Luglio 2009, Anno 1, Numero 3

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Le pubblicazioni

Faraone D, Aguzzi MS, Toletta G, Facchiano AM, Facchiano F, Magenta A, Martelli F, Truffa S, Cesareo E, Ribatti D, Capogrossi MC and Facchiano A. Platelet derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo. Neoplasia, 2009 Aug; 11(8):732-742

Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. Platelet Derived Growth Factor Receptors (PDGF-Rs) expression is reported to be reduced in metastatic melanoma, compared to benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control melanoma cells growth. We show here that melanoma cells over-expressing PDGF-Ralpha respond to serum with a significantly lower proliferation as compared to controls.
Apoptosis, cell cycle arrest, pRb de-phosphorylation and DNA synthesis-inhibition were also observed in cells over-expressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude non-specific toxic effects and indicating that PDGF-Ralpha mediates autocrine anti-proliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the MAP-kinase pathway revealed that melanoma cells over-expressing PDGF-Ralpha show strong reduction of c-Jun phosphorylated in serine 63 and of PP2A/Balpha phosphatase, and marked increase of p38 gamma, MKK3 and SIRPalpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector over-expressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (p<0.001) and a similar inhibition of tumor-angiogenesis. All together these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely via autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

Santarpia L, Sherman SI, Marabotti A, Clayman GL, El-Naggar AK. Detection and molecular characterization of a novel BRAF activated domain mutation in follicular variant of papillary thyroid carcinoma. Human Pathology, 2009 Jun; 40(6):827-33

To assess the mutational status of BRAF in FVPTC, we directly sequenced the genomic DNA of 30 primary FVPTC samples. BRAF mutations were found in only 4 (13%) tumors. We also identified a previously unknown (novel) mutation in the activation kinase domain of the BRAF (A598V), replacing alanine with valine. Functional analysis showed that this mutation led to the up-regulation of the BRAF kinase activity and its downstream signaling factors. The effect of this mutation on the structural formation of the protein is highlighted. Our results confirm the infrequency of BRAF (V600E) mutation in FVPTC and identify a novel (A598V) mutation of this gene.

 

The LIBI Grid Platform Developers*
The LIBI Grid Platform for Bioinformatics
In Handbook of Research on Computational Grid Technologies for Life Sciences, Biomedicine and Healthcare. Volume 2, Edited by: Mario Cannataro, Medical Information Science Reference; Chapter XXIX

The LIBI project (International Laboratory of BioInformatics) aims to develop an advanced bioinformatics and computational biology laboratory, focusing on basic and applied research in modern biology and biotechnologies. The paper presents the core part of the system, a Grid Problem Solving Environment, built on top of EGEE, DEISA and SPACI infrastructures, allowing the submission and monitoring of jobs mapped to complex experiments in bioinformatics. Several case studies on different bioinformatics applications and related results which have been obtained using the LIBI platform are also reported.
*Include anche Giovani Lavorgna (Ospedale San Raffaele, Milano)

Zauli G, di Iasio MG, Secchiero P, Dal Bo M, Marconi D, Bomben R, Del Poeta G, Gattei V.
Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to Nutlin-3 induces a characteristic gene expression profile, which correlates with Nutlin-3-mediated cytotoxicity.
Current Cancer Drug Targets, 2009 June;9(4):510-518(9)

By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 BCLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type BCLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.

Zucchetto A, Benedetti D, Tripodo C, Bomben R, Dal Bo M, Marconi D, Bossi F, Lorenzon D, Degan M, Rossi FM, Rossi D, Bulian P, Franco V, Del Poeta G, Deaglio S, Gaidano G, Tedesco T, Malavasi F, Gattei V.
CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/VCAM-1 are interchained by sequential events sustaining chronic lymphocytic leukemia cell survival.
Cancer Res 2009;69(9):4001–9

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38+CD49d+ versus CD38–CD49d– CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38+CD49d+ but not CD38–CD49d– cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68+ macrophage infiltration was particularly high in BMB from CD38+CD49d+ CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor overproduction. These effects were apparent in BMB from CD38+CD49d+ CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1+ stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38+CD49d+ CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells.

