27 results on '"Carrozzino, F"'
Search Results
2. Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation
- Author
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Benelli, R, Morini, M, Carrozzino, F, Ferrari, N, Minghelli, S, Santi, L, Cassatella, M, Noonan, D, Albini, A, Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, Albini A, Benelli, R, Morini, M, Carrozzino, F, Ferrari, N, Minghelli, S, Santi, L, Cassatella, M, Noonan, D, Albini, A, Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, and Albini A
- Abstract
Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GRO alpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GRO alpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.
- Published
- 2002
3. Generation of expression plasmids for angiostatin, endostatin and TIMP- 2 for cancer gene therapy
- Author
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Indraccolo, S, Minuzzo, S, Gola, E, Habeler, W, Carrozzino, F, Noonan, Douglas, Albini, A, Santi, L, Amadori, A, and CHIECO BIANCHI, L.
- Published
- 1999
4. Generation of angiostatin expression vectors for antiangiogenic gene therapy of Kaposi sarcoma
- Author
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Gola, E., Indraccolo, Stefano, Roni, V., Habeler, W., Minuzzo, S., Rosato, Antonio, Carrozzino, F., Morini, M., Bisacchi, D., Noonan, D., Chieco Bianchi, L., and Albini, A.
- Published
- 1999
5. Generation of Expression Plasmids for Angiostatin, Endostatin and Timp-2 for Cancer Gene Therapy
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Indraccolo, S., Minuzzo, S., Gola, E., Habeler, W., Carrozzino, F., Noonan, D., Albini, A., Santi, L., Amadori, A., and Chieco-Bianchi, L.
- Abstract
Antiangiogenic therapy may represent a promising approach to cancer treatment. Indeed, the efficacy of endogenous angiogenesis inhibitors, including angiostatin, endostatin and TIMPs, has been demonstrated in many types of solid tumors in animal models. In view of the possible problems associated with long-term administration of inhibitors as recombinant proteins, we propose their delivery as nucleic acids through a gene therapy approach. To this end, eukaryotic expression constructs for murine angiostatin and endostatin as well as human TIMP-2 were generated, and characterized in vitro. All constructs carry the relevant cDNAs under the control of the strong HCMV promoter/enhancer, and cleavable leader signals to allow protein secretion. Expression of the angiogenesis inhibitors was detected by in vitro transcription/translation experiments as well as transfection of 293T cells, followed by Western blotting (WB) or radioimmunoprecipitation analysis of both cell lysates and supernatants (SNs). These constructs might be used for in vivo intramuscular delivery of plasmid DNA and as a set of reagents for the development of retroviral as well as adeno-associated viral (AAV) vectors expressing angiogenesis inhibitors.
- Published
- 1999
- Full Text
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6. Two pathways for base excision repair in mammalian cells.
- Author
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Frosina, G, Fortini, P, Rossi, O, Carrozzino, F, Raspaglio, G, Cox, L S, Lane, D P, Abbondandolo, A, and Dogliotti, E
- Abstract
Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.
- Published
- 1996
7. Repair of abasic sites by mammalian cell extracts
- Author
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Frosina, G, Fortini, P, Rossi, O, Carrozzino, F, Abbondandolo, A, and Dogliotti, E
- Abstract
Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.
- Published
- 1994
- Full Text
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8. Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi's sarcoma cells
- Author
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Indraccolo, S., Morini, M., Gola, E., Carrozzino, F., Habeler, W., Minghelli, S., Santi, L., Chieco-Bianchi, L., Cao, Y., Albini, A., and Douglas Noonan
- Subjects
Chemotaxis ,Genetic Vectors ,Plasminogen ,Transfection ,Peptide Fragments ,Cell Line ,Mice ,Cell Movement ,Culture Media, Conditioned ,Tumor Cells, Cultured ,Animals ,Humans ,Angiostatins ,Sarcoma, Kaposi ,Cell Division - Abstract
Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi's sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.
9. Erratum: Generation of Expression plasmids for angiostatin, endostatin and TIMP-2 for cancer gene therapy (The International Journal of Biological Markers (1999) 14, 4 (251-256))
- Author
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Carrozzino, F., Noonan, D. M., Adriana Albini, and Santi, L.
