Publications by authors named "Eleonora Petrucci"

30 Publications

  • Page 1 of 1

Isolation and preliminary characterization of a human 'phage display'-derived antibody against neural adhesion molecule-1 antigen interfering with fibroblast growth factor receptor-1 binding.

Hum Antibodies 2020 Oct 23. Epub 2020 Oct 23.

National Center for Global Health, Istituto Superiore di Sanità, Rome, 00161, Italy.

Background: The NCAM or CD56 antigen is a cell surface glycoprotein belonging to the immunoglobulin super-family involved in cell-cell and cell-matrix adhesion. NCAM is also over-expressed in many tumour types and is considered a tumour associated antigen, even if its role and biological mechanisms implicated in tumour progression and metastasis have not yet to be elucidated. In particular, it is quite well documented the role of the interaction between the NCAM protein and the fibroblast growth factor receptor-1 in metastasis and invasion, especially in the ovarian cancer progression.

Objective: Here we describe the isolation and preliminary characterization of a novel human anti-NCAM single chain Fragment variable antibody able to specifically bind NCAM-expressing cells, including epithelial ovarian cancer cells.

Methods: The antibody was isolate by phage display selection and was characterized by ELISA, FACS analysis and SPR experiments. Interference in EOC migration was analyzed by scratch test.

Results: It binds a partially linear epitope lying in the membrane proximal region of two fibronectin-like domains with a dissociation constant of 3.43 × 10-8 M. Interestingly, it was shown to interfere with the NCAM-FGFR1 binding and to partially decrease migration of EOC cells.

Conclusions: According to our knowledge, this is the first completely human antibody able to interfere with this newly individuated cancer mechanism.
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http://dx.doi.org/10.3233/HAB-200431DOI Listing
October 2020

qSNE: quadratic rate t-SNE optimizer with automatic parameter tuning for large datasets.

Bioinformatics 2020 12;36(20):5086-5092

Research Program in Systems Oncology, Research Programs Unit, Faculty of Medicine, University of Helsinki, 00014 Helsinki, Finland.

Motivation: Non-parametric dimensionality reduction techniques, such as t-distributed stochastic neighbor embedding (t-SNE), are the most frequently used methods in the exploratory analysis of single-cell datasets. Current implementations scale poorly to massive datasets and often require downsampling or interpolative approximations, which can leave less-frequent populations undiscovered and much information unexploited.

Results: We implemented a fast t-SNE package, qSNE, which uses a quasi-Newton optimizer, allowing quadratic convergence rate and automatic perplexity (level of detail) optimizer. Our results show that these improvements make qSNE significantly faster than regular t-SNE packages and enables full analysis of large datasets, such as mass cytometry data, without downsampling.

Availability And Implementation: Source code and documentation are openly available at https://bitbucket.org/anthakki/qsne/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755412PMC
December 2020

Ovarian Cancers: Genetic Abnormalities, Tumor Heterogeneity and Progression, Clonal Evolution and Cancer Stem Cells.

Medicines (Basel) 2018 Feb 1;5(1). Epub 2018 Feb 1.

Department of Oncology and Molecular Medicine, Core Facilities, Istituto Superiore di Sanità, 00161 Rome, Italy.

Four main histological subtypes of ovarian cancer exist: serous (the most frequent), endometrioid, mucinous and clear cell; in each subtype, low and high grade. The large majority of ovarian cancers are diagnosed as high-grade serous ovarian cancers (HGS-OvCas). is the most frequently mutated gene in HGS-OvCas; about 50% of these tumors displayed defective homologous recombination due to germline and somatic mutations, epigenetic inactivation of BRCA and abnormalities of DNA repair genes; somatic copy number alterations are frequent in these tumors and some of them are associated with prognosis; defective NOTCH, RAS/MEK, PI3K and FOXM1 pathway signaling is frequent. Other histological subtypes were characterized by a different mutational spectrum: LGS-OvCas have increased frequency of and mutations; mucinous cancers have mutation in , , , and . Intensive research was focused to characterize ovarian cancer stem cells, based on positivity for some markers, including CD133, CD44, CD117, CD24, EpCAM, LY6A, ALDH1. Ovarian cancer cells have an intrinsic plasticity, thus explaining that in a single tumor more than one cell subpopulation, may exhibit tumor-initiating capacity. The improvements in our understanding of the molecular and cellular basis of ovarian cancers should lead to more efficacious treatments.
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http://dx.doi.org/10.3390/medicines5010016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874581PMC
February 2018

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells.

