, Patient Outcome Assessment, Pedigree, Penetrance, Phenotype, Risk, Young Adult, Cardiomyopathy, Hypertrophic complications, Cardiomyopathy, Hypertrophic genetics, Carrier Proteins genetics, Death, Sudden, Cardiac etiology, Founder Effect, Mutation
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Background: Mutations in the cardiac myosin binding protein C (MYBPC3) gene account for a significant proportion of patients affected with hypertrophic cardiomyopathy (HCM). The aim of this study was to evaluate the penetrance and the impact of a frequent founder MYBPC3 mutation on HCM clinical expression and prognosis., Methods and Results: Mutation screening of MYBPC3 gene was performed in 97 HCM probands. Nineteen (19.5%) resulted to be carriers of the founder p.F305Pfs*27 mutation and other 45 mutation carriers were identified during the evaluation of 14 families. Eleven (38%) mutation carriers were diagnosed between ages 30 years and 40 years. Disease penetrance was incomplete (64.4%), age-related and was greater in men than women (85% vs 48%, p=0.009). Probands carrying the founder mutation exhibited highest prevalence of non-sustained ventricular tachycardia (63% vs 22%, p=0.003; 63% vs 23%, p=0.01) and implantable cardioverter-defibrillator (58% vs 17%, p=0.001; 58% vs 18%, p=0.005) when compared with probands without MYBPC3 mutations or carrying other MYBPC3 mutations. Reduced survival due to sudden cardiac death (SCD) or aborted SCD occurred more frequently after the fourth decade of life in probands carrying p.F305Pfs*27 mutation than those without MYBPC3 mutations (32% vs 15%, p=0.01)., Conclusions: p.F305Pfs*27 mutation carriers have a high probability to develop the disease between ages 30 years and 40 years with a significant major risk if they are men. This founder mutation is associated with an increase of SCD/aborted SCD events after the fourth decade of life.These findings are of relevant importance for management and clinical decision-making in patients with HCM., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)
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- 2015
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212. The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.
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Greco E, Aita A, Galozzi P, Gava A, Sfriso P, Negm OH, Tighe P, Caso F, Navaglia F, Dazzo E, De Bortoli M, Rampazzo A, Obici L, Donadei S, Merlini G, Plebani M, Todd I, Basso D, and Punzi L
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- Adult, Age Factors, Aged, Apoptosis genetics, Case-Control Studies, Cells, Cultured, Familial Mediterranean Fever physiopathology, HEK293 Cells, Humans, Immunoblotting methods, Italy, Male, NF-kappa B metabolism, Polymerase Chain Reaction methods, Reference Values, Risk Assessment, Sampling Studies, Signal Transduction, Familial Mediterranean Fever genetics, Genetic Predisposition to Disease, Mutation, Receptors, Tumor Necrosis Factor genetics, Receptors, Tumor Necrosis Factor, Type I genetics
- Abstract
Introduction: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations., Methods: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively., Results: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation., Conclusions: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.
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- 2015
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213. Autosomal dominant lateral temporal epilepsy (ADLTE): novel structural and single-nucleotide LGI1 mutations in families with predominant visual auras.
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Dazzo E, Santulli L, Posar A, Fattouch J, Conti S, Lodén-van Straaten M, Mijalkovic J, De Bortoli M, Rosa M, Millino C, Pacchioni B, Di Bonaventura C, Giallonardo AT, Striano S, Striano P, Parmeggiani A, and Nobile C
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- Aged, Brain pathology, Brain physiopathology, DNA Copy Number Variations, Epilepsy, Frontal Lobe pathology, Epilepsy, Temporal Lobe pathology, Epilepsy, Temporal Lobe physiopathology, Family, Female, Humans, Intracellular Signaling Peptides and Proteins, Male, Middle Aged, Mutation, Missense, Pedigree, Sequence Deletion, Sleep Wake Disorders pathology, Young Adult, Epilepsy, Frontal Lobe genetics, Epilepsy, Frontal Lobe physiopathology, Epilepsy, Temporal Lobe genetics, Proteins genetics, Sleep Wake Disorders genetics, Sleep Wake Disorders physiopathology
- Abstract
Purpose: Autosomal dominant lateral temporal epilepsy (ADLTE) is a genetic focal epilepsy syndrome characterized by prominent auditory or aphasic symptoms. Mutations in LGI1 account for less than 50% of ADLTE families. We assessed the impact of LGI1 microrearrangements in a collection of ADLTE families and sporadic lateral temporal epilepsy (LTE) patients, and investigated novel ADLTE and LTE patients., Methods: Twenty-four ADLTE families and 140 sporadic LTE patients with no evidence of point mutations in LGI1 were screened for copy number alterations using multiplex ligation-dependent probe amplification (MLPA). Newly ascertained familial and sporadic LTE patients were clinically investigated, and interictal EEG and MRI findings were obtained; probands were tested for LGI1 mutations by direct exon sequencing or denaturing high performance liquid chromatography., Results: We identified a novel microdeletion spanning LGI1 exon 2 in a family with two affected members, both presenting focal seizures with visual symptoms. Also, we identified a novel LGI1 missense mutation (c.1118T > C; p.L373S) in a newly ascertained family with focal seizures with prominent visual auras, and another missense mutation (c.856T > C; p.C286R) in a sporadic patient with auditory seizures., Conclusions: We describe two novel ADLTE families with predominant visual auras segregating pathogenic LGI1 mutations. These findings support the notion that, in addition to auditory symptoms, other types of auras can be found in patients carrying LGI1 mutations. The identification of a novel microdeletion in LGI1, the second so far identified, suggests that LGI1 microrearrangements may not be exceptional., (Copyright © 2014 Elsevier B.V. All rights reserved.)
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- 2015
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214. Biosimilar granulocyte-colony-stimulating factor for mobilization of autologous peripheral blood stem cells in pediatric hematology-oncology patients.
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Cesaro S, Tridello G, Prete A, Dallorso S, Cannata E, Massaccesi E, Risso M, De Bortoli M, and Caselli D
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- Adolescent, Allografts, Antigens, CD34, Autografts, Biosimilar Pharmaceuticals adverse effects, Child, Child, Preschool, Female, Filgrastim, Granulocyte Colony-Stimulating Factor adverse effects, Humans, Infant, Infant, Newborn, Male, Recombinant Proteins administration & dosage, Recombinant Proteins adverse effects, Retrospective Studies, Biosimilar Pharmaceuticals administration & dosage, Granulocyte Colony-Stimulating Factor administration & dosage, Hematopoietic Stem Cell Mobilization methods, Hematopoietic Stem Cells, Neoplasms therapy, Peripheral Blood Stem Cell Transplantation
- Abstract
Background: Recently biosimilars of granulocyte-colony-stimulating factor (G-CSF) became available for prophylaxis and treatment of postchemotherapy neutropenia and for mobilization of peripheral blood CD34+ cells for either autologous or allogeneic hematopoietic stem cell transplant. Most of the data on the mobilization efficacy and safety of biosimilar G-CSF are from adult patients, whereas no data are available in pediatric patients., Study Design and Methods: This was a retrospective study on cases treated at three Italian pediatric transplant centers, from January 2011 to October 2013. Data were collected on all children undergoing first peripheral blood stem cell (PBSC) mobilization after stimulation with biosimilar G-CSF and chemotherapy. The results were compared with a historical control group., Results: Twenty-nine children underwent mobilization with biosimilar G-CSF. Peak peripheral blood CD34+ cell count of 20 × 10(6) /L was achieved in 90% of patients, with a median value of 71 × 10(6) /L. Eighty-three percent reached the desired target (CD34+/kg) dose. The median number of collected CD34+ cells was 10 × 10(6) /kg (range, 4.8 × 10(6) -68.8 × 10(6) /kg). No difference was observed in comparison with historical control group mobilized with originator filgrastim. Moreover, no major and/or unexpected side effects were reported., Conclusion: Biosimilar G-CSF resulted as effective and safe as originator filgrastim molecule in mobilizing PBSCs in children, with the advantage of a reduced cost., (© 2014 AABB.)
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- 2015
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215. Plasma hepcidin in early-stage breast cancer patients: no relationship with interleukin-6, erythropoietin and erythroferrone.
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Ciniselli CM, De Bortoli M, Taverna E, Varinelli L, Pizzamiglio S, Veneroni S, Bonini C, Orlandi R, Verderio P, and Bongarzone I
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- Adult, Aged, Aged, 80 and over, Breast Neoplasms pathology, Case-Control Studies, Female, Humans, Middle Aged, Breast Neoplasms blood, Erythropoietin blood, Hepcidins blood, Interleukin-6 blood, Peptide Hormones blood
- Abstract
Objective: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer., Methods: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays., Results: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE., Conclusion: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.