10. 137 UNSATURATED FATTY ACIDS PROMOTE HEPATOMA PROLIFERATION AND PROGRESSION THROUGH DOWNREGULATION OF THE TUMOR SUPPRESSOR PTEN
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Vinciguerra, M., Carrozzino, F., Carlone, S., Montesano, R., Benelli, R., and Foti, M.
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- 2009
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11. Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation
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Monica Morini, Fabio Carrozzino, Marco A. Cassatella, Douglas M. Noonan, Leonardo Santi, Roberto Benelli, Adriana Albini, Simona Minghelli, Nicoletta Ferrari, Benelli, R, Morini, M, Carrozzino, F, Ferrari, N, Minghelli, S, Santi, L, Cassatella, M, Noonan, D, and Albini, A
- Subjects
Chemokine ,Angiogenesis ,Neutrophils ,Receptors, Cell Surface ,Biochemistry ,Receptors, Interleukin-8B ,Neovascularization ,Chemokine receptor ,Mice ,Cell Movement ,Genetics ,medicine ,Animals ,CXC chemokine receptors ,Molecular Biology ,Angiostatins ,Neutrophils cellular target angiostatin: angiogenesis and inflammation ,Inflammation ,Angiostatin ,biology ,Dose-Response Relationship, Drug ,Neovascularization, Pathologic ,Chemistry ,Interleukin-8 ,Chemotaxis ,Plasminogen ,Angiomotin ,Peptide Fragments ,Cancer research ,biology.protein ,medicine.symptom ,Biotechnology - Abstract
Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GRO alpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GRO alpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.
- Published
- 2002
12. Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression.
- Author
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Carrozzino F, Pugnale P, Féraille E, and Montesano R
- Subjects
- Animals, Anthracenes pharmacology, Butadienes pharmacology, Cell Line, Dogs, Female, Imidazoles pharmacology, MAP Kinase Kinase 4 antagonists & inhibitors, Mammary Glands, Animal cytology, Membrane Proteins genetics, Mice, Nitriles pharmacology, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, Epithelium physiology, MAP Kinase Kinase 4 metabolism, Membrane Proteins metabolism, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.
- Published
- 2009
- Full Text
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13. Unsaturated fatty acids promote hepatoma proliferation and progression through downregulation of the tumor suppressor PTEN.
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Vinciguerra M, Carrozzino F, Peyrou M, Carlone S, Montesano R, Benelli R, and Foti M
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- Animals, Apoptosis drug effects, Base Sequence, Carcinogens toxicity, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, DNA Primers genetics, Down-Regulation drug effects, Hepatocytes drug effects, Hepatocytes metabolism, Hepatocytes pathology, Humans, Liver Neoplasms, Experimental genetics, Liver Neoplasms, Experimental pathology, Mice, Mice, Nude, Neoplasm Transplantation, PTEN Phosphohydrolase antagonists & inhibitors, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase metabolism, RNA, Small Interfering genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Risk Factors, Transfection, Transplantation, Heterologous, Fatty Acids, Unsaturated toxicity, Liver Neoplasms, Experimental etiology
- Abstract
Background/aims: The impact of dietary fatty acids on the development of cancers is highly controversial. We recently demonstrated that unsaturated fatty acids trigger the downregulation of the tumor suppressor PTEN through an mTOR/NF-kappaB-dependent mechanism in hepatocytes. In this study, we investigated whether unsaturated fatty acids promote hepatoma progression by downregulating PTEN expression., Methods: The effects of fatty acids and PTEN-specific siRNAs on proliferation, invasiveness and gene expression were assessed using HepG2 hepatoma cells. The tumor promoting activity of unsaturated fatty acids was evaluated in vivo using HepG2 xenografts in nude mice., Results: Incubation of HepG2 cells with unsaturated fatty acids, or PTEN-specific siRNAs, increased cell proliferation, cell migration and invasiveness, and altered the expression of genes involved in inflammation, epithelial-to-mesenchymal transition and carcinogenesis. These effects were dependent on PTEN expression levels and were prevented by mTOR and NF-kappaB inhibitors. Consistent with these data, the development and size of subcutaneous HepG2-derived tumors in nude mice xenografts were dramatically increased when mice were fed with an oleic acid-enriched diet, even in the absence of weight gain., Conclusions: These data demonstrate that dietary unsaturated fatty acids promote hepatoma progression by reducing the expression of the tumor suppressor PTEN.