PLoS One 2012 25;7(4):e35073. Epub 2012 Apr 25.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Background: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings: LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion: LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338831PMC
September 2012

Hemopoietic and angiogenetic progenitors in healthy athletes: different responses to endurance and maximal exercise.

J Appl Physiol (1985) 2010 Jul 6;109(1):60-7. Epub 2010 May 6.

Biomedical Department, Internal and Specialistic Medicine (DIBIMIS), Section of Pneumology, University of Palermo, Via Trabucco, 180, 90146 Palermo, Italy.

The effects of endurance or maximal exercise on mobilization of bone marrow-derived hemopoietic and angiogenetic progenitors in healthy subjects are poorly defined. In 10 healthy amateur runners, we collected venous blood before, at the end of, and the day after a marathon race (n = 9), and before and at the end of a 1.5-km field test (n = 8), and measured hemopoietic and angiogenetic progenitors by flow cytometry and culture assays, as well as plasma or serum concentrations of several cytokines/growth factors. After the marathon, CD34(+) cells were unchanged, whereas clonogenetic assays showed decreased number of colonies for both erythropoietic (BFU-E) and granulocyte-monocyte (CFU-GM) series, returning to baseline the morning post-race. Conversely, CD34(+) cells, BFU-E, and CFU-GM increased after the field test. Angiogenetic progenitors, assessed as CD34(+)KDR(+) and CD133(+)VE-cadherin(+) cells or as adherent cells in culture expressing endothelial markers, increased after both endurance and maximal exercise but showed a different pattern between protocols. Interleukin-6 increased more after the marathon than after the field test, whereas hepatocyte growth factor and stem cell factor increased similarly in both protocols. Plasma levels of angiopoietin (Ang) 1 and 2 increased after both types of exercise, whereas the Ang-1-to-Ang-2 ratio or vascular endothelial growth factor-A were little affected. These data suggest that circulating hemopoietic progenitors may be utilized in peripheral tissues during prolonged endurance exercise. Endothelial progenitor mobilization after exercise in healthy trained subjects appears modulated by the type of exercise. Exercise-induced increase in growth factors suggests a physiological trophic effect of exercise on the bone marrow.
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http://dx.doi.org/10.1152/japplphysiol.01344.2009DOI Listing
July 2010

The cancer stem cell selective inhibitor salinomycin is a p-glycoprotein inhibitor.

Blood Cells Mol Dis 2010 Jun 4;45(1):86-92. Epub 2010 May 4.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Salinomycin, a polyether antibiotic acting as a highly selective potassium ionophore and widely used as an anticoccidial drug, was recently shown to act as a specific inhibitor of cancer stem cells. In the present study we report that salinomycin acts as a potent inhibitor of multidrug resistance gp170, as evidenced through drug efflux assays in MDR cancer cell lines overexpressing P-gp (CEM-VBL 10 and CEM-VBL 100; A2780/ADR). Conformational P-gp assay provided evidence that the inhibitory effect of salinomycin on P-gp function could be mediated by the induction of a conformational change of the ATP transporter. Treatment of the MDR cell lines with salinomycin restored a normal drug sensitivity of these cells. The observation that salinomycin is a MDR-1 inhibitor may have important implications for the understanding of the mechanisms through which this drug impairs the viability of cancer stem cells. Interestingly, nigericin and abamectin, two additional drugs identified as cancer stem cells inhibitors, also act as potent gp170 inhibitors.
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http://dx.doi.org/10.1016/j.bcmd.2010.03.008DOI Listing
June 2010

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex.

Haematologica 2010 Aug 19;95(8):1253-60. Epub 2010 Mar 19.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy.

Background: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design And Methods: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.
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http://dx.doi.org/10.3324/haematol.2009.018259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913072PMC
August 2010

Primary ovarian cancer cells are sensitive to the proaptotic effects of proteasome inhibitors.

Int J Oncol 2010 Mar;36(3):707-13

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy.

