Publications by authors named "Roberta Riccioni"

26 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 2021 ;29(1):63-84

National Center for Global Health, Istituto Superiore di Sanità, Rome, 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
January 2021

Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification.

Tumori 2020 Feb 14:300891620904412. Epub 2020 Feb 14.

Fondazione IRCCS Istituto Nazionale dei Tumori, Direzione Scientifica, Milan, Italy.

Objective: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL.

Methods: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL.

Results: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified.

Conclusions: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.
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http://dx.doi.org/10.1177/0300891620904412DOI Listing
February 2020

New data from the Italian National Register of Congenital Coagulopathies, 2016 Annual Survey.

Blood Transfus 2020 01 13;18(1):58-66. Epub 2019 Feb 13.

Department of Oncology and Molecular Medicine, Italian National Institute of Health, Rome, Italy.

Background: In Italy, the National Register of Congenital Coagulopathies (NRCC) collects epidemiological and therapeutic data from patients affected by haemophilia A (HA), haemophilia B (HB), von Willebrand's disease (vWD) and other rare coagulation disorders. Here we present data from the 2016 annual survey.

Materials And Methods: Data are provided by the Italian Haemophilia Centres, on a voluntary basis. Information flows from every Centre to a web-based platform of the Italian Association of Haemophilia Centres, shared with the Italian National Institute of Health, in accordance with current privacy laws. Patients are classified by diagnosis, disease severity, age, gender and treatment-related complications.

Results: In 2016, the total number of patients with congenital coagulopathies in the NRCC was 10,360: 39.8% of these patients had HA, 31.5% had vWD, 8.5% had HB, and 20.2% had less common factor deficiencies. The overall prevalence of HA and HB was 13.9/100,000 males and 3.0/100,000 males, respectively. The overall prevalence of vWD was 5.4/100,000 inhabitants. During 2016, 126 patients had current alloantibodies to factor VIII (FVIII) or factor IX (FIX) and were under treatment with bypassing agents and/or immune tolerance induction. Overall, 388 patients with a history of alloantibodies were recorded in the NRCC of whom 337 with severe HA and 12 with severe HB. Coagulation factor use, evaluated from treatment plans, was approximately 451,000,000 IU of FVIII for HA patients (7.5 IU/inhabitant), and approximately 53,000,000 IU of FIX for HB patients (0.9 IU/inhabitant).

Discussion: The prevalences of HA and HB fall within the ranges reported in more developed countries; the consumption of FVIII and FIX was in line with that of other European countries (France, United Kingdom) and Canada. The NRCC, with its bleeding disorder dataset, is a helpful tool for shaping public health policies, as well as planning clinical and epidemiological research projects.
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http://dx.doi.org/10.2450/2019.0211-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053520PMC
January 2020

miR-21 is overexpressed in NPM1-mutant acute myeloid leukemias.

Leuk Res 2015 Feb 18;39(2):221-8. Epub 2014 Nov 18.

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

MicroRNAs (miRs) play a key role in the pathogenesis of human malignancies and particularly in acute myeloid leukemias (AMLs) and are increasingly recognized as potential biomarkers and therapeutic targets. miR-21 is dysregulated in several types of cancers, including some hematologic malignancies, and plays a key role in carcinogenesis, disease recurrence and metastasis. However, no studies have specifically investigated the role of miR-21 in AMLs. In this study we analyzed the expression of miR-21 and of its target PDCD4 (Programmed Cell Death 4) during normal hematopoietic differentiation and in AMLs. Our results showed that: (i) miR-21 expression is strongly up-modulated during normal granulo/monocytic differentiation, while PDCD4 protein level is concomitantly downmodulated; (ii) miR-21 is frequently overexpressed in AML blasts, in association with a marked PDCD4 protein downmodulation; (iii) miR-21 expression level is particularly elevated in NPM1mutant AMLs. Together, these findings suggest that deregulated miR-21 expression may contribute to disease pathogenesis in NPM1-mutated AMLs.
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http://dx.doi.org/10.1016/j.leukres.2014.11.001DOI Listing
February 2015

Immunophenotypic features of acute myeloid leukaemia patients exhibiting high FLT3 expression not associated with mutations.

