Biomedical subjects

Stefano Volinia

Publications and source records attributed to Stefano Volinia.

36 records · Page 2Linked to original sources

PubMed2004

GOAL: automated Gene Ontology analysis of expression profiles.

One of the most common problems encountered while deciphering results from expression profiling experiments is in relating differential expression of genes to molecular functions and cellular processes. A second important problem is that of comparing experiments performed by different labs using different microarray platforms, or even unrelated techniques. Gene Ontology (GO) is now used to describe biological features, since GO terms are associated with genes, to overcome the apparent distance between expression profiles and biological comprehension. Here we describe the development, implementation and use of GOAL (Gene Ontology Automated Lexicon), a web-based application for the identification of functions and processes regulated in microarray and SAGE (serial analysis of gene expression) experiments. We applied GOAL to a range of experimental datasets related to different biological problems, including cancer and the cell cycle. By using GOAL, reported and novel relevant processes were identified in a number of experiments by our collaborators and by us. Different datasets could also be compared with each other to define conserved functional modules. GOAL allows a seamless and high-level analysis of expression profiles and is implemented as a free WWW resource (http://microarrays.unife.it).

Data Interpretation, Statistical↗

PubMed2004

Spectrum of hemojuvelin gene mutations in 1q-linked juvenile hemochromatosis.

Juvenile or type 2 hemochromatosis (JH) is transmitted as a recessive trait that leads to severe iron overload and organ damage typically before age 30 years. Linkage to a locus on chromosome 1q has been found in most patients with JH. The recently identified causal gene encodes hemojuvelin, a protein with a proposed crucial role in iron metabolism. A second, rare type of JH, with clinical expression identical to the 1q-linked form, is due to inactivation of hepcidin, the key regulator of iron homeostasis. Here we report the spectrum of mutations of the hemojuvelin gene (HJV) in 34 patients who did not show hepcidin mutations. This represents the largest cohort of patients with JH collected worldwide. We identified 17 different (16 novel) mutations of HJV, both at the homozygous and at the compound heterozygous state. Mutations either generate premature termination codons or were missense substitutions, affecting highly conserved residues, relevant to the protein structure and/or function.

Adolescent↗

PubMed2004

RNA expression induced by cisplatin in an organ of Corti-derived immortalized cell line.

Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.

Acetylcysteine↗

PubMed2004

An expression atlas of connexin genes in the mouse.

Connexin genes are involved in several human diseases such as hearing and dermatological and peripheral nerve disorders. Connexins are protein units of gap junctions and form homotypic, heterotypic, or heteromeric complexes known as connexons. Data on the expression patterns of members of this family are partial and fragmentary. We therefore cloned all the identifiable murine homologs of human CONNEXIN genes and analyzed their expression patterns in embryonic and neonatal mouse tissues. We found that connexins are preferentially expressed in tissues derived from ectoderm and/or endoderm. Our data provide a comprehensive and detailed atlas of expression of connexin genes and in some cases suggest possible interactions of proteins that are coexpressed in the same tissue. Knowledge of temporal and spatial distribution of connexins also allows the identification of candidate genes for human diseases and provides important insight into mechanisms that lead to human disorders due to mutations in CONNEXIN genes.

Animals↗

PubMed2004

Zirconium oxide: analysis of MG63 osteoblast-like cell response by means of a microarray technology.

Zirconium oxide ceramics have outstanding mechanical properties, a high biocompatibility and a high resistance to scratching. Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured on zirconium oxide discs (Cercon, Degussa Dental, Hanau, Germany) several genes whose expression was significantly up or down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport and (c) cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of a zirconium oxide surface. They can be relevant to better understand the molecular mechanism of biocompatibility and as a model for comparing other materials.

Animals↗

PubMed2004

Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology.

Surface implant modifications have been shown to have a relevant importance in modifying cell response. Expression profiling by DNA microarray is a new molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) on new implant surface (nanoPORE, Out-Link, Sweden and Martina, Due Carrare, Padova, Italy), several genes whose expressions were significantly down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport (c) apoptosis and cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of an implant surface. They can be relevant to better understand the molecular mechanism of implant osseointegration and as a model for comparing other materials.

Apoptosis↗

PubMed2004

P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology.

BACKGROUND: P-15 is an analog of the cell-binding domain of collagen. P-15 has been shown to facilitate physiological processes in a way similar to collagen; to serve as an anchorage for cells; and to promote the binding, migration, and differentiation of cells. METHODS: Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cell line (MG-63) cultured with P-15 several genes whose expression was significantly up- or downregulated. RESULTS: The differentially expressed genes cover a broad range of functional activities: 1) signaling transduction, 2) differentiation, 3) apoptosis, and 4) cell-cycle regulation. It was also possible to detect some genes whose function is unknown. CONCLUSIONS: The data reported are, to our knowledge, the first genetic portrait of P-15 effects. They can help us to better understand the molecular mechanism of osteogenesis and can serve as a model for comparing different cell cultures and/or other materials with similar effect.