Resistance of tumors to cell death signals poses a complex clinical problem. In the present study, we have explored the capacity of proteasome inhibitors to induce cell death of ovarian cancer cells. We explored the sensitivity of primary ovarian cancer cells to a combination of bortezomib (also known as PS-341), a proteasome inhibitor and TRAIL, a death ligand, or mapatumumab or lexatumumab, TRAIL-R1 or TRAIL-R2 targeting agonist monoclonal antibodies, respectively. The results of our study showed that the large majority of primary ovarian cancers are clearly sensitive to the pro-apoptotic action of bortezomib, whose effects are potentiated by the concomitant addition of TRAIL or mapatumumab or lexatumumab. Interestingly, both cisplatin and paclitaxel-chemosensitive and chemoresistant ovarian tumors are equally sensitive to the cytotoxic effect of bortezomib. Bortezomib, combined with TRAIL or TRAIL-R1 or TRAIL-R2 agonist monoclonal antibodies may be a useful treatment for refractory ovarian cancer.
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http://dx.doi.org/10.3892/ijo_00000546DOI Listing
March 2010

Correlations between progression of coronary artery disease and circulating endothelial progenitor cells.

FASEB J 2010 Jun 7;24(6):1981-8. Epub 2010 Jan 7.

Laboratory of Interventional Cardiology, Clinica Mediterranea, Naples, Italy.

The pathophysiology of coronary artery disease (CAD) progression is not well understood. Endothelial progenitor cells (EPCs) may have an important role. In the present observational cohort study we assessed the number of circulating EPCs in 136 patients undergoing elective percutaneous coronary intervention and who had at least one major epicardial vessel with a nonsignificant stenosis [<50% diameter stenosis (DS)], and the relationship between plasma EPC levels and the 24-mo progression of the nonsignificant coronary artery lesion. The following cell populations were analyzed: CD34(+), CD133(+), CD34(+)/KDR(+), CD34(+)/VE cadherin(+), and endothelial cell colony-forming units (CFU-ECs). Progression was defined as a >15% DS increase of the objective vessel at follow-up. At 24 mo, 57 patients (42%) experienced significant progression. Independent predictors of disease progression were LDL cholesterol > 100 mg/dl (OR=1.03; 95% CI 1.01-1.04; P=0.001), low plasma levels of CFU-ECs (OR=3.99; 95% CI 1.54-10.37; P=0.005), and male sex (OR=3.42; 95% CI 1.15-10.22; P=0.027). Circulating levels of EPCs are significantly lower in patients with angiographic CAD progression.
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http://dx.doi.org/10.1096/fj.09-138198DOI Listing
June 2010

Bone marrow-derived progenitors are greatly reduced in patients with severe COPD and low-BMI.

Respir Physiol Neurobiol 2010 Jan 4;170(1):23-31. Epub 2009 Nov 4.

Department of Clinical Medicine, La Sapienza University, Rome, Italy.

Chronic obstructive pulmonary disease (COPD) patients have reduced circulating hemopoietic progenitors. We hypothesized that severity of COPD parallels the decrease in progenitors and that the reduction in body mass index (BMI) could be associated with more severe bone marrow dysfunction. We studied 39 patients with moderate to very severe COPD (18 with low-BMI and 21 with normal-BMI) and 12 controls. Disease severity was associated to a greater reduction in circulating progenitors. Proangiogenetic and inflammatory markers correlated with disease severity parameters. Compared to normal-BMI patients, low-BMI patients showed: greater reduction in circulating progenitors; higher VEGF-A, VEGF-C, HGF, Ang-2, TNF-alpha, IL-6 and MCP-1 levels. Furthermore, among patients with similar pulmonary impairment, those who displayed low-BMI had a more markedly reduced number of CD34(+) cells and late endothelial progenitors. We show that the reduction in hematopoietic and endothelial progenitor cells correlates with COPD severity. Our findings also indicate that, in severe low-BMI COPD patients, bone marrow function seems to be further impaired and may lead to reduced reparative capacity.
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http://dx.doi.org/10.1016/j.resp.2009.10.003DOI Listing
January 2010

High sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide.