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- 2015
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216. Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells.
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Caizzi L, Ferrero G, Cutrupi S, Cordero F, Ballaré C, Miano V, Reineri S, Ricci L, Friard O, Testori A, Corà D, Caselle M, Di Croce L, and De Bortoli M
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- Binding Sites, Breast Neoplasms pathology, Cell Proliferation, Chromatin Immunoprecipitation, Female, Gene Ontology, Humans, Ligands, MCF-7 Cells, Polymerase Chain Reaction, RNA, Small Interfering metabolism, Breast Neoplasms genetics, Estrogen Receptor alpha metabolism, Genome, Human genetics
- Abstract
Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.
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- 2014
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217. Genomic lens on neuroglobin transcription.
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Cutrupi S, Ferrero G, Reineri S, Cordero F, and De Bortoli M
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- Humans, Neuroglobin, Transcription Factors metabolism, Brain metabolism, Epigenomics, Gene Expression Regulation, Genomics, Globins genetics, Nerve Tissue Proteins genetics
- Abstract
Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease., (© 2014 International Union of Biochemistry and Molecular Biology.)
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- 2014
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218. Compound and digenic heterozygosity predicts lifetime arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy.
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Rigato I, Bauce B, Rampazzo A, Zorzi A, Pilichou K, Mazzotti E, Migliore F, Marra MP, Lorenzon A, De Bortoli M, Calore M, Nava A, Daliento L, Gregori D, Iliceto S, Thiene G, Basso C, and Corrado D
- Subjects
- Adult, Arrhythmogenic Right Ventricular Dysplasia physiopathology, Desmocollins genetics, Desmoglein 2 genetics, Desmoplakins genetics, Female, Genotype, Heterozygote, Humans, Kaplan-Meier Estimate, Male, Middle Aged, Plakophilins genetics, Prognosis, Risk Factors, Sex Factors, Tachycardia, Ventricular genetics, Tachycardia, Ventricular physiopathology, Time Factors, Ventricular Fibrillation genetics, Ventricular Fibrillation physiopathology, Young Adult, Arrhythmogenic Right Ventricular Dysplasia genetics, Death, Sudden, Cardiac, Desmosomes genetics, Mutation
- Abstract
Background: Mutations in genes encoding for desmosomal proteins are the most common cause of arrhythmogenic right ventricular cardiomyopathy (ARVC). We assessed the value of genotype for prediction of lifetime major arrhythmic events and sudden cardiac death (SCD) in desmosomal gene-related ARVC., Methods and Results: The overall study population included 134 desmosomal gene mutation carriers (68 men; median age 36 years [22-52]) from 44 consecutive ARVC families undergoing comprehensive genetic screening. The probability of experiencing a first major arrhythmic event or SCD during a lifetime was determined by using date of birth as start point for the time-to-event analysis, and was stratified by sex, desmosomal genes, mutation types, and genotype complexity (single versus multiple mutations). One hundred thirteen patients (84%) carried a single desmosomal gene mutation in desmoplakin (n=44; 39%), plakophilin-2 (n=38; 34%), desmoglein-2 (n=30; 26%), and desmocollin-2 (n=1; 1%), whereas 21 patients (16%) had a complex genotype with compound heterozygosity in 7 and digenic heterozygosity in 14. Over a median observation period of 39 (22-52) years, 22 patients (16%) from 20 different families had arrhythmic events, such as SCD (n=1), aborted SCD because of ventricular fibrillation (n=6), sustained ventricular tachycardia (n=14), and appropriate defibrillator intervention (n=1). Multiple desmosomal gene mutations and male sex were independent predictors of lifetime arrhythmic events with a hazard ratio of 3.71 (95% confidence interval, 1.54-8.92; P=0.003) and 2.76 (95% confidence interval, 1.19-6.41; P=0.02), respectively., Conclusions: Compound/digenic heterozygosity was identified in 16% of ARVC-causing desmosomal gene mutation carriers and was a powerful risk factor for lifetime major arrhythmic events and SCD. These results support the use of comprehensive genetic screening of desmosomal genes for arrhythmic risk stratification in ARVC.
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- 2013
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219. Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy.
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Li Mura IE, Bauce B, Nava A, Fanciulli M, Vazza G, Mazzotti E, Rigato I, De Bortoli M, Beffagna G, Lorenzon A, Calore M, Dazzo E, Nobile C, Mostacciuolo ML, Corrado D, Basso C, Daliento L, Thiene G, and Rampazzo A
- Subjects
- Adult, Aged, Aged, 80 and over, Arrhythmogenic Right Ventricular Dysplasia diagnostic imaging, Chromosomes, Human, Pair 12 genetics, DNA Copy Number Variations, Family, Female, Gene Dosage genetics, Genetic Linkage, Humans, Male, Middle Aged, Pedigree, Real-Time Polymerase Chain Reaction, Ultrasonography, Young Adult, Arrhythmogenic Right Ventricular Dysplasia genetics, Gene Deletion, Plakophilins genetics
- Abstract
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive myocardial loss, with fibro-fatty replacement, and high frequency of ventricular arrhythmias that can lead to sudden cardiac death. ARVC is a genetically determined disorder, usually caused by point mutations in components of the cardiac desmosome. Conventional mutation screening of ARVC genes fails to detect causative mutations in about 50% of index cases, suggesting a further genetic heterogeneity. We performed a genome-wide linkage study and a copy number variations (CNVs) analysis, using high-density SNP arrays, in an ARVC family showing no mutations in any of the desmosomal genes. The CNVs analysis identified a heterozygous deletion of about 122 kb on chromosome 12p11.21, including the entire plakophilin-2 gene and shared by all affected family members. It was not listed on any of available public CNVs databases and was confirmed by quantitative real-time PCR. This is the first SNP array-based genome-wide study leading to the identification of a CNV segregating with the disease phenotype in an ARVC family. This result underscores the importance of performing additional analysis for possible genomic deletions/duplications in ARVC patients without point mutations in known disease genes.
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- 2013
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220. miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1.
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Cimino D, De Pittà C, Orso F, Zampini M, Casara S, Penna E, Quaglino E, Forni M, Damasco C, Pinatel E, Ponzone R, Romualdi C, Brisken C, De Bortoli M, Biglia N, Provero P, Lanfranchi G, and Taverna D
- Subjects
- Adult, Aged, Aged, 80 and over, Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Line, Tumor, Class I Phosphatidylinositol 3-Kinases, Disease Progression, Female, Humans, Integrin alpha5 genetics, Macrophage Colony-Stimulating Factor genetics, MicroRNAs genetics, Middle Aged, Neoplasm Invasiveness, Oncogene Protein p21(ras) genetics, Phosphatidylinositol 3-Kinases genetics, RNA, Neoplasm genetics, rho-Associated Kinases genetics, Breast Neoplasms metabolism, Integrin alpha5 biosynthesis, Macrophage Colony-Stimulating Factor biosynthesis, MicroRNAs metabolism, Oncogene Protein p21(ras) biosynthesis, Phosphatidylinositol 3-Kinases biosynthesis, RNA, Neoplasm metabolism, rho-Associated Kinases biosynthesis
- Abstract
Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.
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- 2013
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221. Desmin mutations and arrhythmogenic right ventricular cardiomyopathy.
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Lorenzon A, Beffagna G, Bauce B, De Bortoli M, Li Mura IE, Calore M, Dazzo E, Basso C, Nava A, Thiene G, and Rampazzo A
- Subjects
- Arrhythmogenic Right Ventricular Dysplasia metabolism, Arrhythmogenic Right Ventricular Dysplasia physiopathology, Chromatography, High Pressure Liquid, DNA Mutational Analysis, Desmin metabolism, Electrocardiography, Female, Humans, Male, Myocardium metabolism, Pedigree, Phenotype, Arrhythmogenic Right Ventricular Dysplasia genetics, DNA genetics, Desmin genetics, Mutation
- Abstract
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease characterized by fibrofatty replacement of the myocardium and ventricular arrhythmias, associated with mutations in the desmosomal genes. Only a missense mutation in the DES gene coding for desmin, the intermediate filament protein expressed by cardiac and skeletal muscle cells, has been recently associated with ARVC. We screened 91 ARVC index cases (53 negative for mutations in desmosomal genes and an additional 38 carrying desmosomal gene mutations) for DES mutations. Two rare missense variants were identified. The heterozygous p.K241E substitution was detected in 1 patient affected with a severe form of ARVC who also carried the p.T816RfsX10 mutation in plakophilin-2 gene. This DES substitution, showing an allele frequency of <0.01 in the control population, is predicted to cause an intolerant amino acid change in a highly conserved protein domain. Thus, it can be considered a rare variant with a possible modifier effect on the phenotypic expression of the concomitant mutation. The previously known p.A213V substitution was identified in 1 patient with ARVC who was negative for mutations in the desmosomal genes. Because a greater prevalence of p.A213V has been reported in patients with heart dilation than in control subjects, the hypothesis that this rare variant could have an unfavorable effect on cardiac remodeling cannot be ruled out. In conclusion, our data help to establish that, in the absence of skeletal muscle involvement suggestive of a desminopathy, the probability of DES mutations in ARVC is very low. These findings have important implications in the mutation screening strategy for patients with ARVC., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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222. Mutations in the area composita protein αT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy.
