Laboratory of Molecular Haematopoiesis and Stem Cell Biology Dept. of Experimental and Clinical Medicine "G. Salvatore" University of Catanzaro Magna Graecia Biosciences Building, Level 7 University Campus 'Salvatore Venuta' - Germaneto 88100 Catanzaro - Italy
Education and training 1983 MD, University of Naples, Naples, Italy - Certificate of the Board of Physicians. 1983-1984 Postdoctoral fellow, Institute of Biochemistry, II Medical School, University of Naples; part-time attending physician, Laboratory of clinical biochemistry, II Medical School, University of Naples. 1984 Visiting fellow, Laboratory of experimental haematology, Dept. of Haematology - Oncology, Albert-Ludwigs University, Freiburg, FRG. 1985-1989 Visiting fellow, Gene structure and regulation programme, European Molecular Biology Laboratory, Heidelberg, FRG.
Positions held and academic responsibilities 1988-1992 Lecturer of Biochemistry, University of Reggio Calabria Medical School, Catanzaro, Italy. 1993-1995 Member of the Steering Committee of the Faculty of Pharmacy, Catanzaro, Italy. 1992-2000 Associate Professor of Biochemistry, University “Magna Graecia”, Catanzaro, Italy. 1995-2000 Project Leader, CEINGE Genetic Engineering Center, Naples, Italy. since 2000 Professor of Biochemistry, University “Magna Graecia”, Catanzaro, Italy. 2000-2003 Visiting Professor, Laboratory of developmental hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York, NY, USA. 1999-2007 Chairman of the PhD programme in Experimental immunology, University “Magna Graecia”, Catanzaro, Italy. 2004-2007 Dean of Research, University “Magna Graecia”, Catanzaro, Italy. since 2007 Chairman, PhD Programme in Molecular oncology, experimental immunology and development of novel therapeutic approaches, University of Catanzaro "Magna Græcia". since 2010 Member of the advisory Board of the Research Consortium "Tumor Stem Cells" of the Deutche Krebshilfe.
Scientific interests 1978-81 Analysis of the surface-membrane transport and intracellular accumulation of radioisotopes used as tumour scanning agents in normal and transformed chick embryo fibroblasts and mammalian cell lines (MD thesis). 1982-85 Study of the regulation of growth in normal and leukaemic haematopoietic cells. Characterisation of cytokine production in human leukocyte subpopulations and identification of autocrine mechanisms supporting the proliferation of leukaemic cells. 1985-89 Characterisation of the role and of the mechanism of action of interleukin-6 and transforming growth factor β in the regulation of genes encoding human acute phase proteins. Identification of cis-acting elements and DNA-binding factors that control the acute phase gene expression. since 1990 Dissection of molecular pathways that control survival/apoptosis, self-renewal, proliferation and gene expression in normal and malignant human haematopoietic stem cells. Design of novel therapeutic approaches for haematopoietic malignancies. Identification, cloning and characterisation of genes differentially expressed in the immature compartment of the haematopoietic system. Production of vectors for efficient gene transfer in haematopoietic stem cells. Functional characterisation of candidate oncogenes and regulatory molecules by enforced expression in haematopoietic stem and progenitor cells and analysis of the resulting phenotypes in vitro and in vivo.
Scientific Societies - European Life Sciences Organisation - International Society for Experimental Hematology - International Society for Stem Cell Research - Italian Society of Biochemistry - Associazione di Biologia Cellulare e del Differenziamento
Abstract: Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.
Abstract: Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells.
Abstract: The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but "physiological" levels of transgene expression in different hematopoietic lineages represents some primary features of a gene-transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic-specific regulatory element of the Wiskott-Aldrich syndrome (WAS) gene-spanning approximately 1,600 and 170 bp-that direct enhanced green fluorescent protein (EGFP) expression. The performance of vectors carrying the 1,600 and 170 bp fragments of the WAS gene promoter was compared with that of a vector carrying the UbiquitinC promoter in human cord blood CD34(+) cells and their differentiated progeny both in vitro and in vivo in non-obese diabetic mice with severe combined immunodeficiency. All vectors displayed a similar transduction efficiency in CD34(+) cells and promoted long-term EGFP expression in different hematopoietic lineages, with an efficiency comparable to, and in some instances (for example, the 170-bp promoter) superior to, that of the UbiquitinC promoter. Our results clearly demonstrate that LV containing fragments of the WAS gene promoter/enhancer region can promote long-term transgene expression in different hematopoietic lineages in vitro and in vivo and represent suitable and highly efficient vectors for gene transfer in gene-therapy applications for different hematological diseases and for research purposes. In particular, the 170-bp carrying vector, for its reduced size, could significantly improve the transduction/expression of large-size genes. STEM CELLS 2009;27:2815-2823.