Apoptosis↗

PubMed2004

Genes transcriptionally modulated by interferon alpha2a correlate with the cytokine activity.

BACKGROUND AND OBJECTIVES: Interferon alpha2a (IFNalpha2a) mediates important antiviral, antiproliferative and immunomodulatory responses and is employed in the treatment of human diseases, including chronic myelogenous leukemia. Here, we report the IFNalpha2a-dependent expression profiles of three malignant cell lines derived from liver, lymphocytes and muscle. DESIGN AND METHODS: The experiments were performed in the presence of cycloheximide, thus our results exclusively reflect direct transcriptional modulation. The short exposure time i.e. 5 hours evidences only the early events, excluding the effects of complex phenotypic changes on the expression. RESULTS: Our findings indicate that IFNalpha2a rapidly up-regulates the expression of STAT1, STAT2 and ISGF3G genes. This activity should result in the amplification of the cellular response to the cytokine. Moreover, IFNalpha2a directly modulates the expression of: (i) important transcriptional factors, e.g. IRF1 and IRF7 which control pivotal cellular events, and (ii) enzymes involved in the IFNalpha2a-dependent antiviral and apoptotic response. Interestingly, we showed that the cytokine induces transcriptional expression of Sjögren's syndrome antigen A1, a protein involved in several autoimmune diseases. INTERPRETATION AND CONCLUSIONS: The observed changes induced by IFNalpha2a could be related to the development of autoimmune syndromes observed during IFNalpha2a treatment. A number of genes transcriptionally regulated by the cytokine have been identified for the first time; these might represent additional effectors of IFNalpha2a activity.

Apoptosis Regulatory Proteins↗

PubMed2003

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells.

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.

Aphidicolin↗

PubMed2003

Genetic profile of clear cell odontogenic carcinoma.

In the head and neck region, clear cell tumors are usually derived from salivary glands, odontogenic tissues, and metastasis. The World Health Organization has classified clear cell odontogenic tumor among benign tumors, but it is now recognized as a more sinister lesion, and current opinion is that it should be designated as a carcinoma. It is characterized by aggressive growth, recurrences, and metastasis. By using complementary DNA microarrays, several genes in clear cell odontogenic tumor were identified that are differentially regulated when compared with non-tumor tissue. In conclusion, the first genetic profiling of clear odontogenic carcinoma is reported. DNA microarrays can potentially help in identifying some genes whose products could be disease-specific targets for cancer therapy as well as a tool for better classifying odontogenic tumor.

Adenocarcinoma, Clear Cell↗

PubMed2003

Analysis of osteoblast-like MG63 cells' response to a rough implant surface by means of DNA microarray.

Several features of the implant surface, such as composition, topography, roughness, and energy, play a relevant role in implant integration with bone. Little is known about the structural and chemical surface properties that may influence biological responses. Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19200 genes, we identified several genes whose expression was significantly down-regulated in osteoblast-like cell line MG63 on a new implant surface (titanium pull spray superficial [TPSS] surface, Oralplant, Cordenons, PN, Italy). The differentially expressed genes cover a broad range of functional activities: (1) signaling transduction, (2) translation, (3) cell cycle regulation, (4) structural and metabolic functions, and (5) apoptosis. It was also possible to detect some genes whose functions are unknown. The data reported can be relevant to better understand the role of the type of surface on the molecular mechanism of implant osseointegration and as a model for comparing other materials.

Apoptosis↗

PubMed2002

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells.

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.

Blotting, Western↗

PubMed2002

The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults.

The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifications and association with other proteins have been implicated in the regulation of its stability and transcriptional activities. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase, which regulates the function of many proteins involved in cell cycle control and apoptosis. The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates conformational changes in p53, enhancing its transactivation activity. Stabilization of p53 is impaired in UV-treated Pin1(-/-) cells owing to its inability to efficiently dissociate from Mdm2. As a consequence, a reduced p53-dependent response was detected in Pin1(-/-) cells, and this correlates with a diminished transcriptional activation of some p53-regulated genes. Our results suggest that, following stress-induced phosphorylation, p53 needs to form a complex with Pin1 and to undergo a conformational change to fulfil its biological roles.

Amino Acid Motifs↗

PubMed2002

Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P.

Animals↗

PubMed2002

Expression profiles of craniosynostosis-derived fibroblasts.