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van Hengel J, Calore M, Bauce B, Dazzo E, Mazzotti E, De Bortoli M, Lorenzon A, Li Mura IE, Beffagna G, Rigato I, Vleeschouwers M, Tyberghein K, Hulpiau P, van Hamme E, Zaglia T, Corrado D, Basso C, Thiene G, Daliento L, Nava A, van Roy F, and Rampazzo A
- Subjects
- Adult, Arrhythmias, Cardiac genetics, Arrhythmogenic Right Ventricular Dysplasia metabolism, Case-Control Studies, Electrocardiography, Female, Heterozygote, Humans, Male, Pedigree, alpha Catenin metabolism, Arrhythmogenic Right Ventricular Dysplasia genetics, Death, Sudden, Cardiac etiology, Gene Deletion, Mutation, Missense genetics, alpha Catenin genetics
- Abstract
Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes., Methods and Results: We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and β-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells., Conclusion: These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.
- Published
- 2013
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223. New insights on cytological and metabolic features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): a multidisciplinary approach.
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Honsell G, Bonifacio A, De Bortoli M, Penna A, Battocchi C, Ciminiello P, Dell'aversano C, Fattorusso E, Sosa S, Yasumoto T, and Tubaro A
- Subjects
- Acrylamides chemistry, Carotenoids chemistry, Chloroplasts ultrastructure, Chromatography, Ion Exchange, Chromatography, Liquid, Cnidarian Venoms, Dinoflagellida genetics, Dinoflagellida ultrastructure, Genotype, Marine Toxins chemistry, Mass Spectrometry, Microscopy, Fluorescence, Pigments, Biological metabolism, Principal Component Analysis, Spectrum Analysis, Raman, Dinoflagellida cytology, Dinoflagellida metabolism
- Abstract
The harmful dinoflagellate Ostreopsis cf. ovata has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as O. cf. ovata, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. In vivo Raman microspectroscopy confirms the presence of the carotenoid peridinin in O. cf. ovata, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, O. cf. ovata may be able to rapidly colonize benthic substrates outcompeting other species.
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- 2013
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224. The role of Transposable Elements in shaping the combinatorial interaction of Transcription Factors.
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Testori A, Caizzi L, Cutrupi S, Friard O, De Bortoli M, Cora' D, and Caselle M
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- Base Sequence, Binding Sites, Genome, Human, Humans, MCF-7 Cells, Molecular Sequence Annotation, Molecular Sequence Data, DNA Transposable Elements, Estrogen Receptor alpha genetics, Gene Expression Regulation, Transcription Factors genetics
- Abstract
Background: In the last few years several studies have shown that Transposable Elements (TEs) in the human genome are significantly associated with Transcription Factor Binding Sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Another important feature of the regulatory network which has been thoroughly studied is the combinatorial organization of transcriptional regulation. In this paper we combine these two observations and suggest that TEs, besides rewiring the network, also played a central role in the evolution of particular patterns of combinatorial gene regulation., Results: To address this issue we searched for TEs overlapping Estrogen Receptor α (ERα) binding peaks in two publicly available ChIP-seq datasets from the MCF7 cell line corresponding to different modalities of exposure to estrogen. We found a remarkable enrichment of a few specific classes of Transposons. Among these a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERα for the MIR class of TEs could be at the origin of the important role assumed by ERα in Mammalians. We then searched for TFBSs within the TEs overlapping ChIP-seq peaks. We found a strong enrichment of a few precise combinations of TFBS. In several cases the corresponding Transcription Factors (TFs) were known cofactors of ERα, thus supporting the idea of a co-regulatory role of TFBS within the same TE. Moreover, most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well-defined "transposon code" within the regulatory network., Conclusions: In this work we tried to shed light into the role of Transposable Elements (TEs) in shaping the regulatory network of higher eukaryotes. To test this idea we focused on a particular transcription factor: the Estrogen Receptor α (ERα) and we found that ERα preferentially targets a well defined set of TEs and that these TEs host combinations of transcriptional regulators involving several of known co-regulators of ERα. Moreover, a significant number of these TEs turned out to be conserved between human and mouse and located in the vicinity (and thus candidate to be regulators) of important estrogen-related genes.
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- 2012
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225. Soluble epidermal growth factor receptor isoforms in non-small cell lung cancer tissue and in blood.
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Maramotti S, Paci M, Miccichè F, Ciarrocchi A, Cavazza A, De Bortoli M, Vaghi E, Formisano D, Canovi L, Sgarbi G, and Bongarzone I
- Subjects
- Carcinoma, Non-Small-Cell Lung blood, Cell Line, Tumor, Culture Media, Conditioned chemistry, ErbB Receptors blood, Humans, Lung metabolism, Lung Neoplasms blood, Protein Isoforms blood, Protein Isoforms metabolism, Carcinoma, Non-Small-Cell Lung metabolism, ErbB Receptors metabolism, Lung Neoplasms metabolism
- Abstract
Epidermal growth factor receptor (EGFR) is implicated in tumor development and is highly expressed in many human tumors. EGFR overexpression has been observed in both premalignant lesions and in malignant lung tumors, as well as in 40-80% of patients with non-small cell lung cancer (NSCLC). EGFR is a 170-kDa transmembrane glycoprotein with an extracellular ligand-binding domain and a cytoplasmic domain with intrinsic tyrosine kinase activity. Soluble forms of EGFR (sEGFR) containing the extracellular domain have been described both in conditioned media from EGFR overexpressing cells as well as in peripheral blood. However, very little is known regarding the molecular function and the biochemical properties of these circulating EGFR isoforms. This study investigates the expression of sEGFR in lung cancer cultured cells and NSCLC patients with the aim of identifying clinically relevant isoforms specifically produced by tumor cells. Proteomic approaches including OFFGEL electrophoresis and Western blotting analysis were used to assess the sEGFR expression pattern in primary lung tumor samples, normal counterparts and matched plasma. We discover that the isoelectric points of sEGFR isoforms in NSCLC biopsy tissue differ from those of the isoforms present in healthy tissue and detected in the plasma of all subjects. These results demonstrate, for the first time, the existence of sEGFR isoforms specifically produced by NSCLC tumor cells which could represent a new potential biomarker for diagnosis and therapy of lung tumors. However, our observations indicate that more highly sensitive and specific quantitative assays are needed in order to reliably detect the tumor-associated sEGFR isoforms in plasma samples., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2012
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226. Effects of oestrogen on microRNA expression in hormone-responsive breast cancer cells.
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Ferraro L, Ravo M, Nassa G, Tarallo R, De Filippo MR, Giurato G, Cirillo F, Stellato C, Silvestro S, Cantarella C, Rizzo F, Cimino D, Friard O, Biglia N, De Bortoli M, Cicatiello L, Nola E, and Weisz A
- Subjects
- Adult, Aged, Binding Sites genetics, Breast Neoplasms metabolism, Breast Neoplasms mortality, Cell Line, Tumor, Cluster Analysis, Estrogen Receptor alpha metabolism, Female, Gene Expression Profiling, Humans, Middle Aged, Response Elements, Breast Neoplasms genetics, Estradiol pharmacology, Gene Expression Regulation, Neoplastic drug effects, MicroRNAs genetics
- Abstract
Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.
- Published
- 2012
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227. Glucocorticoid receptor activity discriminates between progesterone and medroxyprogesterone acetate effects in breast cells.