Abstract: The early hematopoietic zinc finger protein/zinc finger protein 521 (EHZF/ZNF521) is a recently identified, 1131 amino-acid-long nuclear factor that contains 30 zinc fingers distributed in clusters throughout its sequence. A 13-AA motif, that binds to components of the nuclear remodelling and histone deacetylation (NuRD) complex and is conserved in several trascriptional co-repressors, is located at the amino-terminal end of the molecule. EHZF/ZNF521 expression is high in the most immature cells of the haematopoietic system and declines with differentiation. Its transcript is also abundant in brain, particularly in the cerebellum. Its murine counterpart, Evi3/Zfp521, is enriched in haematopoietic and neural stem cells, in cerebellar granule neuron precursors and in the developing striatum. Enforced expression of EHZF/ZNF521 in haematopoietic progenitors results in their expansion and in inhibition of differentiation. EHZF/ZNF521 is a member of the BMP signalling pathway and an inhibitor of the transcription factor OLF1/EBF1, implicated in the differentiation of neural progenitors and in the specification of the B-cell lineage. EHZF expression is observed in most acute myelogenous leukaemias and is particularly high in those with rearrangements of the MLL gene, where EHZF may contribute to the leukaemic phenotype. EHZF/ZNF521 is also abundant in medulloblastomas and other brain tumours. Taken together, the data available suggest a possible role for this factor in development, stem cell regulation and oncogenesis.
Abstract: The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53-NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.
Abstract: Nucleoporin 98 (NUP98) is a component of the nuclear pore complex that facilitates mRNA export from the nucleus. It is mapped to 11p15.5 and is fused to a number of distinct partners, including nine members of the homeobox family as a consequence of leukemia-associated chromosomal translocations. NUP98-HOXA9 is associated with the t(7;11)(p15;p15) translocation in acute myeloid leukemia (AML), myelodysplastic syndrome, and blastic crisis of chronic myeloid leukemia. Expression of NUP98-HOXA9 in murine bone marrow resulted in a myeloproliferative disease progressing to AML by 7-8 months. Transduction of NUP98 fusion genes into human CD34(+) cells confers a proliferative advantage in long-term cytokine-stimulated and stromal cocultures and in NOD-SCID engrafted mice, associated with a five- to eight-fold increase in hematopoietic stem cells. NUP98-HOXA9 expression inhibited erythroid and myeloid differentiation but enhanced serial progenitor replating. NUP98-HOXA9 upregulated a number of homeobox genes of the A and B cluster as well as MEIS1 and Pim-1, and downmodulated globin genes and C/EBPalpha. The HOXA9 component of the NUP98-HOXA9 fusion protein was protected from cullin-4A-mediated ubiquitination and subsequent proteasome-dependent degradation. In NUP98-HOX-transduced CD34(+) cells and cells from AML patients with t(7;11)(p15;p15) NUP98 was no longer associated with the nuclear pore complex but formed intranuclear aggregation bodies. Analysis of NUP98 allelic expression in AML and myelodysplastic syndrome showed loss of heterozygosity observed in 29% of the former and 8% of the latter. This was associated with poor prognosis.
Abstract: OBJECTIVE: To model human leukemogenesis by transduction of human hematopoietic stem cells (HSC) with genes associated with leukemia and expressed in leukemic stem cells. METHODS: Constitutive activation of Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute myeloid leukemia (AML). Retroviral vectors expressing constitutively activated Flt3 and STAT5A were used to transduce human cord blood CD34(+) cells and HSC cell self-renewal and differentiation were evaluated. RESULTS: We have demonstrated that retroviral transduction of Flt3 mutations into CD34(+) cells enhanced HSC self-renewal as measured in vitro in competitive stromal coculture and limiting-dilution week-2 cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased myelopoiesis were noted together with strong activation of STAT5A. Consequently, transduction studies were undertaken with a constitutively active mutant of STAT5A (STAT5A[1( *)6]) and here also a marked, selective expansion of transduced CD34(+) cells was noted, with a massive increase in self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture. Differentiation was biased to erythropoiesis, including erythropoietin independence, with myeloid maturation inhibition. The observed phenotypic changes correlated with differential gene expression, with a number of genes differentially regulated by both the Flt3 and STAT5A mutants. These included upregulation of genes involved in erythropoiesis and downregulation of genes involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also characterized primary Flt3-ITD(+) AML bone marrow samples. Isolation of leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR(-) phenotype was undertaken with Flt3-ITD(+) AML samples resulting in co-purification of early CAFC. Gene expression of LSC relative to the bulk leukemic population revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell proliferation. CONCLUSION: Myeloid leukemogenesis is a multi-stage process that can involve constitutively activated receptors and downstream pathways involving STAT5, HOX genes, and HLF.