- Published
- 2009
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14. cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.
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Montesano R, Ghzili H, Carrozzino F, Rossier BC, and Féraille E
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- Animals, Cell Line, Kidney Diseases, Cystic pathology, Mice, Cell Culture Techniques, Chlorides metabolism, Cyclic AMP metabolism, Kidney Tubules, Collecting cytology
- Abstract
Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.
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- 2009
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15. Induction of CXCL1 by extracellular matrix and autocrine enhancement by interleukin-1 in rat pancreatic beta-cells.
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Ribaux P, Ehses JA, Lin-Marq N, Carrozzino F, Böni-Schnetzler M, Hammar E, Irminger JC, Donath MY, and Halban PA
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- Animals, Cell Movement drug effects, Cells, Cultured, Chemokine CXCL1 metabolism, Chemokine CXCL1 pharmacology, Culture Media, Conditioned pharmacology, Cytokines genetics, Gene Expression Regulation drug effects, Granulocytes cytology, Granulocytes drug effects, Insulin-Secreting Cells metabolism, Male, Models, Biological, Rats, Rats, Wistar, Autocrine Communication drug effects, Chemokine CXCL1 genetics, Extracellular Matrix physiology, Insulin-Secreting Cells drug effects, Interleukin-1 pharmacology
- Abstract
As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.
- Published
- 2007
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16. Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells.
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Montesano R, Carrozzino F, and Soulié P
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- Animals, Blotting, Northern, Cells, Cultured, Culture Media, Enzyme Inhibitors pharmacology, Epithelial Cells drug effects, Female, Mammary Glands, Animal cytology, Mammary Glands, Animal drug effects, Matrix Metalloproteinases metabolism, Mice, Morphogenesis, Nucleic Acid Hybridization, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, Epithelial Cells cytology, Mammary Glands, Animal growth & development, Transforming Growth Factor beta1 physiology
- Abstract
Background: Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS)., Results: Initial experiments showed that the tubulogenesis-inducing activity of FCS was markedly increased by heating (70 degrees C) or transient acidification to pH3. We therefore hypothesized that the tubulogenic agent was transforming growth factor-beta (TGF-beta), a cytokine that is present in serum in latent form and can be activated by heat or acid treatment. We found indeed that the tubulogenic activity of acidified FCS is abrogated by addition of either SB-431542, a selective inhibitor of the TGF-beta type I receptor, or a neutralizing antibody to TGF-beta-1. On the other hand, addition of low concentrations (20-100 pg/ml) of exogenous TGF-beta-1 recapitulated the effect of acidified FCS in inducing morphogenesis of hollow tubes. In contrast, higher concentrations of TGF-beta-1 induced the formation of thin cellular cords devoid of a detectable lumen. To gain insight into the mechanisms underlying TGF-beta-1-induced tube formation, we assessed the potential role of matrix metalloproteinases (MMPs). By western blot and gelatin zymography, we observed a dose-dependent increase in MMP-9 upon TGF-beta-1 treatment. Tube formation was suppressed by a synthetic broad-spectrum metalloproteinase inhibitor, by recombinant tissue inhibitor of metalloproteinases-2 (TIMP-2) and by a selective inhibitor of MMP-9, indicating that this morphogenetic process requires the activity of MMP-9., Conclusion: Altogether, our results provide evidence that, at low concentrations, TGF-beta-1 promotes MMP-dependent branching tubulogenesis by mammary epithelial cells in vitro, and suggest that it plays a similar role during mammary gland development in vivo.
- Published
- 2007
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17. Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.