- Author
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Courtin A, Communal L, Vilasco M, Cimino D, Mourra N, de Bortoli M, Taverna D, Faussat AM, Chaouat M, Forgez P, and Gompel A
- Subjects
- Adolescent, Adult, Breast, Breast Neoplasms, Carrier Proteins pharmacology, Cell Line, Tumor, Cell Proliferation drug effects, Female, Gene Expression Profiling, Gene Expression Regulation, Neoplastic drug effects, High-Throughput Nucleotide Sequencing, Hormone Replacement Therapy methods, Humans, Norpregnadienes pharmacology, RNA Interference, RNA, Small Interfering, Receptors, Estrogen metabolism, Steroids, Transcriptome, Young Adult, Estradiol pharmacology, Medroxyprogesterone Acetate pharmacology, Progesterone pharmacology, Receptors, Glucocorticoid metabolism, Receptors, Progesterone metabolism
- Abstract
The purpose of this article is to determine the tumorigenic potential of estradiol treatment (E2) when combined with either progesterone (P4) or medroxyprogesterone acetate (MPA) in normal luminal human breast cells (HBE) and in human breast cancer cells (T47-D, MCF-7). Proliferation profiles were evaluated, along with the gene transactivation activity between the progesterone and glucocorticoid receptors (PR, GR) in HBE, T47-D, and MCF-7 cells treated by E2 + P4 or E2 + MPA. High throughput transcriptome analysis was performed on RNA from HBE cells treated by E2, E2 + MPA and E2 + P4. GR content was analyzed in normal breast cells as well. In HBE cells, E2 + P4 treatment was antiproliferative and promoted cellular differentiation. In contrast, E2 + MPA displayed mitogenic, antiapoptotic effects in HBE cells and did not influence cellular differentiation. The effect of P4 and MPA on cell proliferation was, however, variable in breast cancer cells. In cells containing GR or/and PR, MPA decreased proliferation whereas P4 antiproliferative effect needed the presence of PR. In HBE cells, the regulation of genes by E2 + P4, and E2 + MPA was significantly different, particularly in cell proliferation and cell death gene families. Further analysis revealed a modulation of the glucocorticoid receptor gene expression pathway by E2 + MPA. Predominant MPA glucocorticoid activity in normal and breast cancer cells was demonstrated using a glucocorticoid antagonist and the down-regulation of the GR by RNA interference. In normal luminal breast cells and in breast cancer cells, P4 and MPA combined with E2 treatment have opposing mitogenic effects due to GR. The consequences of MPA glucocorticoid potencies as well as the importance of GR in breast tissue merit a reappraisal.
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- 2012
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228. Clinical phenotype and diagnosis of arrhythmogenic right ventricular cardiomyopathy in pediatric patients carrying desmosomal gene mutations.
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Bauce B, Rampazzo A, Basso C, Mazzotti E, Rigato I, Steriotis A, Beffagna G, Lorenzon A, De Bortoli M, Pilichou K, Marra MP, Corbetti F, Daliento L, Iliceto S, Corrado D, Thiene G, and Nava A
- Subjects
- Adolescent, Arrhythmogenic Right Ventricular Dysplasia diagnosis, Child, DNA Mutational Analysis, Diagnosis, Differential, Female, Follow-Up Studies, Humans, Male, Pedigree, Phenotype, Prognosis, Retrospective Studies, Arrhythmogenic Right Ventricular Dysplasia genetics, DNA analysis, Desmosomes genetics, Echocardiography, Electrocardiography, Magnetic Resonance Imaging, Cine methods, Mutation
- Abstract
Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease carrying a risk of sudden death. Information about the clinical features during childhood and the age at disease onset is scanty., Objective: The aim of the study was to describe the ARVC phenotype as its initial clinical manifestation in a pediatric population (<18 years) with desmosomal gene mutations., Methods: Fifty-three ARVC desmosomal gene mutation carriers (mean age 12.3 ± 3.9 years) were investigated by electrocardiogram (ECG), signal-averaged ECG, 24-hour Holter, echocardiogram, and contrast-enhanced cardiac magnetic resonance (CMR)., Results: None of the children ≤10 years old fulfilled the 1994 criteria, as opposed to six (33%) aged 11-14 years and eight aged >14 years (42%). At the end of follow-up (9 ± 7 years), 21 (40%) fulfilled the 1994 diagnostic criteria (mean age 16 ± 4 years). By using the 2010 criteria in subjects aged ≤18 years, 53% were unaffected, versus 62% by using the traditional criteria. More than two-thirds of affected subjects had moderate-severe forms of the disease. Contrast-enhanced CMR was performed in 21 (40%); of 13 unaffected gene mutation carriers, six showed ARVC morphological and/or tissue abnormalities., Conclusion: In pediatric ARVC mutation carriers, a diagnosis was achieved in 40% of cases, confirming that the disease usually develops during adolescence and young adulthood. The 2010 modified criteria seem to be more sensitive than the 1994 ones in identifying familial pediatric cases. Contrast-enhanced CMR can provide diagnostic information on gene mutation carriers not fulfilling either traditional or modified criteria. Management of asymptomatic gene mutation carriers remains the main clinical challenge., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2011
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229. Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways.
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Caccia D, Zanetti Domingues L, Miccichè F, De Bortoli M, Carniti C, Mondellini P, and Bongarzone I
- Subjects
- Antineoplastic Agents pharmacology, Cell Line, Tumor, Cluster Analysis, Computational Biology, Dasatinib, Databases, Protein, Humans, Intracellular Space, Models, Biological, Neoplasm Proteins analysis, Neoplasm Proteins chemistry, Neoplasms drug therapy, Phosphotyrosine analysis, Phosphotyrosine chemistry, Pyrimidines pharmacology, Signal Transduction, Thiazoles pharmacology, Transforming Growth Factor beta, Biomarkers, Tumor analysis, Biomarkers, Tumor metabolism, Neoplasms chemistry, Neoplasms metabolism, Proteome analysis, Proteome metabolism
- Abstract
In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and βIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.
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- 2011
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230. Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies.
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Honsell G, De Bortoli M, Boscolo S, Dell'Aversano C, Battocchi C, Fontanive G, Penna A, Berti F, Sosa S, Yasumoto T, Ciminiello P, Poli M, and Tubaro A
- Subjects
- Acrylamides chemistry, Chromatography, Liquid, Cnidarian Venoms, Dinoflagellida classification, Immunohistochemistry, Marine Toxins chemistry, Mass Spectrometry, Oceans and Seas, Time Factors, Acrylamides immunology, Antibodies immunology, Dinoflagellida cytology, Dinoflagellida metabolism, Marine Toxins analysis
- Abstract
Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.
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- 2011
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231. Rimonabant reduces keratinocyte viability by induction of apoptosis and exerts topical anti-inflammatory activity in mice.
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Malfitano AM, Sosa S, Laezza C, De Bortoli M, Tubaro A, and Bifulco M
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- Administration, Topical, Animals, Cell Cycle drug effects, Cell Division drug effects, Cell Line, Keratinocytes cytology, Keratinocytes metabolism, Mice, Rimonabant, Anti-Inflammatory Agents pharmacology, Apoptosis drug effects, Keratinocytes drug effects, Piperidines pharmacology, Pyrazoles pharmacology
- Abstract
Background and Purpose: There is growing evidence that the cannabinoid CB(1) receptor antagonist, rimonabant (SR141716) exerts potential anti-proliferative and anti-inflammatory actions. Here, we have assessed the effects of rimonabant in vitro in murine immortalized keratinocytes and in vivo by assaying the topical anti-inflammatory activity., Experimental Approach: Cell viability and death in a keratinocyte cell line (C5N cells) were measured by Trypan blue exclusion assay and cytotoxicity by sulphorhodamine B test. Cell cycle progression was assessed by flow cytometry and the expression of apoptotic and anti-apoptotic markers, cyclins, pathways of signal transduction and CB1 receptor levels were evaluated by Western blot. The topical anti-inflammatory properties of rimonabant were analysed by inhibition of croton oil-induced ear dermatitis in mice., Key Results: Rimonabant reduced cell viability and induced apoptosis as shown by the enhanced number of cells in the subG0 phase of the cell cycle, the expression of Bax and reduced levels of Bcl-2 and X-inhibitor of apoptosis protein. In addition, reduced levels of phosphorylated serine/threonine protein kinase Akt and nuclear factor-kappa B were detected associated with regulation of total nuclear factor-kappa B and inhibitor of kappa B-α, phosphorylated inhibitor of kappa B-α, cyclins D1, E and A. In croton oil-induced ear dermatitis, rimonabant significantly reduced oedema and leukocyte infiltrate., Conclusions and Implications: Rimonabant reduced cell viability, inducing cell death in keratinocytes and decreased croton oil-induced ear dermatitis. Our findings suggest a potential application of rimonabant as a topical anti-inflammatory drug. We did not assess the involvement of CB(1) receptors in these effects of rimonabant., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)
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- 2011
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232. CircuitsDB: a database of mixed microRNA/transcription factor feed-forward regulatory circuits in human and mouse.