Abstract: The H ferritin promoter spans approximately 150 bp, upstream of the transcription start and is composed by two cis-elements in position -132 (A box) and -62 (B-box), respectively. The A box is recognized by the transcription factor Sp1, and the B-box by a protein complex called Bbf, which includes the CAAT binding factor NF-Y. In this study we performed a functional analysis of an H ferritin promoter allele carrying a G to T substitution adjacent to the Bbf binding site, in position -69. In vitro studies with reporter constructs revealed a significantly reduced transcriptional activity of this allele compared to that of the w.t. promoter that was mirrored by a decrease in Bbf binding. In vivo, this variant genotype is accompanied by a reduced amount of the H mRNA in peripheral blood lymphocytes.
Abstract: The t(7;11)(p15;p15) translocation, observed in acute myelogenous leukemia and myelodysplastic syndrome, generates a chimeric gene where the 5' portion of the sequence encoding the human nucleoporin NUP98 protein is fused to the 3' region of HOXA9. Here, we show that retroviral-mediated enforced expression of the NUP98-HOXA9 fusion protein in cord blood-derived CD34(+) cells confers a proliferative advantage in both cytokine-stimulated suspension cultures and stromal coculture. This advantage is reflected in the selective expansion of hematopoietic stem cells as measured in vitro by cobblestone area-forming cell assays and in vivo by competitive repopulation of nonobese diabetic/severe combined immunodeficient mice. NUP98-HOXA9 expression inhibited erythroid progenitor differentiation and delayed neutrophil maturation in transduced progenitors but strongly enhanced their serial replating efficiency. Analysis of the transcriptosome of transduced cells revealed up-regulation of several homeobox genes of the A and B cluster as well as of Meis1 and Pim-1 and down-modulation of globin genes and of CAAT/enhancer binding protein alpha. The latter gene, when coexpressed with NUP98-HOXA9, reversed the enhanced proliferation of transduced CD34(+) cells. Unlike HOXA9, the NUP98-HOXA9 fusion was protected from ubiquitination mediated by Cullin-4A and subsequent proteasome-dependent degradation. The resulting protein stabilization may contribute to the leukemogenic activity of the fusion protein.
Abstract: To investigate the role of constitutively active internal tandem duplication (ITD) mutants of the Fms-like tyrosine kinase 3 (Flt3) receptor in leukemogenesis, we introduced the Flt3-ITD, W51, into human cord blood CD34+ cells and evaluated their phenotype in diverse hematopoietic assays. W51 expression resulted in a strong proliferative advantage and enhanced erythropoiesis as determined by immunophenotyping, colony assays, and molecular analyses. In MS-5 stromal cocultures, numerous early cobblestone areas (CAs) were generated within 10 to 14 days. Such W51-associated early CAs disappeared by 4 weeks, yet retained self-renewal properties as demonstrated by generation of secondary and tertiary CAs upon replating. This phenotype appears related to the expression of W51 since it was abolished by exposure to the FLT3 inhibitor, AG1295, but not to the c-kit inhibitor PD16. Wild-type Flt3-overexpressing CD34+ cells exposed to high levels of its physiologic ligand did not produce early CAs, highlighting differences in intracellular signaling between wild-type Flt3 and W51. W51-associated signal transducer and activator of transcription 5 (Stat5) activation plays a major role in this phenotype, although additional downstream targets of W51 may be relevant. Flt3-ITD+ acute myeloid leukemia (AML) blasts from patients invariably generated early AG1295-sensitive CAs in MS-5 cocultures, further validating the phenotype observed in transduced CD34+ cells.
Abstract: Activation of the transcription factor signal transducer and activator of transcription (STAT)5 is involved in various aspects of hematopoiesis, affecting cell proliferation, differentiation, and cell survival. Constitutive activation of STAT5 has also been associated with leukemic transformation. We overexpressed the constitutively active mutant STAT5A(1*6) in human cord blood CD34+ cells and evaluated the effects on the hematopoietic potential of stem cells in a variety of in vitro and in vivo systems. The observed phenotypic changes were correlated with differential gene expression patterns induced by STAT5A(1*6). Our data indicate that a persistent activation of STAT5A in human hematopoietic stem and progenitor cells results in their enhanced self-renewal and diverts differentiation to the erythroid lineage.