Cancer Lett 2009 Sep 11;282(2):214-28. Epub 2009 Apr 11.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

In the present study we have explored the sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/ADR and A2780/CISP, OVCAR3, SKOV3 and HEY cancer cell lines and primary ovarian cancer cells, providing evidence that: (i) the majority of these cell lines are highly sensitive to the pro-apoptotic effects induced by CDDO-Im; (ii) TRAIL, added alone exerted only a weak proapoptotic, but clearly potentiated the cytotoxic effect elicited by CDDO-Im; (iii) the apoptotic effect induced by CDDO-Im involves GSH depletion, c-FLIP downmodulation and caspase-8 activation; (iv) CDDO-Im inhibits STAT3 activation and CDDO-Im sensitivity is inversely related to the level of constitutive STAT3 activation. Importantly, studies on primary ovarian cancer cells have shown that these cells are sensitive to the pro-apoptotic effects of CDDO-Im. These observations support the experimental use of synthetic triterpenoids in the treatment of ovarian cancer.
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http://dx.doi.org/10.1016/j.canlet.2009.03.018DOI Listing
September 2009

A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis.

Nat Cell Biol 2008 Jul 22;10(7):788-801. Epub 2008 Jun 22.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy.

MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.
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http://dx.doi.org/10.1038/ncb1741DOI Listing
July 2008

Discovery of a new family of bis-8-hydroxyquinoline substituted benzylamines with pro-apoptotic activity in cancer cells: synthesis, structure-activity relationship, and action mechanism studies.

Eur J Med Chem 2009 Feb 10;44(2):558-67. Epub 2008 Apr 10.

Laboratoire de Chimie Biomoléculaire, IBDML-UMR-6216-CNRS, Faculté des Sciences Luminy, Université de la Méditerranée, Parc Scientifique de Luminy, Case 901, 13288 Marseille Cedex 9, France.

Bis-8-hydroxyquinoline substituted benzylamines have been synthesized and screened for their antitumor activity on KB3 cell line model. Synthesis of this series of new analogues was accomplished using a one pot specific methodology which allows the synthesis of both bis- and mono-8-hydroxyquinoline substituted benzylamines. Among the synthesized compounds two compounds (4a and 5a), respectively, named JLK 1472 and JLK 1486, were particularly potent on KB3 cell line. Their CC(50) values being, respectively, 2.6 and 1.3 nM. Screened on a panel of cell lines showing various phenotype alterations, both compounds were found inactive on some cell lines such as PC3 (prostate cell line) and SF268 (neuroblastoma cell line) while highly active on other different cell lines. Mechanistic studies reveal that these two analogues did not affect tubulin and microtubules neither they exert a proteasomal inhibition effect. In contrast 4a and 5a activate specifically caspase 3/7 and not caspase 8 and 9, suggesting that their biological target should be located upstream from caspase 3/7. Moreover their cytotoxic effect is potentiated by the pro-apoptotic effects of TRAIL.
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http://dx.doi.org/10.1016/j.ejmech.2008.03.042DOI Listing
February 2009

Expression of Tie-2 and other receptors for endothelial growth factors in acute myeloid leukemias is associated with monocytic features of leukemic blasts.

Stem Cells 2007 Aug 19;25(8):1862-71. Epub 2007 Apr 19.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.

We investigated the expression of Tie-2 in primary blasts from 111 patients with acute myeloid leukemia (AML) to evaluate a possible linkage between the expression of this receptor and the immunophenotypic and biologic properties of leukemic blasts. Tie-2 was expressed at moderate and high levels in 39 and 23 of 111 AMLs, respectively. The analysis of the immunophenotype clearly showed that Tie-2 expression in AML was associated with monocytic features. Interestingly, Tie-2 expression on AML blasts was associated with concomitant expression of other receptors for endothelial growth factors, such as vascular endothelial growth factor receptor 1 (VEGF-R1), -R2, and -R3. Tie-2(+) AMLs were characterized by high blast cell counts at diagnosis, a high frequency of Flt3 mutations, and increased Flt3 expression. The survival of Tie-2(+) AMLs is sustained through an autocrine pattern involving Angiopoietin-1 and Tie-2, as suggested by experiments showing induction of apoptosis in Tie-2(+) AMLs by agents preventing the binding of angiopoietins to Tie-2. Finally, the in vitro growth of Tie-2(+) AMLs in endothelial culture medium supplemented with VEGF and angiopoietins resulted in their partial endothelial differentiation. These observations suggest that Tie-2(+) AMLs pertain to a mixed monocytic/endothelial lineage, derived from the malignant transformation of the normal counterpart represented by monocytic cells expressing endothelial markers. The autocrine angiopoietin/Tie-2 axis may represent a promising therapeutic target to improve the outcome of patients with monocytic AML. Disclosure of potential conflicts of interest is found at the end of this article.
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http://dx.doi.org/10.1634/stemcells.2006-0700DOI Listing
August 2007

Thyroid hormone receptor TRbeta1 mediates Akt activation by T3 in pancreatic beta cells.