Secchiero P, Melloni E, di Iasio M.G, Tiribelli M, Rimondi E, Corallini F, Gattei V, Zauli G.
Nutlin-3 upregulates the expression of Notch1 in both myeloid and lymphoid leukemic cells, as part of a negative feed-back anti-apoptotic mechanism.
Blood, 2009 April;113(18):4300-4308

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53wild-type (OCI, SKW6.4) but not in TP53deleted (HL-60) or TP53mutated (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53wild-type leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53wild-type B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53wild-type leukemic cell lines and primary B-CLL cells. A potential drawback of-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of -secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + -secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53wild-type leukemic cells.

Bulian P, Del Poeta G and Gattei V.
How would i manage a sample submitted for flow cytometry analysis for suspicious chronic lymphocytic leukemia?
Hematol Oncol. 2009 Jul 1. [Epub ahead of print]

Samples submitted for a suspect of chronic lymphocytic leukemia are the most frequently observed in flow cytometry laboratories. These cases require not only a precise and prompt diagnosis but also an evaluation on the possibility of performing additional prognostic tests. We will propose two sequential flow cytometry panels and a personal opinion on how to manage these samples for both diagnostic and prognostic assessment, taking into account the published guidelines and recommendations.

Rossi D, Sozzi E, Puma A, De Paoli L, Rasi S, Spina V, Gozzetti A, Tassi M, Cencini E, Raspadori D, Bertoni F, Gattei V, Lauria F, Gaidano G, Forconi F.
The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukemia and is recapitulated by biological risk factors.
Br J Haematol, 2009 Jun;146(1):64-75

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5.0 x 10(9)/l circulating CLL-phenotype B-cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B-cell expansions with CLL-phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22-q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment-free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1.2 x 10(9)/l and >3.7 x 10(9)/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment-free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5.39, 95% confidence interval 1.98-14.44, P = 0.001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.

Rossi D, Cerri M, Capello D, Deambrogi C, Rossi FM, Zucchetto A, De Paoli L, Cresta S, Rasi S, Spina V, Franceschetti S, Lunghi M, Vendramin C, Bomben R, Ramponi A, Monga G, Conconi A, Magnani C, Gattei V, Gaidano G.
Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome.
Br J Haematol, 2008 July;142( 2):202-215(14)

Predictors of chronic lymphocytic leukaemia (CLL) transformation to Richter syndrome (RS) are not established and were investigated in 185 consecutive CLL cases. Actuarial incidence of RS (n = 17; all diffuse large B-cell lymphomas) at 10 years was 16.2% (95% confidence interval: 8.0-24.4%). At CLL diagnosis, prognosticators of RS by univariate analysis were IGHV homology >/=98% (P = 0.006), IGHV4-39 usage (P < 0.001), del13q14 absence (P = 0.004), expression of CD38 (P < 0.001) and ZAP70 (P = 0.004), size (P < 0.001) and number (P < 0.001) of lymph nodes, advanced Binet stage (P = 0.002), and lactate dehydrogenase (P < 0.001). Multivariate analysis, performed separately for biological and clinical variables, identified CD38 expression [Hazard ratio (HR) = 4.26; P = 0.018], IGHV4-39 usage (HR = 4.29; P = 0.018), and lymph node size >/=3 cm (HR = 9.07; P < 0.001) as independent RS prognosticators. A multivariate model simultaneously analysing biological and clinical variables identified lymph node size >/=3 cm (HR = 6.51; P = 0.001) and del13q14 absence (HR = 4.08; P = 0.031) as independent RS prognosticators. Risk factors of CLL transformation differed from risk factors of CLL progression. These results suggest that CD38 and del13q14 may identify biological subsets of CLL with different RS predisposition. Predominant nodal disease, CD38 expression, IGHV4-39 usage, and absence of del13q14 may help in predicting RS at CLL diagnosis. Close monitoring and a careful biopsy policy are needed in patients carrying transformation risk factors.

