Clinical Physiology Institute, CNR Siena unit

Istituto di Fisiologia clinica, CNR
Via Fiorentina 1, 53100 Siena
Tel. 0577-1916294 /295
Fax 0577-280599
website

Clinical Physiology Institute, CNR Siena unit - team members Principal investigator: Caterina Cinti

Team members
Caterina Cinti PhD, first researcher of CNR
Mario Chiariello PhD, first researcher of CNR
Rosalba Casula administrative of CNR
Ilaria Naldi PhD student, CNR fellowship
Monia Taranta PhD, CNR fellowship
Johan Carlos Trivino PhD student, CNR fellowship

Introduction

The Institute of Clinical Physiology- CNR, Siena unit originated as a branch of the Institute of Clinical Physiology – CNR, Pisa unit due to CNR reorganization (D.L. N. 127 del 4 Jun 2003). Its research activity is on the field of biomedical research and in particular the IFC-CNR Siena unit has a central role in basic and technological research in the area of experimental oncology. Its goal is to improve the scientific and technological research through the interaction with national and local scientific institutions. Since 2005 the IFC-CNR of Siena is a “Polo Toscano” Unit of the “CNR Medical Research in Italy” (MERIT) network. The IFC-CNR of Siena is located within the Toscana Life Sciences (TLS) laboratories, which are part of Torre Fiorentina’s NOVARTIS scientific complex in Siena. The IFC-CNR Siena unit has about 160mq of laboratories and 50 mq of administrative offices. Moreover it has access to other facilities as animal house facility, PCL3 laboratory, dedicated rooms for communal analytical equipment, storage of chemicals, radioactive room as well as meeting rooms, a reception and a recreational area. The staff consists of 3 units of CNR personnel employed full-time (2 researchers and 1 administrative) and three CNR grant funded people. From 2006 to present, the Siena Unit of IFC-CNR is under the direction of Dr. Caterina Cinti, CNR head researcher.

The Activity of the CNR of Siena is concentrated on understanding the pathogenetic mechanism of specific tumor histotypes, such as retinoblastoma, osteosarcoma, lung and breast tumors, chemotherapy sensitive and resistant tumors, with the aim to identify tumor specific molecular markers and develop new therapeutic strategies.

Main research themes

Development new therapeutic and diagnostic strategies
Identification of proteins that broaden “gene targeting” in human tumor cells that are sensitive and resistant to chemotherapeutic drugs and development of new therapeutic strategies.

Main achievements:

  1. Multi-drug resistant osteosarcoma cell line have been isolated in our laboratory, starting from a clone of drug sensitive cells, with the aim to develop an experimental model to study the molecular mechanisms of pharmacoresistance in bone tumors. Molecular markers and the mechanisms of regulation of genes typed to the drug resistant phenotype were identified. Several in vitro experiments of combination therapeutic strategies were conducted (hypomethylation of DNA and chemotherapy) with the aim of evaluating possible new therapeutic protocols to cure drug-resistant osteosarcoma.
  2. In vitro experiments on retinoblastoma and non-small lung adenocarcinoma (NSLC) cell lines have identified some possible mechanisms of gene deregulation. Analyses of promoter methylation (MSP) and gene expression profiles (cDNA microarrays) were conducted on both tumor histotypes. Epigenetic modifications due to hypermethylation of the cytosine on the promoters of some genes are necessary to determine their silencing and can compete together with other genetic alterations to neoplastic transformation. Treatments with appropriate doses of demethylating (5-AZA-2-deoxycytidine) and/or histon-deacetylating (HDAC) (Tricostatin A, TSA) agents reactivate the expression of tumorsuppressor genes in retinoblastoma and lung adenocarcinoma tumors, suggesting a possible use of this drug in the therapy of these tumors. Through the identification of tumor markers, it was synthesized specific ribozymes targeting mRNAs of genes over-expressed in retinoblastoma and NSLC tumors. The objective is to develop new therapeutic protocols based on use hypomethylating agents and ribozymes with the aim to induce tumor mass regression and metastasis inhibition in these two tumor histotypes.
  3. New drug delivery systems have been developed in our laboratories to improve the efficiency and efficacy of therapeutic strategies. Newly engineered human erythrocytes and chemical modified magneto-nanopaticles have been developed to specifically target therapeutic agents at the tumor site by using static magnetic field.