Br J Haematol 2011 Apr 20;153(1):33-42. Epub 2011 Feb 20.

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

FMS-related tyrosine kinase 3 (FLT3) mutations are found in 30% of cases of acute myeloid leukaemia (AML). In addition, recent studies have lead to the identification of about 10-15% of AML patients displaying high expression of FLT3, not associated with mutations of the receptor (FLT3 Wild-type High, FLT3WTH). These AMLs, as well as those displaying internal tandem duplication (ITD) are associated with an unfavourable prognosis. However, the biological features of these AMLs are poorly characterized. The present study explored the immunophenotypic features of FLT3WTH AMLs in 94 de novo cases of AML. The levels of FLT3 expression, as assessed by flow cytometry and FLT3 mutational status, was used to identify four AML subgroups: FLT3WTH (14/94); FLT3 Wild-type low (FLT3WTL, 48/94); FLT3 internal tandem duplication (FLT3ITD 26/94); FLT3 aspartic acid 835 (FLT3D835, 6/94). FLT3WTH and FLT3ITD were characterized by: high white blast cell counts; predominance of M4 and M5 French-American-British classification subtypes and associated expression of myelo-monocytic markers; high expression of CD123 and TRAIL-Rs; high expression of receptors for angiogenic growth factors. Addition of FLT3 Ligand to human CD34(+) or monocytic cells stimulated CD123 and TRAIL-R expression. These findings are of potential value for the development of new therapeutic strategies.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08577.xDOI Listing
April 2011

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

Transcriptional silencing of the ETS1 oncogene contributes to human granulocytic differentiation.

Haematologica 2010 Oct 30;95(10):1633-41. Epub 2010 Apr 30.

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

Background: Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid.

Design And Methods: Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA.

Results: NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. Expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells.

Conclusions: Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.
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http://dx.doi.org/10.3324/haematol.2010.023267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948087PMC
October 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

Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation.

Br J Haematol 2009 May 24;145(3):399-411. Epub 2009 Feb 24.

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

Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07627.xDOI Listing
May 2009

Interleukin (IL)-3/granulocyte macrophage-colony stimulating factor/IL-5 receptor alpha and beta chains are preferentially expressed in acute myeloid leukaemias with mutated FMS-related tyrosine kinase 3 receptor.

Br J Haematol 2009 Feb 13;144(3):376-87. Epub 2008 Nov 13.

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

The common beta chain subunit (beta(c)), also known as CDw131, shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding and signal transduction. The present study explored the expression of CDw131 in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131 expression were used to identify two AML subgroups characterized by low (75/105) and high (30/105) expression of this receptor chain. It was observed that (i) the level of CDw131 expression strictly correlated with the level of CD116 (GM-CSFalpha receptor chain) and CD123 (IL-3Ralpha chain); (ii) AMLs with high CDw131 expression were characterized by low CD34 expression and usually high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression frequently co-expressed receptors for angiogenic growth factors (vascular endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression were more cycling than those with low CDw131 expression; (v) AMLs with high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related) tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively activated Signal Transducer and Activator of Transcription-5 (STAT5). In conclusion, the analysis of the level of CDw131 expression enabled the identification of a subset of AMLs characterized by a high cycling status, the expression of myelo-monocytic markers, mutated FLT3 and the co-expression of receptors for angiogenic growth factors. These findings are of value for the development of new therapeutic strategies for the treatment of these AMLs.
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http://dx.doi.org/10.1111/j.1365-2141.2008.07491.xDOI Listing
February 2009

Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways.

Blood 2008 Sep 26;112(6):2450-62. Epub 2008 Jun 26.

Department of Clinical Sciences, University of Parma, Italy.

We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.
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http://dx.doi.org/10.1182/blood-2007-10-114348DOI Listing
September 2008

Resistance of acute myeloid leukemic cells to the triterpenoid CDDO-Imidazolide is associated with low caspase-8 and FADD levels.

Leuk Res 2008 Aug 4;32(8):1244-58. Epub 2008 Mar 4.