BACKGROUND: Craniosynostosis syndromes, a group of connective disorders characterized by abnormalities in vault osteogenesis and premature fusion of bone sutures, are associated with point mutations in FGF receptor family members. The cellular phenotype is characterized by abnormal extracellular matrix turnover. MATERIAL AND METHODS: We used primary cultures of periosteal fibroblasts derived from two different craniosynostosis syndromes, the Apert and Crouzon syndromes. The FGFR2 third immunoglobulin-like domain and its flanking linker regions were analyzed for mutation. DNA microarrays containing 19,200 cDNAs were used to study the gene expression profiles of Apert and Crouzon fibroblasts. The pathologic cells were compared to wild-type human periosteal fibroblasts. RESULTS: The P253R missense mutation and the G338R mutation were observed in Apert and Crouzon fibroblasts, respectively. The genetic profiles, as evaluated by DNA microarrays, yielded different clusters of expressed sequence tag (ESTs) expression within the experiment. Expression profiles from craniosynostosis-derived fibroblasts differ from those of wild-type fibroblasts (288 human ESTs, p< 0.01, pFDR = 0.12). Furthermore, two ESTs clusters discriminate the Crouzon from Apert fibroblasts. The differentially expressed genes cover a broad range of functional activities, including (1) bone differentiation, (2) cell-cycle regulation, (3) apoptotic stimulation, and (4) signaling transduction, cytoskeleton, and vesicular transport. CONCLUSIONS: The transcriptional program of craniosynostosis fibroblasts differs from that of wild-type fibroblasts. Expression profiles of Crouzon and Apert fibroblasts can also be distinguished by two EST expression clusters, thus hinting at a different genetic background.

Acrocephalosyndactylia↗

PubMed2002

Identification of differentially expressed genes in human salivary gland tumors by DNA microarrays.

Differentially expressed genes among different benign and malignant salivary gland tumors were identified by use of cDNA microarrays containing 19,000 human expressed sequence tags. Tumors were classified by using a subset of 486 genes. Benign Warthin's tumor and pleomorphic adenoma showed very distinctive gene expression patterns. One hundred and thirty-three genes differentiated the single malignant clear cell carcinoma from non-tumor salivary glands (P < 0.01), whereas only 16 genes separated it from the highly related benign pleomorphic adenoma (P < 0.01). Fifty-seven cDNAs were associated with mucoepidermoid carcinoma (P < 0.01). The identified genes might help to disclose the molecular mechanisms and processes underlying malignant salivary gland tumors.

DNA, Complementary↗

PubMedDate not supplied

Gene expression analysis in HBV transgenic mouse liver: a model to study early events related to hepatocarcinogenesis.

Hepatitis B virus (HBV) is one of the major etiological factors responsible for the development of hepatocellular carcinoma (HCC). We used a transgenic mouse, containing HBV sequences, as a model system to unravel the molecular mechanisms of hepatocarcinogenesis induced by HBV. We chose this animal model because it consistently develops liver cancer after intermediate steps that mimic the natural history of HBV infection in humans. In this study, we focus our attention on the early events leading to liver cancer. We compared the gene expression profile of 3-month-old transgenic mice with that of 3-month-old wild-type (wt) animals. In the transgenic mouse, microarray data analysis showed a total of 45 significantly differentially expressed genes, 25 highly expressed (fold change > or =2; P = 0.0025), and 20 downregulated (fold change < or =0.5; P = 0.0025). These genes belong to several different functional categories such as the regulation of immunological response, transcription, intracellular calcium ion mobilization, regulation of cell cycle and proliferation, NF-kappab signal transduction cascades, and apoptosis. In particular, the upregulation of the antiapoptotic gene NuprI and the downregulation of the proapoptotic gene Bnip3 were found. This observation was supported by an in vitro apoptosis assay that showed downregulation of apoptosis in hepatocytes of HBV transgenic mouse compared with wt mice treated with staurosporine. In conclusion, our experimental approach allowed identification of new genes modulated by HBV and showed that the apoptotic process was deregulated in transgenic mouse hepatocytes. These data shed light on one possible mechanism by which HBV induces hepatocarcinogenesis.

Animals↗

PubMedDate not supplied

Nanotechnologies and microchips in genetic diseases.

Microarrays or microchips represent a new area of high technology, which will completely change the methodological approach to basic research and clinical diagnostics. This technology can be used for genotyping, expression profiling and proteome analysis. Genetics and molecular medicine have an expanding need for rapid genotyping, mutational analysis and DNA re-sequencing technologies, i.e. microarrays that have a clear potential for miniaturization, parallelization, and automation and enable high-throughput screening. Expression profiling technology is a new tool for investigating expression patterns, identifying new disease genes either for monogenic disorders or for complex traits, identifying new functional and cellular relationships and identifying new pathways and possible related drugs. This technology has been successfully applied to the study of complex traits, i.e. cardiovascular diseases, cancer and type II diabetes, providing new insights into possible pathogenetic mechanisms and new therapeutical approaches. Finally, microarray can further improve proteome analysis. This review discusses these points.

Gene Expression Profiling↗