Current work: Therapeutic strategies based on the use of demethylating agents alone or in combination with chemotherapy drugs or ribozymes are currently tested in vitro in different tumor histotypes sensitive and resistant to chemotherapy drugs. The preliminary results shown that the combination of demethylating agent (5-Aza-dC) with chemotherapy drug (i.e. doxorubicin) or synthetic ribozymes, specifically targeting tumor markers (i.e. HSP1, NFKB, ...), increase the percentage of apoptotic tumor cells. In vitro and in vivo experiments are in progress to set best conditions for our new developed drug delivery systems used to improve the therapeutic effects of anticancer compounds.

Future plans:
With the aim of evaluating the effects of treatments with demethylating/ deacethylating agents alone or in combination with other drugs (chemotherapy or ribozymes) in retinoblastoma and pulmonary adenocarcinoma, it will be conducted in vivo experiments on three different experimental animal models that develop retinoblastoma and pulmonary adenocarcinoma.

Animal models of retinoblastoma: 1) female 6-7 week old CD1 nu/nu mice (nude mice) received an injection in the thigh of approximately 106 Y79 or Weri-Rb1 cells; 2) transgenic murine models that over-express the large T antigen of SV40 (SV40-Tag) and demonstrate spontaneous development of hereditary forms of tumors of the retina, i.e. similar to the human form of retinoblastoma; 3) conditional Ad-Cre Rb -/- transgenic mice that, if induced, develop tumors of the retina.

Animal models of pulmonary adenocarcinoma: mutant LSL-K-Ras G12D murine model that conditionally expresses active K-Ras, after intranasal administration of an adenoviral vector (Ad-Cre), and develop an atypical hyperplasia adenomatosa after 2 weeks of infection, a papillary adenoma after 6 weeks, diffuse adenoma at 12 weeks and adenocarcinoma at 16 weeks.

The therapeutic protocols, based on the use of demethylating agent alone or in combination with: a) chemotherapy drugs, b) new target specific drugs (ribozymes, synthetic peptides, gene and antibody therapy), c) short-interfering RNAs, will be tested in these animal models. New drug delivery systems developed in our laboratories will be used with the objective to improve the efficacy and efficiency of therapeutic protocols.

Development of cDNA tumor-specific microarrays for diagnostic use.

Main achievements:
Various tumor histotypes (renal carcinoma, medullary thyroid carcinoma, malignant mesothelioma, skin melanoma, three different histotypes of non-Hodgkin lymphoma, retinoblastoma, and osteosarcoma) were analyzed using cDNA microarrays (MWG Human Cancer Array) containing 1853 human genes associated with tumor development and progression. The objective was to evaluate the global expression of the genes that characterize each tumor histotype. Several sets of possible molecular markers linked to the genesis of different tumor hystotypes have been identified by using the cDNA microarray analysis.

Current work:
We are currently analyzing the data obtained on the tests of cDNA microarrays, conducted on different tumor cell lines treated and untreated with demethylating/deacetylating agents.
The analysis of Hierarchical Clustering, which takes into consideration the quantity of data regarding differential expression of microarrays reported by the various cellular lines, are being analyzed with the aim of identifying the genetic pathways that influence the epigenetic modifications involved in neoplastic transformation of these tumor histotypes.

Future plans:
A data bank of genes that characterizes each tumor histotype will be constructed with the aim of illustrating cDNA microarrays for diagnostic use. Software that is able to correlate gene profile information with their functional information extrapolated from the data bank (GenBank) will be developed.

References

  1. Macaluso M, Montanari M, Cinti C and Giordano A. Modulation of cell cycle components by epigenetic and genetic events. Sem.Oncol. 32: 452-457 (2005).
  2. G.M. Tosi, C. Trimarchi, M. Macaluso, D. La Sala, A. Ciccodicola, S. Lazzi, M. Massaro-Giordano, A. Caporossi, and C. Cinti. Genetic and epigenetic alterations of RB2/p130 tumor suppressor gene in human sporadic retinoblastoma: implications for pathogenesis and therapeutic approach. Oncogene 24: 5827-5836. (2005).
  3. La Sala D, Roberti A, Macaluso M, Trimarchi C, Cinti C. Effect of demethylating and deacetylating agents on multidrug resistant osteosarcoma HosDXR150 cell line. J. Exper. Clin. Oncol., Suppl. Tumori, 4: 54 (2005)
  4. La Sala D, Magnano AR, Estenoz M, Giordano R and Cinti C. Chromatin remodeling agents for cancer therapy. Reviews on recent clinical trials. 3(3): 192-203 (2008)
  5. Cinti C, Vindigni C, Zamparelli A, La Sala D, Epistolato MC, Marrelli D, Cevenini G and Tosi P. Activated Akt as an indicator of prognosis in gastric cancer. Virchows Arch. 453 (5): 449-455 (2008).