J Mol Endocrinol 2007 Feb;38(1-2):221-33

Chair of Endocrinology, II Faculty of Medicine, University La Sapienza, Rome, Italy.

It has recently been recognized that thyroid hormones may rapidly generate biological responses by non-genomic mechanisms that are unaffected by inhibitors of transcription and translation. The signal transduction pathways underlying these effects are just beginning to be defined. We demonstrated that thyroid hormone T3 rapidly induces Akt activation in pancreatic beta cells rRINm5F and hCM via thyroid hormone receptor (TR) beta1. The phosphorylation of Akt was T3 specific and dependent. Coimmunoprecipitation and colocalization experiments revealed that the phosphatidylinositol 3 kinase (PI3K) p85alpha subunit and the thyroid receptor beta1 were able to form a complex at the cytoplasmic level in both the cell lines, suggesting that a 'cytoplasmic TRbeta1' was implicated. Moreover, we evidenced that T3 treatment was able to induce kinase activity of the TRbeta1-associated PI3K. The silencing of TRbeta1 expression through RNAi confirmed this receptor to be crucial for the T3-induced activation of Akt. This action involved a T3-induced nuclear translocation of activated Akt, as demonstrated by confocal immunofluorescence. In summary, T3 is able to specifically activate Akt in the islet beta cells rRINm5F and hCM through the interaction between TRbeta1 and PI3K p85alpha, demonstrating the involvement of TRbeta1 in this novel T3 non-genomic action in islet beta cells.
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http://dx.doi.org/10.1677/jme.1.02166DOI Listing
February 2007

A small molecule Smac mimic potentiates TRAIL-mediated cell death of ovarian cancer cells.

Gynecol Oncol 2007 May 9;105(2):481-92. Epub 2007 Feb 9.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Objectives: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells.

Methods: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays.

Results: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells.

Conclusions: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.
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http://dx.doi.org/10.1016/j.ygyno.2007.01.011DOI Listing
May 2007

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis.

Apoptosis 2007 Apr;12(4):635-55

Medical Oncology Section, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.
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http://dx.doi.org/10.1007/s10495-006-0025-9DOI Listing
April 2007

Coordinate release of angiogenic growth factors after acute myocardial infarction: evidence of a two-wave production.

J Cardiovasc Med (Hagerstown) 2006 Dec;7(12):872-9

Institute of Heart and Great Vessels Attilio Reale, University of Rome La Sapienza, Rome, Italy.

Background: Previous studies have shown that angiopoietic growth factors, including vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-beta1 are released after acute myocardial infarction (AMI). It was suggested that the release of these factors, triggered by ischemia, may be related to a reparative neoangiogenetic process.

Methods: Plasma VEGF, Ang-2, HGF and TGF-beta levels were measured on admission (baseline) and at various times during the acute (0-48 h) and the subacute (48-240 h) phase in 44 patients with AMI.

Results: In the present study, we have explored in detail the kinetics of release of these growth factors after AMI with the precise aim of evaluating the existence of a double wave of release of these factors: (i) a first wave in the acute and (ii) a second one in the subacute period. The results of these analyses provided evidence for an early (peak at 24-28 h) and late (peak at approximately 170 h) increase of VEGF, Ang-2 and TGF-beta.

Conclusions: According to these data, we suggest that two waves of release of angiogenic factors occur after AMI. The early release makes part of an acute phase response, whereas the late release may underlie the induction of angiogenetic mechanisms involved in tissue reparation.
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http://dx.doi.org/10.2459/01.JCM.0000253831.61974.b9DOI Listing
December 2006

Podocalyxin is expressed in normal and leukemic monocytes.