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

The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X(L) expression in leukemic blasts.
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http://dx.doi.org/10.1016/j.leukres.2007.12.008DOI Listing
August 2008

M4 and M5 acute myeloid leukaemias display a high sensitivity to Bortezomib-mediated apoptosis.

Br J Haematol 2007 Oct;139(2):194-205

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

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib, while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand, when used alone, did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally, analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study, further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug.
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http://dx.doi.org/10.1111/j.1365-2141.2007.06757.xDOI Listing
October 2007

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

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

Deregulation of apoptosis in acute myeloid leukemia.

Haematologica 2007 Jan;92(1):81-94

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

Apoptosis, or programmed cell death, is central to the development and homeostasis of the hematopoietic system. Dysregulation of apoptosis plays an important role in the development of a variety of human pathologies, including cancer, autoimmune diseases and neurodegenerative disorders. Particularly, studies carried out in the last years have shown that leukemia cells invariably have abnormalities in one or more apoptotic pathways, determining a survival advantage of these cells over their normal counterpart. Furthermore, abnormalities in the apoptotic response also play a role in the development of drug resistance by leukemic cells. The identification of the different components of the apoptotic pathways has enabled the detection of various biochemical defects present in leukemic cells compared to their normal counterpart. These defects contribute to the survival advantage of the leukemic clone over the normal hematopoietic cells and are also frequently associated with a low rate of response to standard chemotherapy treatment and with poor survival. Furthermore, these findings have also lead to the identification of many potential apoptotic targets for the development of new drugs targeting anti-apoptotic molecules abnormally expressed or regulated in leukemic cells. Many of these drugs restore the sensitivity of leukemic cells to apoptotic stimuli and some of them are under investigation at a clinical level.
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http://dx.doi.org/10.3324/haematol.10279DOI Listing
January 2007

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

In vitro dual effect of arsenic trioxide on hemopoiesis: inhibition of erythropoiesis and stimulation of megakaryocytic maturation.

Blood Cells Mol Dis 2006 Jan-Feb;36(1):59-76. Epub 2005 Dec 15.

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

Although the arsenic compounds are now widely utilized in clinics in the treatment of various tumors, their effects on normal hematopoiesis do not seem to have been explored. In the present study, we provide evidence that arsenic trioxide (As(2)O(3)) exerts in vitro a potent inhibitory effect on normal erythropoiesis and a stimulatory action on megakaryocytic differentiation. The effect of As(2)O(3) on erythroid and megakaryocytic differentiation was evaluated on both erythroleukemic cell lines K562 and HEL and on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid or megakaryocytic differentiation. The inhibitory effect of As(2)O(3) on erythropoiesis is related to: (a) the inhibition of Stat5 activation with consequent reduced expression of the target genes Bcl-X(L) and glycophorin-A; (b) the activation of an apoptotic mechanism that leads to the cleavage of the erythroid transcription factors Tal-1 and GATA-1, whose integrity is required for erythroid cell survival and differentiation; (c) the reduced expression of heat shock protein 70, required for GATA-1 integrity. The stimulatory effect of As(2)O(3) on normal megakaryocytopoiesis is seemingly related to upmodulation of GATA-2 expression and to stimulation of MAPK activity. These observations may have implications for the patients undergoing anti-leukemic treatment with this compound.
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http://dx.doi.org/10.1016/j.bcmd.2005.10.005DOI Listing
May 2006

Diphtheria toxin fused to variant human interleukin-3 induces cytotoxicity of blasts from patients with acute myeloid leukemia according to the level of interleukin-3 receptor expression.

Blood 2005 Oct 31;106(7):2527-9. Epub 2005 May 31.