Santarpia L, Sherman S.I, Marabotti A, Clayman GL, El-Naggar A.K.
Detection and molecular characterization of a novel BRAF activated domain mutation in follicular variant of papillary thyroid carcinoma.
Hum Pathos, Jun 2009;40(6):827-33

To assess the mutational status of BRAF in FVPTC, we directly sequenced the genomic DNA of 30 primary FVPTC samples. BRAF mutations were found in only 4 (13%) tumors. We also identified a previously unknown (novel) mutation in the activation kinase domain of the BRAF (A598V), replacing alanine with valine. Functional analysis showed that this mutation led to the up-regulation of the BRAF kinase activity and its downstream signaling factors. The effect of this mutation on the structural formation of the protein is highlighted. Our results confirm the infrequency of BRAF (V600E) mutation in FVPTC and identify a novel (A598V) mutation of this gene.

Rocco M, Rosano C, Weisel J.W, Horita D.A. and Hantgan R.R.
Integrin conformational regulation: uncoupling extension/tail separation from changes in the head region by a multi-resolution approach.
Structure, 2008;16:954-964

Integrin-dependent adhesion and signaling are regulated by conformational changes whose details remain controversial. Crystallography revealed bent shapes for resting and primed integrin ectodomains, whereas large, ligand-induced rearrangements in other constructs suggested extension, “opening,” and tail separation. We have used experimental/computed hydrodynamics to discriminate among different αvβ3 and αIIbβ3 atomic models built on X-ray, NMR, and EM data. In contrast with X-ray structures and EM maps, hydrodynamics indicate that resting integrins are already extended. Furthermore, the hydrodynamics of an αvβ3 ectodomain-fibronectin fragment complex support opening via additional head region conformational changes (hybrid domain swing-out), but without tail separation. Likewise, frictional changes induced by priming agents in full-length αIIbβ3 correlate well with the swing-out coupled to a simple transmembrane helix shift in an extended, electron tomography-based model. Extension and immediate tail separation are then uncoupled from head region rearrangements following activation, thus underscoring integrins' delicate, finely tuned plasticity.

Brookes E, Demeler B, Rosano C. and Rocco M.
The implementation of SOMO (SOlution MOdeller) in the UltraScan analytical ultracentrifugation data analysis suite: enhanced capabilities allow the reliable hydrodynamic modeling of virtually any kind of biomacromolecule.
Eur Biophys J. 2009 Feb 21. [Epub ahead of print]

The interpretation of solution hydrodynamic data in terms of macromolecular structural parameters is not a straightforward task. Over the years, several approaches have been developed to cope with this problem, the most widely used being bead modeling in various flavors. We report here the implementation of the SOMO (SOlution MOdeller; Rai et al. in Structure 13:723-734, 2005) bead modeling suite within one of the most widely used analytical ultracentrifugation data analysis software packages, UltraScan (Demeler in Modern analytical ultracentrifugation: techniques and methods, Royal Society of Chemistry, UK, 2005). The US-SOMO version is now under complete graphical interface control, and has been freed from several constraints present in the original implementation. In the direct beads-per-atoms method, virtually any kind of residue as defined in the Protein Data Bank (e.g., proteins, nucleic acids, carbohydrates, prosthetic groups, detergents, etc.) can be now represented with beads whose number, size and position are all defined in user-editable tables. For large structures, a cubic grid method based on the original AtoB program (Byron in Biophys J 72:408-415, 1997) can be applied either directly on the atomic structure, or on a previously generated bead model. The hydrodynamic parameters are then computed in the rigid-body approximation. An extensive set of tests was conducted to further validate the method, and the results are presented here. Owing to its accuracy, speed, and versatility, US-SOMO should allow to fully take advantage of the potential of solution hydrodynamics as a complement to higher resolution techniques in biomacromolecular modeling.

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