Blood Cells Mol Dis 2006 Nov-Dec;37(3):218-25. Epub 2006 Oct 23.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

We have investigated the expression of podocalyxin in primary cultures of leukemic blast cells from 73 patients with acute myeloid leukemia. Podocalyxin was expressed at moderate levels in 15 patients and at high levels in 13 patients. The analysis of membrane markers showed that Podocalyxin expression in leukemic blasts was associated with a monocytic immunophenotype. Cases of podocalyxin-positive acute myelogenous leukemia had high blast cell counts at diagnosis and elevated CD123, CD135, VLA-4 and CXCR4 expression, features associated with poor prognosis. Podocalyxin expression in leukemic blasts was coupled with the concomitant expression of VEGF-R1, -R2, -R3 and Tie-2, the capacity to release VEGF-A and angiopoietin1 and the ability to differentiate into endothelial cells under appropriate culture conditions. These findings show that podocalyxin is a marker of acute myeloid leukemia with a monocytic phenotype and suggest that podocalyxin-positive cases of acute myeloid leukemia originate from the malignant transformation of progenitors common to the myeloid and endothelial lineages. These observations suggest a possible relationship between the monocytic lineage and podocytes.
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http://dx.doi.org/10.1016/j.bcmd.2006.09.001DOI Listing
February 2007

Relation of various plasma growth factor levels in patients with stable angina pectoris and total occlusion of a coronary artery to the degree of coronary collaterals.

Am J Cardiol 2006 Feb 20;97(4):472-6. Epub 2005 Dec 20.

Laboratory of Interventional Cardiology and Department of Cardiology, Clinica Mediterranea, Naples, Italy.

We assessed (1) angiogenic factors in patients with stable angina and longstanding (> or =24 months) total occlusion of a single coronary artery and (2) the relation between plasma levels of angiogenic factors and the development of collateral vessels as evaluated by coronary angiography. Plasma concentrations of vascular endothelial growth factor (VEGF(165)), fibroblast growth factor, placenta-derived growth factors (PlGFs), and hepatocyte growth factor were assessed in 96 patients with stable angina and longstanding (> or =24 months) total occlusion of a single coronary artery. According to coronary angiographic results, 18 patients had no visible collaterals (group 0), 21 patients had visible collaterals but no filling of the recipient epicardial vessel (group 1), and 57 patients showed filling (partial or complete) of the recipient epicardial vessel by collaterals (group 2). Plasma VEGF(165) and PlGF concentrations were higher in group 1 than in groups 0 and 2 (VEGF(165) 75 pg/ml, range 24 to 105, vs 23 pg/ml, range 15 to 29, and 19 pg/ml, range 10 to 41, respectively, F = 5.53, p = 0.006; PlGF 35 pg/ml, range 3.5 to 105, vs 1 pg/ml, range 1 to 38, and 1 pg/ml, range 1 to 5, respectively, F = 7.09, p = 0.008). Plasma VEGF(165) and PlGF levels were similar in groups 0 and 2. There was no significant difference in plasma levels of fibroblast and hepatocyte growth factor concentrations across the 3 groups. In conclusion, plasma levels of angiogenic growth factors differ among patients with stable angina pectoris and longstanding total coronary occlusion.
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http://dx.doi.org/10.1016/j.amjcard.2005.09.076DOI Listing
February 2006

Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes.

J Cell Sci 2006 Feb 31;119(Pt 4):744-52. Epub 2006 Jan 31.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.

The megakaryocyte is a paradigm for mammalian polyploid cells. However, the mechanisms underlying megakaryocytic polyploidization have not been elucidated. In this study, we investigated the role of Shc-Ras-MAPK and PI3K-AKT-mTOR pathways in promoting megakaryocytic differentiation, maturation and polyploidization. CD34+ cells, purified from human peripheral blood, were induced in serum-free liquid suspension culture supplemented with thrombopoietin (TPO) to differentiate into a virtually pure megakaryocytic progeny (97-99% CD61+/CD41+ cells). The early and repeated addition to cell cultures of low concentrations of PD98059, an inhibitor of MEK1/2 activation, gave rise to a population of large megakaryocytes showing an increase in DNA content and polylobated nuclei (from 45% to 70% in control and treated cultures, respectively). Conversely, treatment with the mTOR inhibitor rapamycin strongly inhibited cell polyploidization, as compared with control cultures. Western blot analysis of PD98059-treated progenitor cells compared with the control showed a downmodulation of phospho-ERK 1 and phospho-ERK 2 and a minimal influence on p70S6K activation; by contrast, p70S6K activation was completely inhibited in rapamycin-treated cells. Interestingly, the cyclin D3 localization was nuclear in PD98059-induced polyploid megakaryocytes, whereas it was completely cytoplasmic in those treated with rapamycin. Altogether, our results are in line with a model in which binding of TPO to the TPO receptor (mpl) could activate the rapamycin-sensitive PI3K-AKT-mTOR-p70S6K pathway and its downstream targets in promoting megakaryocytic cell polyploidization.
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http://dx.doi.org/10.1242/jcs.02784DOI Listing
February 2006

Multiple members of the TNF superfamily contribute to IFN-gamma-mediated inhibition of erythropoiesis.