Research grants

  • 2003-2004 Strategic Project of in the Biomedical Area, Ministry of Health, "New combination therapeutic strategies: DNA hypomethylates and bioimmunotherapy". (Euro 30.000,00)
  • 2003-2004 ISPESL-Research Project (Annual Modal-Research Contribution) "Immunological markers of alterations caused by non-ionized radiation" (Euro 50.000,00)
  • 2003-2008 NIH Project (NIH 2 RO1 CA 060999-09) "pRb2/p130: from the mechanisms to gene therapy". (USA Dollar: 1.856.000,00)
  • 2008-2011 Modulo Commessa CNR ME.P06.012.007 “Sviluppo di un sistema di veicolazione specifica di farmaci antiblastici tramite campi magnetici ed elettromagneticii” all’interno della Commessa ME PO6.012 “Studio dell'interazione tra campi elettromagnetici e cellule eucariotiche” nell’ambito del progetto ME.PO6 “Innovazione-integrazione tecnologica in medicina” del Dipartimento di Medicina CNR. (Euro 84.250,00)
  • 2008-2011 Commessa CNR ME PO3.013 “Regolazione genetica ed epigenetica nella patogenesi di malattie complesse: sviluppo di strategie terapeutiche, di sistemi diagnostici e di "drug delivery" innovativi”, nell’ambito del progetto ME.PO3 “Oncologia: meccanismi e tecnologie applicate” del Dipartimento di Medicina CNR. (Euro 750.000,00)
  • 2008-2011 Modulo Commessa CNR ME.P03.013.001 “Identificazione di target molecolari tumore specifici, sviluppo di strategie terapeutiche di combinazione (demetilanti e bioterapia) e di sistemi innovativi di drug delivery” (Euro 84.250,00)
  • 2010-2013 Progetto Fase1 srl: “Sviluppo di sistema terapeutico innovativo basato su una efficiente veicolazione di farmaci antiblastici legati a nanoparticelle magnetiche”. (Euro 168.500,00)

Main collaborations

With units within ITT

  • CRL-Trasduzione del segnale, ITT Siena

With other Italian and foreign units

  • Istituto Neurobiologia e Medicina Molecolare, CNR, Tor Vergata Roma
  • Istituto di Neuroscienze, CNR, Pisa
  • Fase1 srl, Cagliari
  • Marshall University, Dept. Microbiology and Biothecnology, Hungtinton, WV (USA)