Abstract: Little is known about the molecular mechanisms that direct the transition from primitive to definitive hematopoiesis. In this study, we cocultured murine embryonic stem (ES) cells on OP9 stroma to induce hematopoietic differentiation as a model to study factors involved in the generation of adult hematopoietic stem cells (HSCs). Overexpression of the constitutively activated mutant signal transducer and activator of transcription (STAT) 5A(1*6) in ES cells facilitated the generation of cells that expressed the endothelial-hemangioblast marker Flk-1 within 5 days of coculture on OP9. The first CD41+/ CD45+/c-Kit+/(Flk-1)- hematopoietic cells arose in our culture conditions between days 5 and 7. Persistent activation of STAT5A greatly enhanced the generation of hematopoietic progenitors compared with controls, as determined by colony assays in methylcellulose. Moreover, whereas controls generated only a short transient wave of hematopoiesis lasting less than 3 weeks, expression of STAT5A(1*6) resulted in the generation of hematopoietic cobblestone area-forming cells (CAFCs) on OP9 that could be serially passaged onto new OP9, giving rise to second and third CAFCs that generated hematopoietic progenitors for > or = 5 weeks, indicating a role for STAT5A in HSC self-renewal in vitro. Several definitive hematopoietic genes were upregulated by STAT5A (1*6), as well as Runx1/AML1, vascular endothelial growth factor, oncostatin M receptor, HoxB4, Wnt5A, Delta-like-1, and Bmi-1. Furthermore, ES-derived hematopoietic cells expressing STAT5A(1*6) contributed to myeloid-lymphoid hematopoiesis in primary and secondary nonobese diabetic-severe combined immunodeficiency recipients, although no donor-derived cells could be detected after 7 weeks in the secondary recipients. These data indicate that a persistent activation of STAT5A allows the generation of ES-derived HSCs that can, at least for an intermediate period, contribute to hematopoiesis in vivo.
Abstract: The Notch family of transmembrane receptors has been implicated in the regulation of many developmental processes. In this study, we evaluated the role of Notch4 in immature hematopoietic progenitors by inducing, with retroviral transduction, enforced expression of Int-3, the oncogenic and constitutively active form of mouse Notch4. Int-3-transduced human myeloid leukemia (HL-60) cells demonstrated significantly delayed expression of differentiation markers following retinoic acid and 12-0-tetradecanoylphorbol 13-acetate treatment. Furthermore, HL-60 cells expressing Int-3 displayed a slower growth rate than cells infected with void virus, and accumulation in the G0/G1 phases of cell cycle. Transduction with deletion mutants of Int-3 defined the importance of individual domains of the protein (in particular, the ANK domain and the C-terminal domain) in the inhibition of differentiation and growth arrest of HL-60 cells. When mouse bone marrow enriched for stem cells (5-fluorouracil-resistant, lineage negative) was transduced and cultured for two weeks, the Int-3-transduced population displayed a lower expression of differentiation markers and a three- to five-fold higher frequency of colony-forming cells (CFU-GM/BFU-E) than control cultures. These results strongly support the notion that Notch signaling inhibits differentiation and promotes expansion of hematopoietic stem/progenitor cells.
Abstract: Comparison of the gene expression repertoire in human hematopoietic progenitors and mature leukocytes led to identification of a transcript expressed in CD34+cells and undetectable in differentiated cells. Sequencing of the cDNA (termed EHZF: early hematopoietic zinc finger) revealed 30 zinc fingers with 96% homology to mouse Evi3, a recently identified gene associated with the retroviral integration site in AKXD-27 B-cell lymphomas. EHZF and Evi3 share high homology with the transcription cofactor OAZ, implicated in the control of olfactory epithelium and B-lymphocyte differentiation and in the bone morphogenic protein (BMP) signal transduction. Here we show that (1) EHZF expression is abundant in human CD34+ progenitors and declines rapidly during cytokine-driven differentiation; (2) significant mRNA levels are found in most acute myelogenous leukemias; (3) in response to BMPs EHZF complexes SMADs 1 and 4, binds to, and enhances the transcriptional activity of, a BMP2/4 responsive element; (4) EHZF inhibits the transcriptional activity of early B-cell factor (EBF), a transcription factor essential for specification of the B-cell lineage. Taken together, our data suggest that EHZF is likely to play a relevant role in the control of human hematopoiesis and might be implicated in the development of hematopoietic malignancies.