J Immunol 2005 Aug;175(3):1464-72

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

IFN-gamma inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-gamma up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-gamma in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-gamma on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-gamma inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-gamma-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-gamma-mediated regulation of hematopoiesis and show that the effects of IFN-gamma on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.
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http://dx.doi.org/10.4049/jimmunol.175.3.1464DOI Listing
August 2005

Supramaximal exercise mobilizes hematopoietic progenitors and reticulocytes in athletes.

Am J Physiol Regul Integr Comp Physiol 2005 Nov 14;289(5):R1496-503. Epub 2005 Jul 14.

Department of Experimental Medicine, University of Palermo, Italy.

Marathon runners show increased circulating CD34+ cell counts and postexercise release of interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF) and flt3-ligand (Bonsignore MR, Morici G, Santoro A, Pegano M, Cascio L, Bonnano A, Abate P, Mirabella F, Profita M, Insalaco G, Gioia M, Vignola AM, Majolino I, Testa U, and Hogg JC. J Appl Physiol 93: 1691-1697, 2002). In the present study we hypothesized that supramaximal ("all-out") exercise may acutely affect circulating progenitors and reticulocytes and investigated possible mechanisms involved. Progenitor release was measured by flow cytometry (n = 20) and clonogenic assays (n = 6) in 20 young competitive rowers (13 M, 7 F, age +/- SD: 17.1 +/- 2.1 yr, peak O2 consumption: 56.5 +/- 11.4 ml.min(-1).kg(-1)) at rest and shortly after 1,000 m "all-out." Release of reticulocytes, cortisol, muscle enzymes, neutrophil elastase, and several cytokines/growth factors was measured. Supramaximal exercise doubled circulating CD34+ cells (rest: 7.6 +/- 3.0, all-out: 16.3 +/- 9.1 cells/mul, P < 0.001), and increased immature reticulocyte fractions; AC133+ cells doubled, suggesting release of angiogenetic precursors. Erythrocyte burst forming units and colony forming units for granulocytes-monocytes and all blood series increased postexercise by 3.4-, 5.5-, and 4.8-fold, respectively (P < 0.01 for all). All-out rowing acutely increased plasma cortisol, neutrophil elastase, flt3-ligand, hepatocyte growth factor, VEGF, and transforming growth factor-beta1, and decreased erythropoietin; K-ligand, stromal-derived factor-1, IL-6, and G-CSF were unchanged. Therefore, all-out exercise is a physiological stimulus for progenitor release in athletes. Release of reticulocytes and proangiogenetic cells and mediators suggests tissue hypoxia as possibly involved in progenitor mobilization.
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http://dx.doi.org/10.1152/ajpregu.00338.2005DOI Listing
November 2005

Transferrin receptor 2 protein is not expressed in normal erythroid cells.

Biochem J 2004 Aug;381(Pt 3):629-34

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.
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http://dx.doi.org/10.1042/BJ20040230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133871PMC
August 2004

Interferon regulatory factor-2 drives megakaryocytic differentiation.

Biochem J 2004 Jan;377(Pt 2):367-78

Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.

IRFs [IFN (interferon) regulatory factors] constitute a family of transcription factors involved in IFN signalling and in the development and differentiation of the immune system. IRF-2 has generally been described as an antagonist of IRF-1-mediated transcription of IFN and IFN-inducible genes; however, it has been recently identified as a transcriptional activator of some genes, such as those encoding histone H4, VCAM-1 (vascular cell adhesion molecule-1) and Fas ligand. Biologically, IRF-2 plays an important role in cell growth regulation and has been shown to be a potential oncogene. Studies in knock-out mice have also implicated IRF-2 in the differentiation and functionality of haematopoietic cells. Here we show that IRF-2 expression in a myeloid progenitor cell line leads to reprogramming of these cells towards the megakaryocytic lineage and enables them to respond to thrombopoietin, as assessed by cell morphology and expression of specific differentiation markers. Up-regulation of transcription factors involved in the development of the megakaryocytic lineage, such as GATA-1, GATA-2, FOG-1 (friend of GATA-1) and NF-E2 (nuclear factor-erythroid-2), and transcriptional stimulation of the thrombopoietin receptor were also demonstrated. Our results provide evidence for a key role for IRF-2 in the induction of a programme of megakaryocytic differentiation, and reveal a remarkable functional diversity of this transcription factor in the regulation of cellular responses.
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http://dx.doi.org/10.1042/BJ20031166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223861PMC
January 2004

HbF reactivation in sibling BFU-E colonies: synergistic interaction of kit ligand with low-dose dexamethasone.