Publications

  1. La Sala D, Magnano AR, Estenoz M, Giordano R and Cinti C. Chromatin remodeling agents for cancer therapy. Reviews on recent clinical trials. 3(3): 192-203 (2008)
  2. Cinti C, Vindigni C, Zamparelli A, La Sala D, Epistolato MC, Marrelli D, Cevenini G and Tosi P. Activated Akt as an indicator of prognosis in gastric cancer . Virchows Arch. 453 (5): 449-455 (2008).
  3. Macaluso M, Montanari M, Cinti C and Giordano A. Modulation of cell cycle components by epigenetic and genetic events. Sem.Oncol. 32: 452-457 (2005).
  4. G.M. Tosi, C. Trimarchi, M. Macaluso, D. La Sala, A. Ciccodicola, S. Lazzi, M. Massaro-Giordano, A. Caporossi, A. Giordano and C. Cinti. Genetic and epigenetic alterations of RB2/p130 tumor suppressor gene in human sporadic retinoblastoma: implications for pathogenesis and therapeutic approach. Oncogene 24: 5827-5836. (2005).
  5. Roberti A, La Sala D, Tosi GM, Trimarchi C, Cinti C. Genetic and epigenetic alterations of Rb2/p130 tumor suppressor gene in human sporadic retinoblastoma: implications for pathogenesis and therapeutic approach. J. Exper. Clin. Oncol., Suppl. Tumori, 4: 31 (2005).
  6. La Sala D, Roberti A, Macaluso M, Trimarchi C, Cinti C. Effect of demethylating and deacetylating agents on multidrug resistant osteosarcoma HosDXR150 cell line. J. Exper. Clin. Oncol., Suppl. Tumori, 4: 54 (2005)
  7. M. Massaro–Giordano, M. Montanari, C.M. Marshall, A. Gambone, C. Cinti, G. Tosi, A. Giordano, and M. Macaluso: pRb2/p130 and PAI–2 Interaction in the Cytoplasm and Nucleus of Normal Human Corneal and Conjunctival Cells. Invest. Ophthalmol. Vis. Sci. 46: 2114 (2005).
  8. M Macaluso, C Cinti, G Russo, A Russo & A.Giordano. pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1 multimolecular complexes mediate the transcription of Estrogen Receptor- α in breast cancer. Oncogene 22:3511-3517 (2003).
  9. G. De Falco, C. Bellan, S. Lazzi, P.P. Claudio, D. La Sala, C. Cinti, P. Tosi, A. Giordano and L. Leoncini. The interaction between HIV-1 Tat and pRb2/p130: a possible mechanism in the pathogenesis of AIDS-related neoplasms. Oncogene 22: 6214-6219 (2003).
  10. D.-D Gan, M. Macaluso, C. Cinti,K. Khalili, A. Giordano. How a normal human cell becomes a cancer cell. J.Exp.Clin.Cancer Res. 22: 421-427 (2003).
  11. C. Bellan, S. Lazzi, M. Zazzi, AV. Lalinga, N. Palummo, P. Galieni, T. Marafioti, T. Tonini, C.Cinti, L. Leoncini, S.Pileri, P.Tosi. Immunoglobulin gene rearrangement analysis in composite Hodgkin’s disease and large B-cell lymphoma: evidence for receptor revision of immunoglobulin heavy chain variable region genes in Hodgkin-Reed-Sternberg cells? . Diagnostic Mol. Pathol. 11: 2-8 (2002).
  12. Macaluso M, Russo G, Cinti C, Russo A. The Ras family genes: an interesting link between cell cycle and cancer. J.Cell.Physiol. 192: 125-130 (2002). Review
  13. S. Lazzi, C Bellan, G De Falco, C Cinti, F Ferrari, A Nyongo, PP Claudio, GM Tosi, R Vatti, A Gloghini, A Carbone, A Giordano, L Leoncini and P Tosi.Expression of RB2/p130 tumor suppressor gene in AIDS-related non-Hodgkin's lymphomas. Implications for disease pathogenesis. Human Pathol 33:723-731(2002)
  14. C Bellan, G De Falco, GM Tosi, S Lazzi, F Ferrari, G Morbini, S Bartolomei, P Toti, P Mangiavacchi, G Cevenini, C Trimarchi, C Cinti, A Giordano, L Leoncini, P Tosi H Cottier. Missing expression of pRb2/p130 in human retinoblastomas is associated with reduced apoptosis and lesser differentiation. Inv.Ophthal.Visual Sci. 43:3602-3608 (2002).
  15. Leoncini L, C Bellan, Cinti C, Giordano A. From Cancer Drug Discovery and development: Cell cycle inhibitors in cancer therapy, Tumor suppressor genes as diagnostic tools. Edit by A.Giordano and KJ Soprano, Humana Press Inc.,Totowa, NJ. Chapter 4, p: 49-82 (2002).
  16. De Falco G, Bellan C, Lazzi S, Claudio PP, Bagella L, La Sala D, Carbone A, Tosi P, Giordano A, Cinti C, Leoncini L. The interaction between HIV-1 TAT and pRB2/p130: a possible mechanism in the pathogenesis of AIDS-related neoplasms. Journal of Clinical Pathology Vol. 55, suppl. 1, p.A23. (2002)
  17. P.P.Claudio, C. Cinti, A. Giordano. Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction. Anal. Biochem. 29: 96-101 (2001).
  18. Zamparelli A, Masciullo V, Bovicelli A, Santini D, Ferrandina G, Minimo C, Terzano P, Costa S, Cinti C, Ceccarelli C, Mancuso S, Scambia G, Bovicelli L, Giordano A. Expression of cell-cycle-associated proteins pRb2/p130 and p27kip in vulvar squamous cell carcinomas. Hum Pathol. 32:4-9. (2001).
  19. Maraldi NM, Giordano A, Manzoli L, Falconi M, Pol AD, Cinti C. Genetic alterations at the nuclear localization signal of the RB2/p130 gene occur in lymphoid tumor but not in osteosarcoma cell lines. Adv Enzyme Regul. 41:31-55 (2001).
  20. Claudio PP, Zamparelli A, Zupi A, Califano L, Bellan C, Minimo C, Howard C, Micheli P, Cinti C, Leoncini L, De Rosa G, Giardino C, Giordano A. Expression of cell-cycle regulated prb2/p130, p107, E2F4, p27 and of PCNA in salivary gland tumors. Prognostic and diagnostic implications. Rivista Italiana di Chirurgia Maxillo-Facciale XII . 1:37-48 (2001)

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