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Friard O, Re A, Taverna D, De Bortoli M, and Corá D
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- Animals, Base Sequence, Gene Regulatory Networks, Genome, Humans, Internet, Mice, Neoplasms genetics, Sequence Alignment, Databases, Genetic, Gene Expression Regulation, MicroRNAs metabolism, Transcription Factors metabolism
- Abstract
Background: Transcription Factors (TFs) and microRNAs (miRNAs) are key players for gene expression regulation in higher eukaryotes. In the last years, a large amount of bioinformatic studies were devoted to the elucidation of transcriptional and post-transcriptional (mostly miRNA-mediated) regulatory interactions, but little is known about the interplay between them., Description: Here we describe a dynamic web-accessible database, CircuitsDB, supporting a genome-wide transcriptional and post-transcriptional regulatory network integration, for the human and mouse genomes, based on a bioinformatic sequence-analysis approach. In particular, CircuitsDB is currently focused on the study of mixed miRNA/TF Feed-Forward regulatory Loops (FFLs), i.e. elementary circuits in which a master TF regulates an miRNA and together with it a set of Joint Target protein-coding genes. The database was constructed using an ab-initio oligo analysis procedure for the identification of the transcriptional and post-transcriptional interactions. Several external sources of information were then pooled together to obtain the functional annotation of the proposed interactions. Results for human and mouse genomes are presented in an integrated web tool, that allows users to explore the circuits, investigate their sequence and functional properties and thus suggest possible biological experiments., Conclusions: We present CircuitsDB, a web-server devoted to the study of human and mouse mixed miRNA/TF Feed-Forward regulatory circuits, freely available at: http://biocluster.di.unito.it/circuits/
- Published
- 2010
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233. The p.A897KfsX4 frameshift variation in desmocollin-2 is not a causative mutation in arrhythmogenic right ventricular cardiomyopathy.
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De Bortoli M, Beffagna G, Bauce B, Lorenzon A, Smaniotto G, Rigato I, Calore M, Li Mura IE, Basso C, Thiene G, Lanfranchi G, Danieli GA, Nava A, and Rampazzo A
- Subjects
- Alternative Splicing genetics, Amino Acid Sequence, Base Sequence, Cell Line, DNA Mutational Analysis, Desmocollins chemistry, Desmocollins metabolism, Female, Haplotypes genetics, Heterozygote, Humans, Male, Middle Aged, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Pedigree, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Arrhythmogenic Right Ventricular Dysplasia genetics, Desmocollins genetics, Frameshift Mutation genetics
- Abstract
Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), an autosomal-dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 112 ARVC/D probands for mutations in desmocollin-2 (DSC2) gene and detected two different amino-acid substitutions (p.E102K, p.I345T) and a frameshift variation (p.A897KfsX4) in 7 (6.2%) patients. DSC2a variant p.A897KfsX4, previously reported as a p.E896fsX900 mutation, was identified in five unrelated probands. Four of them were found to carry one or two mutations in different ARVC/D genes. Unexpectedly, p.A897KfsX4 variation was also found in 6 (1.5%) out of 400 control chromosomes. In vitro functional studies showed that, unlike wild-type DSC2a, this C-terminal mutated protein was localised in the cytoplasm. p.A897KfsX4 variation affects the last five amino acids of the DSC2a isoform but not of DSC2b. In contrast with what we found in other human tissues, in the heart DSC2b is more expressed than DSC2a, suggesting that relative deficiency of DSC2a might be compensated by isoform b. In conclusion, DSC2 gene mutations are not frequently involved in ARVC/D. The p.A897KfsX4 variation, identified in several Italian healthy control subjects, which affects only one of the two DSC2 isoforms, may be considered a rare variant, though possibly affecting phenotypic expression of concomitant ARVC/D mutations.
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- 2010
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234. Estrogen receptor alpha controls a gene network in luminal-like breast cancer cells comprising multiple transcription factors and microRNAs.
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Cicatiello L, Mutarelli M, Grober OM, Paris O, Ferraro L, Ravo M, Tarallo R, Luo S, Schroth GP, Seifert M, Zinser C, Chiusano ML, Traini A, De Bortoli M, and Weisz A
- Subjects
- Binding Sites, Cell Line, Tumor, Chromatin Immunoprecipitation, Estradiol metabolism, Estrogen Receptor alpha metabolism, Humans, Kinetics, MicroRNAs metabolism, Models, Biological, Oligonucleotide Array Sequence Analysis, RNA metabolism, Breast Neoplasms metabolism, Estrogen Receptor alpha physiology, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, MicroRNAs genetics, Transcription Factors metabolism
- Abstract
Luminal-like breast tumor cells express estrogen receptor alpha (ERalpha), a member of the nuclear receptor family of ligand-activated transcription factors that controls their proliferation, survival, and functional status. To identify the molecular determinants of this hormone-responsive tumor phenotype, a comprehensive genome-wide analysis was performed in estrogen stimulated MCF-7 and ZR-75.1 cells by integrating time-course mRNA expression profiling with global mapping of genomic ERalpha binding sites by chromatin immunoprecipitation coupled to massively parallel sequencing, microRNA expression profiling, and in silico analysis of transcription units and receptor binding regions identified. All 1270 genes that were found to respond to 17beta-estradiol in both cell lines cluster in 33 highly concordant groups, each of which showed defined kinetics of RNA changes. This hormone-responsive gene set includes several direct targets of ERalpha and is organized in a gene regulation cascade, stemming from ligand-activated receptor and reaching a large number of downstream targets via AP-2gamma, B-cell activating transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and RXRalpha transcription factors. MicroRNAs are also integral components of this gene regulation network because miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated by 17beta-estradiol along with other microRNAs that can target a significant number of transcripts belonging to one or more estrogen-responsive gene clusters.
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- 2010
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235. Elevated levels of the acute-phase serum amyloid are associated with heightened lung cancer risk.
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Cremona M, Calabrò E, Randi G, De Bortoli M, Mondellini P, Verri C, Sozzi G, Pierotti MA, La Vecchia C, Pastorino U, and Bongarzone I
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- Aged, C-Reactive Protein analysis, Case-Control Studies, Female, Humans, Inflammation complications, Male, Middle Aged, Risk, Sensitivity and Specificity, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Biomarkers, Tumor blood, Lung Neoplasms blood, Serum Amyloid A Protein analysis
- Abstract
Background: The authors investigated whether early stage lung cancer could be identified by proteomic analyses of plasma., Methods: For the first case-control study, plasma samples from 52 patients with lung cancer and from a group of 51 controls were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. In a second case-control study, a classifier of 4 markers (mass-to-charge ratio, 11,681, 6843, 5607, and 8762) also was tested for validation on plasma from 16 consecutive patients with screen-detected cancer versus 406 healthy individuals. The most relevant marker was identified, and an enzyme-linked immunosorbent assay-based analysis revealed that signal intensity was correlated with concentration., Results: The classifier had a sensitivity of 94.23% and a specificity of 76.47% in the first study but lost predictive value in the second study. Nevertheless, the 11,681 cluster, which was identified as serum amyloid protein A (SAA), resulted in a multiple logistic regression model that indicated a strong association with lung cancer. When both studies were considered as a together, the odds ratio (OR) for an SAA intensity > or =0.5 was 10.27 (95% confidence interval [CI], 4.64-22.74), whereas an analysis restricted to stage I cancers (TNM classification) revealed an OR of 8.45 (95% CI, 2.76-25.83) for T1 lung cancer and 21.22 (95% CI, 5.62-80.14) for T2 lung cancer., Conclusions: SAA levels were predictive of an elevated risk of lung cancer, supporting the general view that inflammation is implicated in lung cancer development.
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- 2010
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236. Valproic acid restores ER alpha and antiestrogen sensitivity to ER alpha-negative breast cancer cells.