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Carrozzino F, Soulié P, Huber D, Mensi N, Orci L, Cano A, Féraille E, and Montesano R
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- Animals, Cell Line, Dogs, Gene Expression Regulation drug effects, Mice, Snail Family Transcription Factors, Tetracycline pharmacology, Transcription Factors biosynthesis, Gene Expression Regulation physiology, Ion Transport physiology, Tight Junctions metabolism, Transcription Factors physiology
- Abstract
Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.
- Published
- 2005
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18. Tumour necrosis factor alpha confers an invasive, transformed phenotype on mammary epithelial cells.
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Montesano R, Soulié P, Eble JA, and Carrozzino F
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- Animals, Cell Line, Collagen metabolism, Epithelial Cells cytology, Epithelial Cells metabolism, Extracellular Matrix metabolism, Integrin alpha2beta1 metabolism, Mammary Glands, Animal cytology, Mammary Glands, Animal metabolism, Metalloproteases antagonists & inhibitors, Metalloproteases metabolism, Mice, Phenotype, Protease Inhibitors pharmacology, Receptors, Tumor Necrosis Factor, Type I metabolism, Cell Movement drug effects, Cell Proliferation drug effects, Epithelial Cells drug effects, Mammary Glands, Animal drug effects, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor alpha (TNF-alpha) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-alpha abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-alpha induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-alpha-induced 3D scattering. TNF-alpha also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the alpha2 integrin subunit. Addition of a blocking antibody to beta1-integrin or of rhodocetin (a specific alpha2beta1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and alpha2beta1 integrin in the invasive response of 31EG4-2A4 cells to TNF-alpha. We propose that the biological activities described in this study contribute to the ability of TNF-alpha to promote tumour progression and cancer-cell dissemination.
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- 2005
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19. Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.
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Soulié P, Carrozzino F, Pepper MS, Strongin AY, Poupon MF, and Montesano R
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- Animals, Cell Line, Dogs, Epithelial Cells pathology, Humans, Matrix Metalloproteinase 14, Matrix Metalloproteinases, Membrane-Associated, Mice, Mice, Nude, Neoplasm Invasiveness, Transplantation, Heterologous, Metalloendopeptidases physiology, Neoplasms, Experimental etiology
- Abstract
Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.
- Published
- 2005
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20. Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation.
- Author
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Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, and Albini A
- Subjects
- Angiostatins, Animals, Cell Movement drug effects, Dose-Response Relationship, Drug, Inflammation prevention & control, Interleukin-8 metabolism, Interleukin-8 pharmacology, Mice, Neovascularization, Pathologic prevention & control, Neutrophils cytology, Neutrophils metabolism, Receptors, Cell Surface metabolism, Receptors, Interleukin-8B metabolism, Neutrophils drug effects, Peptide Fragments pharmacology, Plasminogen pharmacology
- Abstract
Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GROalpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GROalpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.
- Published
- 2002
- Full Text
- View/download PDF
21. Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi's sarcoma cells.
- Author
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Indraccolo S, Morini M, Gola E, Carrozzino F, Habeler W, Minghelli S, Santi L, Chieco-Bianchi L, Cao Y, Albini A, and Noonan DM
- Subjects
- Angiostatins, Animals, Cell Division, Cell Line, Cell Movement drug effects, Chemotaxis drug effects, Culture Media, Conditioned pharmacology, Genetic Vectors genetics, Humans, Mice, Peptide Fragments genetics, Plasminogen genetics, Sarcoma, Kaposi genetics, Transfection, Tumor Cells, Cultured, Peptide Fragments physiology, Plasminogen physiology, Sarcoma, Kaposi pathology
- Abstract
Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi's sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.