Abstract: High-mobility group A (HMGA) proteins are non-histone nuclear proteins that bind DNA and several transcription factors. They are involved in the regulation of chromatin structure and function. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several human tumours. Rearrangements of the HMGA genes have been frequently detected in human benign tumours of mesenchymal origin, e.g. lipomas, lung hamartomas and uterine leiomiomas. HMGA proteins have been implicated in the control of cell growth and differentiation of the pre-adipocytic cell line 3T3-L1. In an attempt to better understand the role of HMGA1 proteins in haematological neoplasias and in the differentiation of haematopietic cells, we have investigated their expression in human leukaemias and in leukaemic cell lines induced to terminal differentiation. Here we report HMGA1 overexpression in most fresh human leukaemias of different origin and in several leukaemic cell lines. Moreover, differentiation of three cell lines towards the megakaryocytic phenotype was associated with HMGA1 protein induction, whereas induction of erythroid and monocytic differentiation generally resulted in reduced HMGA1 expression.
Abstract: The HOXA9 homeodomain protein is a key regulator of hematopoiesis and embryonic development. HOXA9 is expressed in primitive hematopoietic cells, and its prompt downregulation is associated with myelocytic maturation. Although transcriptional inactivation of HOXA9 during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOXA9 protein. Here we report that the CUL-4A ubiquitylation machinery controls the stability of HOXA9 by promoting its ubiquitylation and proteasome-dependent degradation. The homeodomain of HOXA9 is responsible for CUL-4A-mediated degradation. Interfering CUL-4A biosynthesis by ectopic expression or by RNA-mediated interference resulted in alterations of the steady-state levels of HOXA9, mirrored by impairment of the ability of 32D myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. These results revealed a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis.
Abstract: Bisphosphonates (BPs) are commonly used in the treatment of myeloma-associated osteolytic lesions. Recent reports have suggested that BPs may also exert direct antitumor effects on myeloma cells. Here, we show that the treatment of myeloma cell lines with the combination of the potent BP zoledronate and dexamethasone inhibits cell growth and synergistically induces apoptotic cell death, providing a rationale for potential applications in vivo.
Abstract: The monoclonal antibody UN1 was previously produced in our laboratory on the basis of selective reactivity with human thymocytes and has been classified as unclustered by the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. The antigen recognized by mAb UN1 was found to be expressed on the cell surface of immature human thymocytes, a subpopulation of peripheral T lymphocytes and on several fetal tissues including thymus. The UN1 antigen is purified from children's thymus by ion-exchange and affinity chromatography. Two-dimensional electrophoresis shows that the purified antigen displays microheterogeneity appearing as multiple spots over a pI range 4.4-5.0 at 100-120 kDa. Treatment with neuraminidase results in a retarded migration in SDS-PAGE, an increase in isoelectric point and a reduction in carbohydrate content, indicating a substantial content of sialic acid. Glycosidase digestion and lectin-binding analysis indicate that the carbohydrate residues are essentially O-linked. A preliminary analysis has detected the UN1 antigen in human breast carcinoma tissues but not in normal breast. The biochemical features and the pattern of expression of the UN1 antigen indicate that this molecule may have the characteristics typical of the family of cell-membrane-associated mucin-like glycoproteins; a number of these molecules are thought to have a role in cell-cell interaction, tumor progression and metastasis.
Abstract: The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5' deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.
Abstract: The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension cofirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.
Abstract: BACKGROUND & AIMS: Cell-mediated immunity is a feature of Crohn's disease (CD). The heterodimer interleukin (IL)-12, produced by phagocytes, induces T-cell cytokines, primarily interferon (IFN)-gamma. This study examined whether CD lamina propria mononuclear cells (LPMCs) express and release bioactive IL-12. METHODS: LPMCs were isolated from 13 patients with CD, 9 with ulcerative colitis (UC), and 13 controls. Messenger RNA for p40 and p35 IL-12 subunits was evaluated by reverse-transcription polymerase chain reaction. IL-12 was measured by enzyme-linked immunosorbent assay in LPMC culture supernatants. The INF-gamma-inducing effect of unstimulated LPMC supernatants was evaluated. RESULTS: Messenger RNA for both IL-12 subunits was detected in LPMCs of 11 of 13 patients with CD, 1 of 9 patients with UC, and 1 of 13 controls (P < 0.001). IL-12 was measured (10.5 +/- 2 pg/mL at 24 hours) in unstimulated CD LPMCs and was enhanced by pokeweed mitogen, lipopolysaccharide, and staphylococcal enterotoxin B. No IL-12 was detectable in 8 of 9 patients with UC and 12 of 13 control-unstimulated LPMCs. IL-12 induced by pokeweed mitogen and staphylococcal enterotoxin B in UC was lower than in CD and did not differ from controls. An IFN-gamma-inducing effect was restricted to unstimulated CD LPMC supernatants and was inhibited by an anti-IL-12 antibody in a dose-dependent fashion. CONCLUSIONS: IL-12 transcripts are expressed in CD intestinal tissues. CD LPMCs are up-regulated in their capability of releasing bioactive IL-12. Expression and release of bioactive IL-12 seem to differentiate CD from UC.
