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Ugo Testa

Publications and source records attributed to Ugo Testa.

31 records · Page 2Linked to original sources
PubMed2003

Stem cell factor protects erythroid precursor cells from chemotherapeutic agents via up-regulation of BCL-2 family proteins.

Suppression of red blood cell production is a common complication of chemotherapy, causing anemia in a significant number of cancer patients. We have evaluated the sensitivity of human hematopoietic progenitors and erythroid precursor cells to chemotherapeutic drugs and found that probasophilic erythroblasts represent the stage of erythroid differentiation more vulnerable to the cytotoxic effects of myelosuppressive agents. Stem cell factor (SCF) supports proliferation and survival of early hematopoietic cells by binding to the c-kit receptor. In unilineage erythropoietic culture of CD34+ progenitors, short-term pretreatment of immature erythroid precursors with SCF results in protection from apoptosis induced by chemotherapeutic agents and restores normal proliferation and differentiation after removal of the cytotoxic stimulus. The levels of drug-induced caspase processing are significantly reduced in erythroblasts treated with SCF, indicating that activation of the c-kit receptor generates antiapoptotic signals acting before amplification of the caspase cascade. Accordingly, we found that SCF up-regulates Bcl-2 and Bcl-X L in erythroid precursors and that exogenous expression of these proteins protects erythroblasts from caspase activation and death induced by chemotherapeutic agents. These results suggest a possible mechanism for SCF-mediated protection of erythroid precursor cells from apoptosis and may contribute to devise new strategies for prevention and treatment of chemotherapy-induced anemia.

Antineoplastic Agents↗
PubMed2002

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

Base Sequence↗
PubMed2002

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

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.

Cell Culture Techniques↗
PubMed2002

Identification of the hemangioblast in postnatal life.

Postnatal CD34(+) cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34(+)KDR(+) cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34(+)KDR(+) cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34(+)KDR(-) cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription-polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34(+)KDR(+) cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds-that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34(+)KDR(+) cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors.

Adult↗
PubMed2002

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

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.

Antibodies, Monoclonal↗
PubMed2002

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

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.

Adult↗
PubMed2002

PLZF induces megakaryocytic development, activates Tpo receptor expression and interacts with GATA1 protein.

We investigated the expression of the PLZF gene in purified human hematopoietic progenitors induced to unilineage erythroid, granulocytic or megakaryocytic differentiation and maturation in serum-free culture. PLZF is expressed in quiescent progenitors: the expression level progressively rises through megakaryocytic development, whereas it gradually declines in erythroid and granulopoietic culture. To investigate the role of PLZF in megakaryopoiesis, we transduced the PLZF gene into the erythro-megakaryocytic TF1 cell line. PLZF overexpression upmodulates the megakaryocytic specific markers (CD42a, CD42b, CD61, PF4) and induces the thrombopoietin receptor (TpoR). The proximal promoter of the TpoR gene is activated in PLZF-expressing TF1 cells: in this promoter region, a PLZF DNA-binding site was identified by deletion constructs studies. Interestingly, PLZF and GATA1 proteins coimmunoprecipitate in PLZF-expressing TF1 cells: enforced expression of both PLZF and GATA1 in TF1 cells results in increased upregulation of megakaryocytic markers, as compared to exogenous PLZF or GATA1 alone, suggesting a functional role for the PLZF/GATA1 complex. Our data indicate that PLZF plays a significant stimulatory role in megakaryocytic development, seemingly mediated in part by induction of TpoR expression at transcriptional level. This stimulatory effect is potentiated by physical interaction of PLZF and GATA1, which are possibly assembled in a multiprotein transcriptional complex.

Binding Sites↗
PubMed2002

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

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.

Acute Disease↗
PubMed2002

Modulation by growth factors of the expression of interleukin 3 and granulocyte-macrophage colony-stimulating factor receptor common chain beta c.

Interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 5 (IL-5) exert their biological activities through interaction with cell-surface receptors that consist of two subunits, a specific a subunit and a common beta transducing subunit (beta c). We have evaluated the effect of growth factors on the expression of beta c in normal monocytes. Addition of either GM-CSF or M-CSF to monocytes elicited a marked increase of beta c chain expression, a phenomenon seemingly related to a stimulation of the transcriptional activity of this gene mediated through an enhancement of the PU.1 DNA binding activity. Interestingly, during the activation of beta c chain expression by growth factors a switch from the synthesis of the truncated betaIT to the full-length beta c was observed. Similar observations have been made also in the growth factor-dependent erythroleukemic cell line TF-1, showing that GM-CSF deprivation elicited a marked decrease of beta c chain expression.

Cells, Cultured↗
PubMed2002

Circulating hematopoietic progenitor cells in runners.

Because endurance exercise causes release of mediators and growth factors active on the bone marrow, we asked whether it might affect circulating hematopoietic progenitor cells (HPCs) in amateur runners [n = 16, age: 41.8 +/- 13.5 (SD) yr, training: 93.8 +/- 31.8 km/wk] compared with sedentary controls (n = 9, age: 39.4 +/- 10.2 yr). HPCs, plasma cortisol, interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), and the growth factor fms-like tyrosine kinase-3 (flt3)-ligand were measured at rest and after a marathon (M; n = 8) or half-marathon (HM; n = 8). Circulating HPC counts (i.e., CD34(+) cells and their subpopulations) were three- to fourfold higher in runners than in controls at baseline. They were unaffected by HM or M acutely but decreased the morning postrace. Baseline cortisol, flt3-ligand, IL-6, and G-CSF levels were similar in runners and controls. IL-6 and G-CSF increased to higher levels after M compared with HM, whereas cortisol and flt3-ligand increased similarly postrace. Our data suggest that increased HPCs reflect an adaptation response to recurrent, exercise-associated release of neutrophils and stress and inflammatory mediators, indicating modulation of bone marrow activity by habitual running.

Adult↗
PubMedDate not supplied

Role of Ets-1 in erythroid differentiation.

Members of the Ets gene family are known to be expressed in the hematopoietic tissue and some of them play a pivotal role in normal hematopoietic cell development. Ets-1 gene expression was analyzed in Friend Leukemia Cells (FLC) induced to erythroid differentiation by DMSO. We show that the level of Ets-1 protein and its binding activity decreases in FLC along erythroid differentiation of primary human progenitors. The same behavior was observed during normal erythroid differentiation. Moreover, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. These data indicate that a decrease in Ets-1 binding activity is required for a normal erythroid maturation and that a deregulated expression of this transcription factor may interfere with terminal erythroid differentiation.

Cell Differentiation↗