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

Leukemic blasts from patients with acute myeloid leukemia (AML) frequently express high levels of the interleukin-3 receptor alpha chain (IL-3Ralpha). In the present study, we have explored the sensitivity of primary leukemic blasts obtained from 34 patients with AML to a diphtheria toxin (DT) composed of the catalytic and translocation domains of DT (DT388) fused to IL-3 (DT388IL-3) and to DT388 fused to a variant IL-3 with increased binding affinity (DT388IL-3[K116W]). On a molar basis, DT388IL-3[K116W] was significantly more active than DT388IL-3 in mediating leukemic cell killing. The rate of cell killing induced by the 2 DT/IL-3 fusion proteins was significantly correlated with the level of IL-3Ralpha/IL-3Rbeta expressed on leukemic blasts. These observations support a potential use of DT388IL-3[K116W] in the treatment of refractory AMLs and provide a simple biochemical parameter for the selection of eligible patients.
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http://dx.doi.org/10.1182/blood-2005-02-0540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895267PMC
October 2005

TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.

Haematologica 2005 May;90(5):612-24

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

Background And Objectives: The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, many cancer cells are resistant to apoptosis induction by TRAIL. The present study was designed to evaluate the sensitivity to TRAIL-induced apoptosis in acute myeloblastic leukemias (AML).

Design And Methods: TRAIL/TRAIL receptor (TRAIL-R) expression and sensitivity to TRAIL-mediated apoptosis were explored in 79 AML patients, including 17 patients with acute promyelocytic leukemia (APL).

Results: In non-APL AML we observed frequent expression of TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4), while TRAIL-R1 and TRAIL-R2 expression was restricted to AML exhibiting monocytic features. Total leukemic blasts, as well as AML colony-forming units (AML-CFU), were invariably resistant to TRAIL-mediated apoptosis. APL express membrane-bound TRAIL on their surface and exhibit a pattern of TRAIL-R expression similar to that observed in the other types of AML. Before, during and after retinoic acid treatment APL cells are TRAIL-resistant. The induction of granulocytic maturation of APL cells by retinoic acid was associated with a marked decline of TRAIL expression.

Interpretation And Conclusions: The analysis of experimental APL models (i.e., U937 cells engineered to express PML/RAR-Eo and NB4 cells) provided evidence that PML/RAR-Eo expression was associated with downmodulation of TRAIL-R1 and with resistance to TRAIL-mediated apoptosis. We suggest that AML blasts, including APL blasts, are resistant to TRAIL-mediated apoptosis, a phenomenon seemingly related to the expression of TRAIL decoy receptors on these cells. Finally, APL blasts express membrane-bound TRAIL that could confer an immunologic privilege to these cells.
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May 2005

Immunophenotypic features of acute myeloid leukemias overexpressing the interleukin 3 receptor alpha chain.

Leuk Lymphoma 2004 Aug;45(8):1511-7

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

In a previous study we have shown that the interleukin-3 receptor alpha chain (IL-3Ralpha) is over expressed in about 45% of acute myeloid leukemias (AMLs) and this phenomenon was associated with high blast cell counts at diagnosis, high rate of cycling of leukemic blasts and with a worse prognosis. Here we have investigated the immunophenotypic features of 125 AML patients subdivided into three groups (IL-3R(high), IL-3R(middle) and IL-3R(low)) according to the level of IL-3Ralpha expression. AMLs over expressing the IL-3Ralpha represent a subgroup of AMLs with a peculiar immunophenotype mainly consisting in the elevated expression of CD34 and several receptor membrane tyrosine kinases, such as c-kit and flt3, and in a usually low expression of myeloid-associated antigens such as CD11b, CD14 and CD15. These findings suggest that IL-3Ralpha + + + AMLs are blocked at an early stage of differentiation and express at elevated levels several growth factor receptors. It is proposed that these findings may further help to understand the mechanisms involved in the development of high-risk acute leukemias.
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http://dx.doi.org/10.1080/104281090310001646031DOI Listing
August 2004

A new complex rearrangement involving the ETV6, LOC115548, and MN1 genes in a case of acute myeloid leukemia.

Genes Chromosomes Cancer 2004 Nov;41(3):272-7

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.