Abstract: CD69 is an early activation antigen of peripheral blood lymphocytes and is constitutively expressed on a wide variety of bone marrow-derived cells. To further characterize the distribution and understand the potential biological role of the molecule in normal and malignant hematopoiesis, we used a novel high affinity anti-CD69 mAb (UN6) and analyzed hematopoietic progenitor cells together with a panel of myeloid and lymphoid malignancies. We report that mobilized peripheral blood CD34+ cells display detectable levels of CD69 and that the density of membrane expression correlates with the immature phenotype CD34bright Thy-1bright cells. Furthermore, during cytokine-induced differentiation, the expression of CD69 is moderately down-regulated. Analysis of hematopoietic malignancies revealed that CD69 expression correlates with the immature myeloid phenotype. Taken together these data suggest a role of CD69 during the early phase of hematopoiesis and in the leukemic transformation.
Abstract: The common gamma-chain (gamma c) is a shared component of cell-surface receptors for the interleukins- 2, -4 and -7, and possibly others. We studied its expression in cells and cell lines of myeloid origin and found ubiquitous presence of gamma c mRNA in all cells examined. Differential regulation of gamma c expression was observed in myeloid cell lines induced to differentiate in vitro. In K-562 erythromyeloid cells, a sharp rise in the levels of gamma c mRNA and protein accompanied megakaryocytic, but not erythroid, differentiation. Surface binding of interleukin-2, as well as the transcripts for cognate receptor chains, were scarcely detectable in K-562 cells, whereas a significant increase in the binding of granulocyte-macrophage colony-stimulating factor specifically occurred during their megakaryocytic maturation. Our data indicate that expression of gamma c is a common feature of human myeloid cells, and suggest that its expression may be a requirement for human myelopoiesis.
Abstract: The monoclonal antibody (mAb) UN2 was generated upon immunization of a Balb/c mouse with human thymocytes. mAb UN2 recognized an antigen expressed by a subpopulation of human thymocytes and peripheral blood lymphocytes. In thymus, mAb UN2 recognized cortical cells; its expression was higher on CD3bright than on CD3dim thymocytes. This antigen was also detected on peripheral blood granulocytes, monocytes, platelets and on cell lines MOLT4, U937 and KG1. mAb UN2 was submitted to the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens, Boston, MA, 1993, and was assigned to the CD31. Expression of the UN2-recognized antigen in malignant lymphoid cells from 57 cases of B-cell chronic lymphoproliferative disease and 4 of B-cell acute lymphoblastic leukemia was analysed in flow cytometry. Among the 57 cases of B-cell chronic lymphoproliferative malignancies studied, 49 were classified as B-cell chronic lymphocytic leukemia. These showed high (86 +/- 8%) UN2 antigen expression. In 8 cases of hairy-cell leukemia the percentage of cells reacting with mAb UN2 was 42 +/- 4%; the fluorescence intensity of labelled cells was lower than that displayed by cells of B-cell chronic lymphocytic leukemia and comparable to that of normal lymphoid cells. mAb UN2 could prove useful in analysis of the lymphoid development and diagnostics of B-cell chronic lymphoproliferative disorders.
Abstract: A murine monoclonal antibody (mAb) UN1 was produced on the basis of selective reactivity with human thymocytes. Characterization of UN1 by immunofluorescence gave a high intensity of labeling with the majority of human thymocytes. Expression was preferentially associated with immature thymocytes (CD3dim) compared to mature cells, whereas only a subpopulation of peripheral blood lymphocytes was weakly stained. No specific binding to monocytes or granulocytes was detected. The T-cell lines HPB-ALL, H9 and MOLT-4 were all positively bound by UN1. Immunohistological staining of thymic tissues showed that mAb UN1 detected cells in both the cortex and medulla of fetal thymus, whereas the reaction in thymus samples from young children was mainly with medullar cells. By western blotting analysis, the antigen recognized by mAb UN1 corresponds to a membrane polypeptide with a molecular weight of approximately 120 kDa present on thymocytes and HPB-ALL cells. The mAb UN1 was submitted to the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens, Boston, 1993. UN1 did not cluster in any of the old or new clusters of differentiation discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggests that the UN1 antibody defines a previously undescribed molecule present on the cell surface of thymocytes and a minority of peripheral blood lymphocytes.
