Biomedical subjects

Carla Cicchini

Publications and source records attributed to Carla Cicchini.

5 recordsLinked to original sources

PubMed2006

Snail controls differentiation of hepatocytes by repressing HNF4alpha expression.

Epithelial-to-mesenchymal transition (EMT) is a coordinated process, occurring both during morphogenesis and tumor progression, that allows epithelial cells to dissociate from initial contacts and migrate to secondary sites. The transcriptional repressors of the Snail family induce EMT in different epithelial cell lines and their expression is strictly correlated with EMT during the development and progression of carcinomas. We have previously shown that EMT in hepatocytes correlates with the downregulation of hepatic differentiation key factors HNFs (hepatocyte nuclear factors), and in particular of HNF4alpha. Here, we demonstrate that Snail overexpression is sufficient (i) to induce EMT in hepatocytes with conversion of morphology, downregulation of several epithelial adhesion molecules, reduction of proliferation and induction of matrix metalloproteinase 2 expression and, (ii) most relevantly, to repress the transcription of the HNF4alpha gene through a direct binding to its promoter. These finding demonstrate that Snail is at the crossroads of the regulation of EMT in hepatocytes by a dual control of epithelial morphogenesis and differentiation.

Animals↗

PubMed2004

Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice.

To assess the effects of constitutive hepatitis C virus (HCV) gene expression on liver, transgenic mice carrying the entire HCV open reading frame inserted in the alpha1 antitrypsin (A1AT) gene were generated. Expression of A1AT/HCV mRNA was found to be mainly limited to perivascular areas of the liver as indicated by in situ hybridization analysis. HCV core protein was detected in Western blots of liver extracts, whereas the expression of E2, NS3 and NS5 proteins was revealed by immunostaining of liver samples using HCV-specific antisera. Histological analysis of HCV transgenic mice showed that these animals develop extensive steatosis, but very little necrosis of liver tissue. Moreover, a consistent T cell infiltrate and a slight hepatocyte proliferation were observed. Phenotypic analysis of cells infiltrating the liver indicated that recruitment and/or expansion of residing CD8(+), NK, NKT and gammadelta T cells occurred in transgenic animals. Among these cells, a large fraction of CD8(+) T lymphocytes released mainly IL-10 and, to a lesser extent, IFN-gamma upon mitogenic stimulation in vitro. Furthermore, both intrahepatic lymphocytes and splenocytes did not produce cytokines in response to HCV antigens. Thus, these data indicate that constitutive expression of HCV proteins may be responsible for intrahepatic lymphocyte recruitment in absence of viral antigen recognition. This response is likely to be driven by virus-induced cellular factors and may play a significant role in the immunopathology of chronic HCV infection and liver disease.

Animals↗

PubMed2002

Searching for DNA-protein interactions by lambda phage display.

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

Adenosine Triphosphatases↗

PubMed2002

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands.

cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody-antigen, protein- protein and DNA-protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.

Amino Acid Sequence↗

PubMed2002

Synergy between truncated c-Met (cyto-Met) and c-Myc in liver oncogenesis: importance of TGF-beta signalling in the control of liver homeostasis and transformation.

The c-Met tyrosine kinase receptor and its ligand, Hepatocyte Growth Factor/ Scatter Factor, have been implicated in human cancer. We have previously described that the transgenic expression of a truncated form of human c-Met (cyto-Met) in the liver confers resistance to several apoptotic stimuli. Here we show the impact of cyto-Met expression on liver proliferation and transformation. Despite a sixfold increase of hepatocyte proliferation, adult transgenic livers displayed normal size and architecture. We present evidence showing that activation of TGF-beta1 signalling controls the liver mass in cyto-Met mice. The oncogenic potential of cyto-Met was further assessed in the context of c-Myc-induced hepatocarcinogenesis, using WHV/c-Myc transgenic mice. Co-expression of cyto-Met and c-Myc further enhanced hepatocyte proliferation and caused a dramatic acceleration of the Myc-induced tumorigenesis, leading to the emergence of hepatocarcinomas in 3-4-month-old animals. Importantly, the TGF-beta receptor type II expression was strongly downregulated in most tumours, indicating that impairment of TGF-beta1-mediated growth inhibition plays a major role in accelerated neoplastic development. The strong potential of cyto-Met for oncogenic cooperation without direct transforming activity designates cyto-Met mice as an ideal tool for studying the early steps of multistage hepatocarcinogenesis and for identification of prognostic markers of transformation.

Animals↗