Showing papers by "Anna Rita Migliaccio published in 2007"

Molecular Profiling of CD34+ Cells in Idiopathic Myelofibrosis Identifies a Set of Disease-Associated Genes and Reveals the Clinical Significance of Wilms' Tumor Gene 1 (WT1)

Abstract: This study was aimed at the characterization of a gene expression signature of the pluripotent hematopoietic CD34(+) stem cell in idiopathic myelofibrosis (IM), which would eventually provide novel pathogenetic insights and/or diagnostic/prognostic information. Aberrantly regulated genes were revealed by transcriptome comparative microarray analysis of normal and IM CD34(+) cells; selected genes were also assayed in granulocytes. One-hundred seventy four differentially expressed genes were identified and in part validated by quantitative polymerase chain reaction. Altered gene expression was corroborated by the detection of abnormally high CD9 or CD164, and low CXCR4, membrane protein expression in IM CD34(+) cells. According to class prediction analysis, a set of eight genes (CD9, GAS2, DLK1, CDH1, WT1, NFE2, HMGA2, and CXCR4) properly recognized IM from normal CD34(+) cells. These genes were aberrantly regulated also in IM granulocytes that could be reliably differentiated from control polycythemia vera and essential thrombocythemia granulocytes in 100% and 81% of cases, respectively. Abnormal expression of HMGA2 and CXCR4 in IM granulocytes was dependent on the presence and the mutational status of JAK2(V617F) mutation. The expression levels of both CD9 and DLK1 were associated with the platelet count, whereas higher WT1 expression levels identified IM patients with more active disease, as revealed by elevated CD34(+) cell count and higher severity score. In conclusion, molecular profiling of IM CD34(+) cells uncovered a limited number of genes with altered expression that, beyond their putative role in disease pathogenesis, are associated with patients' clinical characteristics and may have potential prognostic application.

Inhibition of the SH3 domain-mediated binding of Src to the androgen receptor and its effect on tumor growth

Abstract: In human mammary and prostate cancer cells, steroid hormones or epidermal growth factor (EGF) trigger association of the androgen receptor (AR)-estradiol receptor (ER) (alpha or beta) complex with Src. This interaction activates Src and affects the G1 to S cell cycle progression. In this report, we identify the sequence responsible for the AR/Src interaction and describe a 10 amino-acid peptide that inhibits this interaction. Treatment of the human prostate or mammary cancer cells (LNCaP or MCF-7, respectively) with nanomolar concentrations of this peptide inhibits the androgen- or estradiol-induced association between the AR or the ER and Src the Src/Erk pathway activation, cyclin D1 expression and DNA synthesis, without interfering in the receptor-dependent transcriptional activity. Similarly, the peptide prevents the S phase entry of LNCaP and MCF-7 cells treated with EGF as well as mouse embryo fibroblasts stimulated with androgen or EGF. Interestingly, the peptide does not inhibit the S phase entry and cytoskeletal changes induced by EGF or serum treatment of AR-negative prostate cancer cell lines. The peptide is the first example of a specific inhibitor of steroid receptor-dependent signal transducing activity. The importance of these results is highlighted by the finding that the peptide strongly inhibits the growth of LNCaP xenografts established in nude mice.

p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival.

Abstract: Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.

Identification of two new synthetic histone deacetylase inhibitors that modulate globin gene expression in erythroid cells from healthy donors and patients with thalassemia.

Abstract: We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of γ-globin and/or β-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the γ/(γ+β) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of γ-globin mRNA and the γ/(γ+β) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of γ-globin and decreased by 2-fold that of β-globin mRNA, increasing the γ/(γ+β) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as γ/(γ+β) mRNA inducers in erythroblasts obtained from patients with β0 thalassemia. Progenitor cells from patients with β0 thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low β-globin levels but 10 times higher-than-normal levels of the α hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in β0 thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the γ/(γ+β) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with β0 thalassemia.

Pericyte coverage of abnormal blood vessels in myelofibrotic bone marrows

Abstract: Background and Objectives Myelofibrotic bone marrow displays abnormal angiogenesis but the pathogenic mechanisms of this are poorly understood. Since pericyte abnormalities are described on solid tumor vessels we studied whether vessel morphology and pericyte coverage in bone marrow samples from patients with myelofibrosis differed from that in samples from controls.Design and Methods We assessed the microvascular density (MVD), vessel morphology and pericyte coverage in bone marrows from 19 myelofibrosis patients and nine controls. We also studied the same parameters in two mouse models of myelofibrosis, with genetic alterations affecting megakaryocyte differentiation (i.e. one model with low GATA-1 expression and the other with over-expression of thrombopoietin).Results In myelofibrotic marrows, MVD was 3.8-fold greater than in controls (p

RPC cosmic ray tests in the ATLAS experiment

Abstract: Extensive tests with cosmic rays were performed with Resistive Plate Chamber (RPC) trigger chambers belonging to 6 muon stations of sector 13 installed in the ATLAS muon spectrometer. We illustrate the results of this pre-commissioning phase, which represents a test bench for the final commissioning of the ATLAS RPC system with cosmic rays.