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Fortunati N, Bertino S, Costantino L, De Bortoli M, Compagnone A, Bandino A, Catalano MG, and Boccuzzi G
- Subjects
- Animals, Cell Proliferation drug effects, Estradiol metabolism, Estradiol pharmacology, Estrogen Antagonists pharmacology, Estrogen Receptor alpha genetics, Estrogen Receptor beta genetics, Estrogen Receptor beta metabolism, Female, Gene Expression Regulation, Neoplastic drug effects, Hepatocyte Nuclear Factor 3-alpha genetics, Hepatocyte Nuclear Factor 3-alpha metabolism, Humans, Tamoxifen pharmacology, Transcription, Genetic drug effects, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Breast Neoplasms metabolism, Cell Line, Tumor drug effects, Enzyme Inhibitors pharmacology, Estrogen Receptor Modulators metabolism, Estrogen Receptor Modulators therapeutic use, Estrogen Receptor alpha metabolism, Valproic Acid pharmacology
- Abstract
Histone deacetylase inhibitors (HDIs) are valuable drugs in breast cancer where estrogen receptor alpha (ER alpha) can be silenced by epigenetic modifications. We report the effect of the clinically available HDI, valproic acid (VPA), on ER alpha expression and function in ER-negative breast cancer cells, MDA-MB-231. VPA induced ER alpha mRNA and protein, while did not modify ER beta. In VPA-treated cells, we also observed: (1) a correct transcriptional response to estradiol after transfection with the luciferase gene under the control of an estrogen-responsive minimal promoter (ERE-TKluc); (2) increased expression of the ER-related transcription factor FoxA1; (3) estradiol-induced up-regulation of several estrogen-regulated genes (e.g. pS2, progesterone receptor); (4) inhibitory effect of tamoxifen on cell growth. In conclusion, the HDI VPA, inducing ER alpha and FoxA1, confers to MDA-MB 231 cells an estrogen-sensitive "phenotype", restoring their sensitivity to antiestrogen therapy.
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- 2010
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237. Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia.
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Bauce B, Nava A, Beffagna G, Basso C, Lorenzon A, Smaniotto G, De Bortoli M, Rigato I, Mazzotti E, Steriotis A, Marra MP, Towbin JA, Thiene G, Danieli GA, and Rampazzo A
- Subjects
- Adult, Chromatography, High Pressure Liquid, Cohort Studies, Death, Sudden, Cardiac etiology, Desmoglein 2 genetics, Desmoplakins genetics, Desmosomes chemistry, Female, Genetic Testing, Humans, Male, Middle Aged, Mutation, Pedigree, Plakophilins genetics, Risk Assessment, Young Adult, gamma Catenin, Arrhythmogenic Right Ventricular Dysplasia genetics, Cytoskeletal Proteins genetics, Desmocollins genetics, Desmosomes genetics
- Abstract
Background: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a progressive cardiomyopathy showing a wide clinical spectrum in terms of clinical expressions and prognoses., Objective: This study sought to estimate the occurrence of compound and double heterozygotes for mutations in desmosomal proteins encoding genes in a cohort of ARVC/D Italian index cases, and to assess the clinical phenotype of mutations carriers., Methods: Fourty-two consecutive ARVC/D index cases who fulfilled the International Task Force diagnostic criteria were screened for mutations in PKP2, DSP, DSG2, DSC2, and JUP genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing., Results: Three probands (7.1%) showing a family history of sudden death carried multiple mutations. Family screening identified an additional 7 multiple-mutation carriers. Among the 7 double heterozygotes for mutations in different genes, 2 were clinically unaffected, 2 were affected, and 3 showed some clinical signs of ARVC/D even if they did not fulfill the diagnostic criteria. Two compound heterozygotes for mutations in the same gene and 1 subject carrying 3 different mutations showed a severe form of the disease with heart failure onset at a young age. Moreover, multiple-mutation carriers showed a higher prevalence of left ventricular involvement (P = .025) than single-mutation carriers., Conclusion: Occurrence of compound and double heterozygotes in ARVC/D index cases is particularly relevant to mutation screening strategy and to genetic counseling. Even if multiple-mutation carriers show a wide variability in clinical expression, the extent of the disease is higher compared to that in single-mutation carriers.
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- 2010
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238. Stereostructure and biological activity of 42-hydroxy-palytoxin: a new palytoxin analogue from Hawaiian Palythoa subspecies.
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Ciminiello P, Dell'aversano C, Dello Iacovo E, Fattorusso E, Forino M, Grauso L, Tartaglione L, Florio C, Lorenzon P, De Bortoli M, Tubaro A, Poli M, and Bignami G
- Subjects
- Animals, Cells, Cultured, Chromatography, High Pressure Liquid, Cnidarian Venoms chemistry, Hawaii, Magnetic Resonance Spectroscopy, Mice, Pyrans chemistry, Spectrometry, Mass, Electrospray Ionization, Stereoisomerism, Acrylamides chemistry, Anthozoa chemistry, Cnidarian Venoms therapeutic use, Pyrans therapeutic use
- Abstract
This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.
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- 2009
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239. AP-2alpha regulates migration of GN-11 neurons via a specific genetic programme involving the Axl receptor tyrosine kinase.
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Orso F, Jäger R, Calogero RA, Schorle H, Sismondi P, De Bortoli M, and Taverna D
- Subjects
- Animals, Binding Sites, Cell Line, Cell Proliferation, Clone Cells, Embryo, Mammalian cytology, Fibroblasts cytology, Fibroblasts enzymology, Gene Expression Profiling, Gene Expression Regulation, Gene Knockdown Techniques, Gene Regulatory Networks, Humans, Mice, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic, Protein Binding, Proto-Oncogene Proteins, Reproducibility of Results, Transcription, Genetic, Axl Receptor Tyrosine Kinase, Cell Movement, Neurons cytology, Neurons enzymology, Oncogene Proteins metabolism, Receptor Protein-Tyrosine Kinases metabolism, Transcription Factor AP-2 metabolism
- Abstract
Background: Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2alpha is a transcription factor essential for neural crest cell migration and its mutation results in apoptosis within this cell population, as demonstrated by genetic models., Results: We down-modulated AP-2alpha expression in GN-11 neurons by RNA interference and observe reduced neuron migration following the activation of a specific genetic programme including the Adhesion Related Kinase (Axl) gene. We prove that Axl is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2alpha via the binding to one or more functional AP-2alpha binding sites present in its regulatory region. Analysis of migration in AP-2alpha null mouse embryo fibroblasts also reveals an essential role for AP-2alpha in cell movement via the activation of a distinct genetic programme., Conclusion: We show that AP-2alpha plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2alpha regulated gene Axl is an essential player in GN-11 neuron migration.
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- 2009
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240. ERalpha as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells.
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Cardamone MD, Bardella C, Gutierrez A, Di Croce L, Rosenfeld MG, Di Renzo MF, and De Bortoli M
- Subjects
- Amino Acid Sequence, Antigens, CD, Breast Neoplasms genetics, Breast Neoplasms metabolism, Cell Line, Tumor, Epithelial Cells metabolism, Humans, Ligands, Molecular Sequence Data, Promoter Regions, Genetic, Transcription, Genetic, Breast Neoplasms pathology, Cadherins genetics, Epithelial Cells pathology, Estrogen Receptor alpha metabolism, Gene Expression Regulation, Neoplastic
- Abstract
Estrogen receptor alpha (ERalpha) and E-cadherin are primary markers of luminal epithelial breast cancer cells with E-cadherin being a main caretaker of the epithelial phenotype. E-cadherin repression is needed for cancer cells to acquire motile and invasive properties, and it is known that in ER-positive breast cancer cells, estrogen down-regulate E-cadherin gene transcription. We report here that ERalpha is bound to the E-cadherin promoter in both the presence and the complete absence of estrogen, suggesting an unexpected role for unliganded ERalpha in E-cadherin transcription. Indeed, our data reveal that activation by unliganded ERalpha and repression by estrogen-activated ERalpha require direct binding to a half-estrogen response element within the E-cadherin promoter and exchange from associated coactivators to corepressors. Therefore, these results suggest a pivotal role for unliganded ERalpha in controlling a fundamental caretaker of the epithelial phenotype in breast cancer cells. Here, we show that ERalpha-positive breast cancer T47D cells transduced with the sfRON kinase undergo a full epithelial-mesenchymal conversion and lose E-cadherin and ERalpha expression. Our data show that, although the E-cadherin gene becomes hypermethylated and heterochromatic, kinase inhibitors can restore E-cadherin expression, together with an epithelial morphology in an ERalpha-dependent fashion. Similarly, transfection of ERalpha, in the absence of ligands, was sufficient to restore E-cadherin transcription in both sfRON-T47D and other ERalpha-, E-cadherin-negative cells. Therefore, our results suggest a novel role for the ERalpha that plays the dual role of ligand-independent activator and ligand-dependent repressor of E-cadherin in breast cancer cells.
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- 2009
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241. Identification of new genes associated with breast cancer progression by gene expression analysis of predefined sets of neoplastic tissues.