Abstract: We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins.
Abstract: Although several reports indicate proliferative and functional effects of human GH (hGH) on peripheral blood lymphocytes (PBL), no information is available about hGH receptor (GHR) expression in PBL subsets. Here, the surface membrane GHR levels were investigated in different human PBL subpopulations using a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody specific for the GHR (mAb263) in dual fluorochrome flow cytometric assays. Strong GHR expression was found in B-cells (CD20+), whereas CD2+ lymphocytes, including T-cells as well as natural killer cells, exhibited considerably lower levels of receptor expression. Similarly, using FITC-labeled recombinant hGH, receptor expression on CD20+ cells was significantly higher than that on CD2+ cells. Abundant expression of GHR in B-lymphocytes was confirmed by reverse transcriptase-polymerase chain reaction analysis of GHR messenger ribonucleic acid from isolated B-cells. Accordingly, the B-cell merits greater consideration as a GH target cell. The use of FITC-labeled mAb263 and hGH is of potential use for the study of GHR levels in patients exhibiting different types of growth disorders. Because of its high specificity for GHR, FITC-labeled mAb263 is also of considerable value for specifically demonstrating the presence of GHR, because hGH may interact with and act through PRL receptor, as shown previously in human neutrophils.
Abstract: This paper describes a new murine monoclonal antibody, UN5, raised against human thymocytes. This antibody recognizes a molecule of approximately 45 kDa on thymocytes. Flow cytometric analysis reveals a high intensity of labeling with the majority of thymocytes, whereas only CD20+ cells from peripheral whole-blood samples are weakly stained. Peripheral T cells, granulocytes, platelets and red blood cells do not express this antigen, while monocytes are only weakly labeled by UN5. Furthermore, the UN5 antibody discriminates between different types of B-cell malignancies, reacting with a subgroup of B-cell chronic lymphocytic leukemias and hairy cell leukemias, but not with the other kinds of hematopoietic malignancies tested. Antibody UN5 should prove a useful tool for the study of T-cell precursors and for analysis of both normal and neoplastic B cells.
Abstract: The effect of transforming growth factor beta (TGF beta) on the expression of a group of liver genes has been investigated in the hepatoma cell line Hep 3B. TGF beta induces a decrease of the basal level of apolipoprotein A-II (ApoA-II), retinol binding protein (RBP) and alpha-fetoprotein (alpha Fp). Furthermore, TGF beta efficiently antagonizes the IL-6-induction of hemopexin (Hpx) and haptoglobin (Hp) and alpha 1-acid glycoprotein (AGP). These effects of TGF beta are apparently mediated by post-transcriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGF beta on acute phase genes (e.g. ApoA-I and albumin), suggest a role for TGF beta in the regulation of expression of liver genes.
Abstract: High-density lipoprotein (HDL) is shown by ligand blotting to bind membrane-associated polypeptides with sizes of 60, 100 and 210 kDa. Binding was concentration-dependent and competed by excess unlabelled HDL. All the major apolipoproteins of HDL, apoA-I, apoA-II and apoA-IV, bound independently. The 100 kDa and 210 kDa HDL-binding activities were purified from membranes of Hep3B tumour cells by ion-exchange chromatography and gel filtration. The binding activities at 100 kDa and 210 kDa co-purified. After treatment with disulphide-reducing reagent, the 210 kDa band was no longer present and an increase was observed in the amount and binding ability of the 100 kDa polypeptide. The 100 kDa binding protein labelled at the cell surface with 125I could be immunoprecipitated after cross-linking to cell-surface-bound HDL. It is proposed that this HDL-binding activity, a putative cell-surface receptor for HDL, exists totally or in part as a high-molecular-mass complex composed of 100 kDa subunits.
Abstract: Human C-reactive protein (CRP) is the major acute phase reactant during acute inflammation. The human CRP promoter is expressed in an inducible and cell-specific manner when linked to the bacterial CAT gene and transfected into human hepatoma cell cultures. In this paper we analyze the effect of several recombinant cytokines or CRP promoter inducibility in human Hep3B cells. When cytokines are tested singly the major inducer of CRP-CAT fusions is interleukin-6 (IL-6). Maximal CAT gene expression, however, is only achieved when both interleukin-1 beta (IL-1 beta) and IL-6 are present. The response to the two cytokines is cooperative. Cooperativity is maintained when the CRP promoter is linked to a different coding region, that of the bacterial neomycin phosphotransferase II gene. With a series of 5' and 3' deletions we show the existence of two distinct and independent regions responsive to IL-6 and located upstream to the TATA box. The IL-1 effect is exerted at the level of downstream sequences that are probably important for optimal mRNA translatability or nuclear-cytoplasmic transport. Inducibility is not influenced by the activation of protein kinases C or A and does not require new protein synthesis.