- Published
- 2001
22. Analysis of repair of abasic sites in early onset breast cancer patients.
- Author
-
Rossi O, Carrozzino F, Cappelli E, Carli F, and Frosina G
- Subjects
- Adult, Age of Onset, Binding Sites genetics, Breast Neoplasms enzymology, Breast Neoplasms pathology, Carbon-Oxygen Lyases metabolism, Carcinoma, Ductal, Breast enzymology, Carcinoma, Ductal, Breast pathology, DNA Replication genetics, DNA-(Apurinic or Apyrimidinic Site) Lyase, Deoxyribonuclease IV (Phage T4-Induced), Female, Humans, Leukocytes enzymology, Middle Aged, Plasmids metabolism, Tissue Extracts metabolism, Breast Neoplasms genetics, Carcinoma, Ductal, Breast genetics, DNA Repair genetics, DNA, Neoplasm metabolism
- Abstract
Defective DNA repair has been suggested as a possible predisposing factor for breast cancer. We have investigated the repair of the frequent endogenous lesions abasic sites in sporadic early onset breast cancer patients and matched control individuals. No significant difference was observed between the abasic site repair capacities of peripheral blood lymphocytes from cases and controls. Repair of abasic sites was also studied in tumor and surrounding normal tissues of the patients. The 2 tissues showed marked differences in histology and protein composition with a fibro-collagenous component varying from sample to sample but invariably higher in normal tissues as compared with the adjacent tumor. These differences involved the need to calculate the repair activities of tissues on the basis of cellular DNA content for comparison purposes. After doing so, tumor and normal tissues exhibited similar abasic site repair capacities, whereas lymphocytes showed a repair capacity significantly lower than tissues. We conclude that early onset sporadic breast cancer patients show no evident defect in repair of abasic sites at the level of both lymphocytes and tumor., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
- Full Text
- View/download PDF
23. The DNA helicases acting in nucleotide excision repair, XPD, CSB and XPB, are not required for PCNA-dependent repair of abasic sites.
- Author
-
Cappelli E, Carrozzino F, Abbondandolo A, and Frosina G
- Subjects
- Animals, CHO Cells, Cricetinae, DNA Repair Enzymes, DNA-Binding Proteins metabolism, Humans, Poly-ADP-Ribose Binding Proteins, Proteins metabolism, Radiation Tolerance, Ultraviolet Rays, Xeroderma Pigmentosum Group D Protein, Cockayne Syndrome metabolism, DNA Helicases metabolism, DNA Repair, Proliferating Cell Nuclear Antigen metabolism, Transcription Factors, Xeroderma Pigmentosum metabolism
- Abstract
DNA repair of abasic sites is accomplished in mammalian cells by two distinct base excision repair (BER) pathways: a single nucleotide insertion pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway involving a resynthesis patch of 2-10 nucleotides 3' to the lesion. The latter pathway shares some enzymatic components with the nucleotide excision repair (NER) pathway acting on damage induced by ultraviolet light: both pathways are strictly dependent on PCNA and several observations suggest that the polymerization and ligation phases may be carried out by common enzymatic activities (DNA polymerase delta/epsilon and DNA ligase I). Furthermore, it has been postulated that the transcription-NER coupling factor Cockayne syndrome B has a role in BER. We have investigated whether three NER proteins endowed with DNA helicase activities (the xeroderma pigmentosum D and B gene products and the Cockayne syndrome B gene product) may also be involved in repair of natural abasic sites, by using the Chinese hamster ovary mutant cell lines UV5, UV61 and 27-1. No defect of either the PCNA-dependent or the single nucleotide insertion pathways could be observed in UV5, UV61 or 27-1 mutant cell extracts, thus showing that the partial enzymatic overlap between PCNA-dependent BER and NER does not extend to DNA helicase activities.
- Published
- 1999
- Full Text
- View/download PDF
24. Age-independency of AP site incision capacity in women.
- Author
-
Rossi O, Carrozzino F, and Frosina G
- Subjects
- Adult, Age Factors, Female, Genetic Techniques, Humans, Lymphocytes physiology, Middle Aged, Aging physiology, DNA Repair physiology
- Abstract
Women in the age range 40-59 years have a >4 fold higher risk to develop cancer as compared to women in the age range birth-39 years. This age-related increase in cancer incidence might be partially linked to reduced efficiency of the DNA repair machinery. We have investigated the abasic (AP) site incision capacity of peripheral blood lymphocytes (PBL) from 23 women in the age range 27-57 years. The AP sites incision capacity was determined in relation to protein or DNA content of PBL extracts. In either case, no significant correlation was found between AP sites incision capacity and age, thus suggesting that no decline occurs in the age range investigated., (Copyright 1999 Wiley-Liss, Inc.)
- Published
- 1999
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