Blood 2003 Apr 7;101(7):2826-32. Epub 2002 Nov 7.

Department of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy.

Mechanisms underlying fetal hemoglobin (HbF) reactivation in stress erythropoiesis have not been fully elucidated. We suggested that a key role is played by kit ligand (KL). Because glucocorticoids (GCs) mediate stress erythropoiesis, we explored their capacity to potentiate the stimulatory effect of KL on HbF reactivation, as evaluated in unilineage erythropoietic culture of purified adult progenitors (erythroid burst-forming units [BFU-Es]). The GC derivative dexamethasone (Dex) was tested in minibulk cultures at graded dosages within the therapeutical range (10(-6) to 10(-9) M). Dex did not exert significant effects alone, but synergistically it potentiated the action of KL in a dose-dependent fashion. Specifically, Dex induced delayed erythroid maturation coupled with a 2-log increased number of generated erythroblasts and enhanced HbF synthesis up to 85% F cells and 55% gamma-globin content at terminal maturation (ie, in more than 80%-90% mature erythroblasts). Equivalent results were obtained in unicellular erythroid cultures of sibling BFU-Es treated with KL alone or combined with graded amounts of Dex. These results indicate that the stimulatory effect of KL + Dex is related to the modulation of gamma-globin expression rather than to recruitment of BFU-Es with elevated HbF synthetic potential. At the molecular level, Id2 expression is totally suppressed in control erythroid culture but is sustained in KL + Dex culture. Hypothetically, Id2 may mediate the expansion of early erythroid cells, which correlates with HbF reactivation. These studies indicate that GCs play an important role in HbF reactivation. Because Dex acts at dosages used in immunologic disease therapy, KL + Dex administration may be considered to develop preclinical models for beta-hemoglobinopathy treatment.
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http://dx.doi.org/10.1182/blood-2002-05-1477DOI Listing
April 2003

Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor.

Blood 2003 Feb 24;101(4):1316-23. Epub 2002 Oct 24.

Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.

The expression/function of vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt1 and VEGFR2/KDR/Flk1) in hematopoiesis is under scrutiny. We have investigated the expression of Flt1 and kinase domain receptor (KDR) on hematopoietic precursors, as evaluated in liquid culture of CD34(+) hematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), megakaryocytic (Mk), granulocytic (G), or monocytic (Mo) lineage. KDR, expressed on 0.5% to 1.5% CD34(+) cells, is rapidly downmodulated on induction of differentiation. Similarly, Flt1 is present at very low levels in HPCs and is downmodulated in E and G lineages; however, Flt1 is induced in the precursors of both Mo and Mk series; ie, its level progressively increases during Mo maturation, and it peaks at the initial-intermediate culture stages in the Mk lineage. Functional experiments indicate that Mk and E, but not G and Mo, precursors release significant amounts of VEGF in the culture medium, particularly at low O(2) levels. The functional role of VEGF release on Mk maturation is indicated by 2 series of observations. (1) Molecules preventing the VEGF-Flt1 interaction on the precursor membrane (eg, soluble Flt1 receptors) significantly inhibit Mk polyploidization. (2) Addition of exogenous VEGF or placenta growth factor (PlGF) markedly potentiates Mk maturation. Conversely, VEGF does not modify Mo differentiation/maturation. Altogether, our results suggest that in the hematopoietic microenvironment an autocrine VEGF loop contributes to optimal Mk maturation through Flt1. A paracrine loop involving VEGF release by E precursors may also operate. Similarly, recent studies indicate that an autocrine loop involving VEGF and Flt1/Flk1 receptors mediates hematopoietic stem cell survival and differentiation.
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http://dx.doi.org/10.1182/blood-2002-07-2184DOI Listing
February 2003