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Cimino D, Fuso L, Sfiligoi C, Biglia N, Ponzone R, Maggiorotto F, Russo G, Cicatiello L, Weisz A, Taverna D, Sismondi P, and De Bortoli M
- Subjects
- Disease Progression, Disease-Free Survival, Female, Humans, Kaplan-Meier Estimate, Middle Aged, Oligonucleotide Array Sequence Analysis, Prognosis, Reverse Transcriptase Polymerase Chain Reaction, Breast Neoplasms genetics, Breast Neoplasms mortality, Gene Expression, Gene Expression Profiling, Neoplasm Recurrence, Local genetics
- Abstract
Gene expression profiles were studied by microarray analysis in 2 sets of archival breast cancer tissues from patients with distinct clinical outcome. Seventy-seven differentially expressed genes were identified when comparing 30 cases with relapse and 30 cases without relapse within 72 months from surgery. These genes had a specific ontological distribution and some of them have been linked to breast cancer in previous studies: AIB1, the two keratin genes KRT5 and KRT15, RAF1, WIF1 and MSH6. Seven out of 77 differentially expressed genes were selected and analyzed by qRT-PCR in 127 cases of breast cancer. The expression levels of 6 upregulated genes (CKMT1B, DDX21, PRKDC, PTPN1, SLPI, YWHAE) showed a significant association to both disease-free and overall survival. Multivariate analysis using the significant factors (i.e., estrogen receptor and lymph node status) as covariates confirmed the association with survival. There was no correlation between the expression level of these genes and other clinical parameters. In contrast, SERPINA3, the only downregulated gene examined, was not associated with survival, but correlated with steroid receptor status. An indirect validation of our genes was provided by calculating their association with survival in 3 publicly available microarray datasets. CKMT1B expression was an independent prognostic marker in all 3 datasets, whereas other genes confirmed their association with disease-free survival in at least 1 dataset. This work provides a novel set of genes that could be used as independent prognostic markers and potential drug targets for breast cancer., (Copyright 2008 Wiley-Liss, Inc.)
- Published
- 2008
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242. AP-2alpha and AP-2gamma regulate tumor progression via specific genetic programs.
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Orso F, Penna E, Cimino D, Astanina E, Maione F, Valdembri D, Giraudo E, Serini G, Sismondi P, De Bortoli M, and Taverna D
- Subjects
- Animals, Base Sequence, Breast Neoplasms etiology, Breast Neoplasms genetics, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Death, Cell Division, Cell Line, Tumor, Cell Movement, Chemokine CXCL2 genetics, Epidermal Growth Factor genetics, Epiregulin, Female, HeLa Cells, Humans, Membrane Proteins genetics, Mice, Mice, Nude, Neoplasm Invasiveness, Neoplasm Transplantation, Neoplasms etiology, Neoplasms metabolism, Neoplasms pathology, Oligonucleotide Array Sequence Analysis, RNA Interference, RNA, Small Interfering genetics, Transcription Factor AP-2 antagonists & inhibitors, Transcription Factor AP-2 metabolism, Transplantation, Heterologous, Neoplasms genetics, Transcription Factor AP-2 genetics
- Abstract
The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP-2 proteins consists of five different transcription factors (alpha, beta, gamma, delta, and epsilon) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP-2alpha and AP-2gamma proteins play an essential role in tumorigenesis. Down-modulation of AP-2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy-induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP-2 overexpression. We observed that increased xenotransplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP-2alpha knockdown cells and observed that AP-2alpha regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP-2 controlled migration, as ablation of any of these genes severely altered migration.
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- 2008
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243. Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays.
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Ravo M, Mutarelli M, Ferraro L, Grober OM, Paris O, Tarallo R, Vigilante A, Cimino D, De Bortoli M, Nola E, Cicatiello L, and Weisz A
- Subjects
- Biopsy, Cell Line, Tumor, Female, Formaldehyde, Humans, Oligonucleotide Array Sequence Analysis, Paraffin Embedding, Reproducibility of Results, Breast Neoplasms pathology, Carcinoma, Ductal, Breast pathology, Gene Expression Profiling methods, RNA, Neoplasm analysis
- Abstract
Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, particularly formalin-fixed, paraffin-embedded (FFPE) archived tissues, is limited by the poor quality of the RNA recovered. This represents a serious drawback, as FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer (MT) biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17beta-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.
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- 2008
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244. Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D.
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Castellino RC, De Bortoli M, Lu X, Moon SH, Nguyen TA, Shepard MA, Rao PH, Donehower LA, and Kim JY
- Subjects
- Adolescent, Apoptosis drug effects, Cell Line, Tumor, Child, Child, Preschool, Chromosomes, Human, Pair 17, Female, Green Fluorescent Proteins biosynthesis, Humans, In Situ Hybridization, Fluorescence methods, In Situ Nick-End Labeling, Infant, Male, Medulloblastoma genetics, Phosphoprotein Phosphatases genetics, Protein Phosphatase 2C, Retrospective Studies, Transfection methods, Tumor Suppressor Protein p53 metabolism, Gene Expression Regulation, Neoplastic physiology, Medulloblastoma metabolism, Phosphoprotein Phosphatases metabolism
- Abstract
Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-type p53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.
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- 2008
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245. Psychometric and comparative study of an Argentine version of the morningness composite and the early/late preference scales.
- Author
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Gil E, Abdo PL, Rodríguez M, Zanín L, and De Bortoli M
- Subjects
- Adolescent, Adult, Aged, Aging physiology, Argentina, Female, Humans, Male, Middle Aged, Photoperiod, Psychometrics, Seasons, Time Factors, Circadian Rhythm physiology
- Abstract
This study examined the psychometric properties of an Argentine Version of the Morningness Composite (CS) and the Early/Late Preference (PS) Scales. During summer (long photoperiod in Argentina), 304 subjects (69.1% women; mean age=33.64 yrs, SD=14 yrs) completed the scales for the first time. In winter (short photoperiod), 100 of the same individuals (71% women; mean age=37.17 yrs, SD=14.63 yrs) were retested. The total scores ranged within the values reported by previous studies and were independent of gender. Older subjects showed higher morningness scores. The internal consistencies were good (CS=0.86, PS=0.82). Item 7 from the CS and items 7 and 9 from the PS showed low item-scale correlation. Factor analysis produced a three-factor solution for both scales. However, the inconsistency of the evening items suggests that the single-solution may be more acceptable. Test-retest correlations were satisfactory (CS=0.88, PS=0.78), but the two-related-sample test revealed significant differences between test and retest scores, suggesting relative temporal stabilities. Both scales presented similar and acceptable psychometric properties and good correlation, indicating construct validity.
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- 2008
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246. Missense mutations in desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro.
- Author
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Beffagna G, De Bortoli M, Nava A, Salamon M, Lorenzon A, Zaccolo M, Mancuso L, Sigalotti L, Bauce B, Occhi G, Basso C, Lanfranchi G, Towbin JA, Thiene G, Danieli GA, and Rampazzo A
- Subjects
- Adolescent, Adult, Animals, Cells, Cultured, Female, Genetic Carrier Screening, Green Fluorescent Proteins, Humans, Male, Middle Aged, Myocytes, Cardiac, Rats, Transfection, Arrhythmogenic Right Ventricular Dysplasia genetics, Desmocollins genetics, Mutation, Missense
- Abstract
Background: Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue., Methods: Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing. To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells., Results: We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions. In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm., Conclusion: The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.
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- 2007
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247. Regulation of the microsomal prostaglandin E synthase-1 in polarized mononuclear phagocytes and its constitutive expression in neutrophils.