Abstract: During the acute phase (AP) reaction the expression of a series of liver-specific genes coding for secretory proteins is either stimulated or suppressed by different cytokines released by activated monocytes. Transforming growth factor beta (TGF-beta) is a cytokine that, first identified for its ability to regulate cellular growth, has been gradually recognized to modulate several other functions. We have investigated the effect of TGF-beta on the expression of acute phase genes in liver cells. We found that TGF-beta selectively induces a specific decreases in the amount of mRNAs of genes negatively regulated during AP reaction, like albumin and apolipoprotein A-I (ApoA-I). The inhibitory effect of TGF-beta on the expression of negative AP genes is primarily post-transcriptional and it is very likely to be mediated via an enhancement of the turnover of both albumin and ApoA-I mRNAs.
Abstract: The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention has been focused on interleukin 6 (IL-6). We have previously shown that part of the AP response consists of the increase in the rate of transcription of the AP genes. Here we have treated the human hepatoma cell line Hep 3B with either crude MoCM or recombinant IL-6 and compared the effect of the two stimulants on the expression of both endogenous AP genes and recombinant plasmids introduced into the cells by transfection. The transfected plasmids contained the 5'-flanking region of AP genes fused to the coding region of the bacterial chloramphenicol acetyltransferase gene. We observe that the induction of mRNA accumulation of the endogenous genes corresponds to the transcriptional activation of the chloramphenicol acetyltransferase fusions. This is good evidence that the effect of IL-6 is totally or partially exerted at the level of transcription and that short segments of the 5'-flanking sequences of the inducible genes contain IL-6-responsive elements. Our results show that IL-6 is fully effective only on some of all the genes induced or repressed by MoCM, whereas others are only partially affected or totally nonresponsive.
Abstract: Haptoglobin is a plasma protein scarcely present in fetal but abundant in adult serum, where it is present at a concentration of approximately 150 mg/100 ml. In this paper we show by run-on experiments that the haptoglobin (Hp) gene is actively transcribed in adult but not in fetal liver nuclei. Studies with established cell lines indicate that the Hp gene is expressed in the hepatoma cells HepG2 but not in the hepatoma cell line Hep3B nor in HeLa cells. Plasmids carrying various segments of the 5' flanking region of the Hp gene fused to the chloramphenicol acetyl transferase (CAT) gene direct CAT transcription when introduced into HepG2 but are inactive in Hep3B and in HeLa cells, thus behaving like the resident chromosomal Hp gene. Deletion analysis defines a region, upstream to the transcription initiation site, essential for cell-specific expression. The Hp gene is induced in Hep3B cells by treatment with supernatant from LPS-stimulated monocytes (SMS), in a manner mimicking the acute phase reaction. We characterize the DNA segment necessary and sufficient for cell-specific expression of the Hp-CAT constructions in HepG2 and show that the same segment is also sufficient for acute phase induction in Hep3B.
Abstract: In this study we report that the ROHA-9 cell line, an IL 1-secreting EBV-transformed human B cell line, exhibits an autocrine pathway of growth. In fact, ROHA-9 cells spontaneously secreted an autoregulatory growth factor that co-purified with the constitutively secreted IL 1-like molecules. Accordingly, monocyte-derived human IL 1, free of other known biological activities, also stimulated the growth of ROHA-9 cells in a dose-dependent way. Human recombinant interleukin 2, recombinant IFN-alpha or IFN-gamma and purified IFN-beta were ineffective when used at concentrations up to 1 X 10(3) U/ml. Furthermore, mouse recombinant IL 1, HPLC-purified multi-colony stimulating factor and partially purified preparations of BCGF were ineffective when assayed for growth-promoting activity on ROHA-9 cells. Moreover, a rabbit polyclonal antibody and a mouse monoclonal antibody to human IL 1 molecules blocked the growth of ROHA-9 cells induced by the autologous growth factor and by human IL 1. Lastly, purified human IL 1 increased the clonal efficiency of ROHA-9 cells seeded at a low cell concentration, allowing the isolation of the ROHA-9MC3 subclone, which showed similar growth response specificity and was particularly sensitive to the mitogenic activity of human IL 1.
Abstract: The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.