Protein kinase Cα is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the Aγ globin-promoter in cellular models of hemoglobin switching

Abstract: PKCα was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the γ/γ + β globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 ± 12 vs. 7 ± 3, respectively), we tested the hypothesis that PKCα might affect γ-globin expression by measuring the levels of Aγ- or β-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual µLCRβprRlucAγprFluc reporter in the presence of transient expression of either the constitutively active (sPKCα) or catalytically inactive (iPKCα) PKCα. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 µM). In all the cells analyzed, sPKCα significantly increased (by two- to sixfold) the levels of luciferase activity driven by the Aγ-promoter and the Aγ-F/(Aγ-F + 2β-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the Aγ-driven luciferase activity and the Aγ-F/(Aγ-F + 2β-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCα might affect the activity of Aγ-promoter-driven reporters. J. Cell. Biochem. 101: 411–424, 2007. © 2007 Wiley-Liss, Inc.

Role of Thrombopoietin in Mast Cell Differentiation

Abstract: Mast cells are important elements of the body response to foreign antigens, being those represented either by small molecules (allergic response) or harbored by foreign microorganisms (response to parasite infection). These cells derive from hematopoietic stem/progenitor cells present in the marrow. However, in contrast with most of the other hematopoietic lineages, mast cells do not differentiate in the marrow but in highly vascularized extramedullary sites, such as the skin or the gut. Mast cell differentiation in the marrow is activated as part of the body response to parasites. We will review here the mast cell differentiation pathway and what is known of its major intrinsic and extrinsic control mechanisms. It will also be described that thrombopoietin, the ligand for the Mpl receptor, in addition to its pivotal rule in the control of thrombocytopoiesis and of hematopoietic stem/progenitor cell proliferation, exerts a regulatory function in mast cell differentiation. Some of the possible implications of this newly described biological activity of thrombopoietin will be discussed.

Status of the ATLAS Level-1 Central Trigger and Muon Barrel Trigger and First Results from Cosmic-Ray Data

Abstract: The ATLAS detector at CERN's Large Hadron Collider (LHC) will be exposed to proton-proton collisions from beams crossing at 40 MHz. A three-level trigger system will select potentially interesting events in order to reduce the readout rate to about 200 Hz. The first trigger level is implemented in custom-built electronics and makes an initial fast selection based on detector data of coarse granularity. It has to reduce the rate by a factor of 104 to less than 100 kHz. The other two consecutive trigger levels are in software and run on PC farms. We present an overview of the first-level central trigger and the muon barrel trigger system and report on the current installation status. Moreover, we show analysis results of cosmic-ray data recorded in situ at the ATLAS experimental site with final or close-to-final hardware.

The ATLAS level-1 trigger: Status of the system and first results from cosmic-ray data

Abstract: The ATLAS detector at CERN's Large Hadron Collider (LHC) will be exposed to proton–proton collisions from beams crossing at 40 MHz. At the design luminosity of 10 34 cm - 2 s - 1 there are on average 23 collisions per bunch crossing. A three-level trigger system will select potentially interesting events in order to reduce the readout rate to about 200 Hz. The first trigger level is implemented in custom-built electronics and makes an initial fast selection based on detector data of coarse granularity. It has to reduce the rate by a factor of 10 4 to less than 100 kHz. The other two consecutive trigger levels are in software and run on PC farms. We present an overview of the first-level trigger system and report on the current installation status. Moreover, we show analysis results of cosmic-ray data recorded in situ at the ATLAS experimental site with final or close-to-final hardware.

The ATLAS level-1 trigger: Status of the system and experience from commissioning with cosmic ray muons

Abstract: The detector at CERN's large hadron collider (LHC) was exposed to proton-proton collisions from beams crossing at 40 MHz A three-level trigger system will select potentially interesting events in order to reduce this rate to 100- 200 Hz A trigger decision is made by the Level-1 central trigger processor (CTP) reducing the incoming rate to less than 100 kHz The Level-1 decision is based on calorimeter information and hits in dedicated muon trigger detectors The final Level-1 trigger system is currently being installed in the experiment with completion expected in autumn 2007 Cosmic ray data are regularly recorded as an increasing fraction of the trigger system comes online We present an overview of the Level-1 trigger system architecture and report on the installation and commissioning process at the ATLAS experimental site Emphasis is put on the integration of the CTP with the calorimeter and muon trigger systems We show results from analyses of cosmic ray data recorded in situ and verify, where possible, that the Level-1 trigger meets the requirements and will be ready for data taking