A new complex rearrangement involving chromosome bands 5q13, 12p13, 22q11, and 3q12 was identified and characterized in a patient with acute myeloid leukemia. Fluorescence in situ hybridization showed the involvement of the ETV6 gene in 12p13. ETV6 primers were specifically designed for 3'- and 5'-RACE-PCR experiments, which led to the identification of the other two rearranged genes. The derivative chromosome 5 harbored a fusion of the ETV6 sequence with that of the LOC115548 gene. The two genes were placed in opposite orientation and did not encode a fusion protein. On the derivative chromosome 12, ETV6 was fused to the MN1 gene on chromosome 22. Also in this case, the insertion, within the MN1 sequence, of a portion of chromosome 3 prevented the formation of a fusion protein. Finally, the derivative chromosome 22 contained the 3' portions of both LOC115548 and MN1, and no fusion transcript with coding potential could be predicted. In conclusion, all chromosome breakpoints led to the truncation of the three involved genes in the absence of predicted fusion proteins. This study lends further support to the hypothesis that gene disruption resulting in either loss of function or haploinsufficiency may be relevant in acute myeloid leukemia pathogenesis.
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http://dx.doi.org/10.1002/gcc.20081DOI Listing
November 2004

Elevated expression of IL-3Ralpha in acute myelogenous leukemia is associated with enhanced blast proliferation, increased cellularity, and poor prognosis.

Blood 2002 Oct;100(8):2980-8

Departments of Hematology and Oncology, Virology, and Immunology, Istituto Superiore di Sanità, Rome, Italy.

We have investigated the expression of interleukin-3 receptor alpha (IL-3Ralpha) chain in primary blasts from 79 patients with acute myeloid leukemia (AML), 25 patients with B-acute lymphoid leukemia (B-ALL), and 7 patients with T-acute lymphoid leukemia (T-ALL) to evaluate a linkage between the expression of this receptor chain, blast proliferative status, and disease prognosis. Although IL-3Ralpha chain was scarcely expressed in most patients with T-ALL, it was overexpressed in 40% and 45% of patients with B-ALL and AML, respectively, compared with the levels observed in normal CD34(+) progenitors. The biological and clinical significance of this overexpression pattern was investigated in AML. At the biological level, elevated IL-3Ralpha expression was associated with peculiar properties of leukemic blasts, specifically in 3 areas. First, in all patients the blasts expressing elevated IL-3Ralpha levels exhibited higher cycling activity and increased resistance to apoptosis triggered by growth factor deprivation. Second, spontaneous signal transducer and activator of transcription 5 (Stat5) phosphorylation was observed in 13% of AML patients, all pertaining to the group of patients exhibiting high IL-3Ralpha expression. Third, following IL-3 treatment, Stat5 was activated at higher levels in blasts with elevated IL-3Ralpha expression. At the clinical level, a significant correlation was observed between the level of IL-3Ralpha expression and the number of leukemic blasts at diagnosis, and patients exhibiting elevated IL-3Ralpha levels had a lower complete remission rate and survival duration than those showing normal IL-3Ralpha levels. These findings suggest that in AML, deregulated expression of IL-3Ralpha may contribute to the proliferative advantage of the leukemic blasts and, hence, to a poor prognosis.
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http://dx.doi.org/10.1182/blood-2002-03-0852DOI Listing
October 2002

Human acute stem cell leukemia with multilineage differentiation potential via cascade activation of growth factor receptors.

Blood 2002 Jun;99(12):4634-7

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

The morphologic, immunophenotypic, genotypic, genomic, and functional features of an undifferentiated acute leukemia with stem cell features are reported. At light and electron microscopy, the leukemic population was represented by primitive progenitor cells with no evidence of differentiation. The blasts were CD34(+), AC133(+), CD71(-), HLA-DR(-), CD38(-/dim+), CD90(+), CD117(dim+), flt3(+); did not express B, T, or myeloid-associated antigens; and showed a germline configuration of the immunoglobulin and T-cell receptor. Genomic profiling documented the expression of early stem cell and myeloid-associated genes. Receptors for early-acting hemopoietic growth factors (HGFs) were detected, while receptors for unilineage HGF were not expressed. Incubation with the flt3 or Kit ligand induced the expression of unilineage HGF receptors, allowing these cells to respond to their respective ligands. Growth without differentiation was sustained only in the presence of early-acting HGF, namely flt3 ligand, while early and unilineage HGF gave rise to all types of hemopoietic colonies.
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http://dx.doi.org/10.1182/blood.v99.12.4634DOI Listing
June 2002