- Author
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Mosca M, Polentarutti N, Mangano G, Apicella C, Doni A, Mancini F, De Bortoli M, Coletta I, Polenzani L, Santoni G, Sironi M, Vecchi A, and Mantovani A
- Subjects
- Animals, Cell Line, Tumor, Cells, Cultured, Enzyme Induction genetics, Enzyme Induction immunology, Humans, Intramolecular Oxidoreductases biosynthesis, Intramolecular Oxidoreductases genetics, Intramolecular Oxidoreductases metabolism, Isoenzymes biosynthesis, Isoenzymes genetics, Isoenzymes metabolism, Macrophages, Peritoneal cytology, Mice, Mice, Inbred C57BL, Prostaglandin-E Synthases, RNA, Messenger metabolism, Cell Polarity, Gene Expression Regulation, Enzymologic, Microsomes enzymology, Neutrophils enzymology, Neutrophils metabolism, Phagocytes physiology
- Abstract
PGs are potent mediators of pain and inflammation. PGE synthases (PGES) catalyze the isomerization of PGH(2) into PGE(2). The microsomal (m)PGES-1 isoform serves as an inducible PGES and is responsible for the production of PGE(2), which mediates acute pain in inflammation and fever. The present study was designed to investigate the regulation of expression of mPGES-1 in polarized phagocytes, which represent central, cellular orchestrators of inflammatory reactions. Here, we report that human peripheral blood monocytes did not express mPGES-1. Exposure to LPS strongly induced mPGES-1 expression. Alternatively activated M2 monocytes-macrophages exposed to IL-4, IL-13, or IL-10 did not express mPGES-1, whereas in these cells, IL-4, IL-13, and to a lesser extent, IL-10 or IFN-gamma inhibited LPS-induced, mPGES-1 expression. It is unexpected that polymorphonuclear leukocytes expressed high basal levels of mPGES-1, which was up-regulated by LPS and down-regulated by IL-4 and IL-13. Induction of mPGES-1 and its modulation by cytokines were confirmed at the protein level and correlated with PGE(2) production. Cyclooxygenase 2 expression tested in the same experimental conditions was modulated in monocytes and granulocytes similarly to mPGES-1. Thus, activated M1, unlike alternatively activated M2, mononuclear phagocytes express mPGES-1, and IL-4, IL-13, and IL-10 tune expression of this key enzyme in prostanoid metabolism. Neutrophils, the first cells to enter sites of inflammation, represent a ready-made, cellular source of mPGES-1.
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- 2007
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248. Overexpressed TP73 induces apoptosis in medulloblastoma.
- Author
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Castellino RC, De Bortoli M, Lin LL, Skapura DG, Rajan JA, Adesina AM, Perlaky L, Irwin MS, and Kim JY
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- Biomarkers, Tumor genetics, Blotting, Western, Cell Line, Tumor, Cell Survival genetics, Cerebellar Neoplasms metabolism, Cerebellar Neoplasms pathology, Child, Child, Preschool, Disease Progression, Flow Cytometry, Follow-Up Studies, Gene Silencing, Humans, Immunohistochemistry, In Situ Nick-End Labeling, Infant, Male, Medulloblastoma metabolism, Medulloblastoma pathology, Prognosis, Retrospective Studies, Reverse Transcriptase Polymerase Chain Reaction, Severity of Illness Index, Time Factors, Apoptosis genetics, Cerebellar Neoplasms genetics, DNA-Binding Proteins genetics, Gene Expression Regulation, Neoplastic, Medulloblastoma genetics, Nuclear Proteins genetics, RNA, Neoplasm genetics, Tumor Suppressor Proteins genetics
- Abstract
Background: Medulloblastoma is the most common malignant brain tumor of childhood. Children who relapse usually die of their disease, which reflects resistance to radiation and/or chemotherapy. Improvements in outcome require a better understanding of the molecular basis of medulloblastoma growth and treatment response. TP73 is a member of the TP53 tumor suppressor gene family that has been found to be overexpressed in a variety of tumors and mediates apoptotic responses to genotoxic stress. In this study, we assessed expression of TP73 RNA species in patient tumor specimens and in medulloblastoma cell lines, and manipulated expression of full-length TAp73 and amino-terminal truncated DeltaNp73 to assess their effects on growth., Methods: We analyzed medulloblastoma samples from thirty-four pediatric patients and the established medulloblastoma cell lines, Daoy and D283MED, for expression of TP73 RNA including the full-length transcript and the 5'-terminal variants that encode the DeltaNp73 isoform, as well as TP53 RNA using quantitative real time-RTPCR. Protein expression of TAp73 and DeltaNp73 was quantitated with immunoblotting methods. Clinical outcome was analyzed based on TP73 RNA and p53 protein expression. To determine effects of overexpression or knock-down of TAp73 and DeltaNp73 on cell cycle and apoptosis, we analyzed transiently transfected medulloblastoma cell lines with flow cytometric and TUNEL methods., Results: Patient medulloblastoma samples and cell lines expressed full-length and 5'-terminal variant TP73 RNA species in 100-fold excess compared to non-neoplastic brain controls. Western immunoblot analysis confirmed their elevated levels of TAp73 and amino-terminal truncated DeltaNp73 proteins. Kaplan-Meier analysis revealed trends toward favorable overall and progression-free survival of patients whose tumors display TAp73 RNA overexpression. Overexpression of TAp73 or DeltaNp73 induced apoptosis under basal growth conditions in vitro and sensitized them to cell death in response to chemotherapeutic agents., Conclusion: These results indicate that primary medulloblastomas express significant levels of TP73 isoforms, and suggest that they can modulate the survival and genotoxic responsiveness of medulloblastomas cells.
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- 2007
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249. Influence of estrogens and antiestrogens on the expression of selected hormone-responsive genes.
- Author
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Sismondi P, Biglia N, Ponzone R, Fuso L, Scafoglio C, Cicatiello L, Ravo M, Weisz A, Cimino D, Altobelli G, Friard O, and De Bortoli M
- Subjects
- Breast Neoplasms pathology, Carcinoma pathology, Down-Regulation, Female, Fulvestrant, Gene Expression Regulation, Neoplastic drug effects, Gene Expression Regulation, Neoplastic genetics, Humans, Tamoxifen pharmacology, Tumor Cells, Cultured, Up-Regulation, Breast Neoplasms genetics, Carcinoma genetics, Estradiol analogs & derivatives, Estradiol pharmacology, Estrogen Antagonists pharmacology, Raloxifene Hydrochloride pharmacology, Selective Estrogen Receptor Modulators pharmacology, Tamoxifen analogs & derivatives
- Abstract
Estrogen exerts a primary regulatory role on a wide variety of physiological processes in different tissues and organs. Agonistic ad antagonistic compounds are widely used in human health and, therefore, a deep understanding of their mechanisms of action at the molecular level is mandatory. The effect of 17beta-estradiol and three antiestrogenic drugs, comprising two selective estrogen receptor modulator (SERM, 4-OH-tamoxifen, Raloxifene) and the pure antiestrogen ICI 182,780, on genome-wide gene expression levels was evaluated in breast carcinoma cell lines by DNA microarray analysis. Different clusters of genes, showing specific coregulation patterns, were found. First, several groups of genes displaying temporal-specific up- or down-regulation were characterized. Second, clusters of genes responding to different antiestrogenic drugs in either antagonstic or agonistic fashion, were found. Genes responding specifically to antiestrogens, but not to estrogen, were also identified. In addition, each individual compound exhibited a very specific gene regulation. Bioinformatic analysis was applied to the regulatory sequences of different groups of genes and confirmed that specific pathways and secondary responses are activated at each temporal point and in response to different compounds. Our results underline the complexity of genomic responses to estrogen in breast cancer cells and strongly suggest that the molecular characterization of estrogen agonists and antagonists used in human therapy should be carefully studied.
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- 2007
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250. Diacylglycerol kinase is required for HGF-induced invasiveness and anchorage-independent growth of MDA-MB-231 breast cancer cells.
- Author
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Filigheddu N, Cutrupi S, Porporato PE, Riboni F, Baldanzi G, Chianale F, Fortina E, Piantanida P, De Bortoli M, Vacca G, Graziani A, and Surico N
- Subjects
- Cell Culture Techniques, Cell Line, Tumor, Cell Movement, Diacylglycerol Kinase analysis, Humans, Neoplasm Invasiveness, Receptors, Estrogen analysis, Receptors, Estrogen metabolism, Breast Neoplasms enzymology, Breast Neoplasms pathology, Diacylglycerol Kinase metabolism, Hepatocyte Growth Factor pharmacology
- Abstract
Background: Estrogen receptor (ER)-negative breast cancers have a worse prognosis than ER-positive cancers, being more aggressive and overexposed to stimuli leading to their progression. Hepatocyte growth factor (HGF) has been associated with proliferation, migration and invasion of tumor cells, and several tumors, including those of breast cancer, produce HGF and overexpress its receptor. Diacylglycerol kinases (Dgks), which phosphorylate diacylglycerol to phosphatidic acid, are key regulators of cell signaling. Our research was focused on their role in HGF-induced invasion of MDA-MB-231 cells, a model of ER-negative breast cancer., Materials and Methods: Dgk activity was evaluated with a kinase assay, MDA-MB-231 cell invasion via culturing of cells in matrigel-coated transwells, and anchorage-independent growth was assessed using a soft agar assay., Results: HGF induces Dgk activation in MDA-MB-231 cells that is required for cell invasiveness. Moreover, Dgks are involved in MDA-MB-231 anchorage-independent growth., Conclusion: Dgks could be a target for ER-negative breast cancer therapy.
- Published
- 2007
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