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SERGIO FERRARI

Professore emerito
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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Pubblicazioni

2019 - Loss of expression of μ-protocadherin and protocadherin-24 in sporadic and hereditary nonpolyposis colorectal cancers [Articolo su rivista]
Losi, Lorena; Lancellotti, Cesare; Parenti, Sandra; Scurani, Letizia; Zanocco-Marani, Tommaso; Buffoli, Federico; Grassia, Roberto; Ferrari, Sergio; Grande, Alexis
abstract

Colorectal cancer (CRC) is a neoplastic disease in which normal mucosa undergoes a process of malignant transformation due to the progressive accumulation of molecular alterations affecting proto-oncogenes and oncosuppressor genes. Some of these modifications exert their carcinogenic potential by promoting a constitutive activation of the β-catenin signaling proliferation pathway, and when present, loss of cadherin expression also significantly contributes to the same effect. Using a combined approach of molecular and immunohistochemical analysis, we have previously demonstrated that most sporadic CRCs exhibit a down-regulated expression of a cadherin, named μ-protocadherin, that is generally observed in association with a higher proliferation rate and a worse prognosis. The aim of this report was to perform a comparative immunohistochemical assessment of μ-protocadherin and a similar cadherin, named protocadherin-24, in sporadic CRC and hereditary nonpolyposis colorectal cancer. The data obtained put in evidence that double-negative CRCs, lacking both the analyzed protocadherins, are more represented among sporadic tumors, whereas double-positive CRCs, maintaining their expression, exhibit an opposite trend. As expected, loss of protocadherin expression was accompanied by nuclear localization of β-catenin and increased positivity of the Ki-67 proliferation marker. This finding is consistent with the different clinical evolution of the 2 considered CRC sets according to which patients with hereditary nonpolyposis colorectal cancer experience a better prognosis as compared with those affected by a sporadic CRC.

2019 - Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation [Articolo su rivista]
Mammoli, F.; Parenti, S.; Lomiento, M.; Gemelli, C.; Atene, C. G.; Grande, A.; Corradini, R.; Manicardi, Agnese; Fantini, S.; Zanocco-Marani, T.; Ferrari, S.
abstract

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

2018 - Chromosome positioning in interphase nuclei of hematopoietic stem cell and myeloid precursor [Articolo su rivista]
Lomiento, Mariana; Mammoli, Fabiana; Mazza, Emilia Maria Cristina; Bicciato, Silvio; Ferrari, Sergio
abstract

Human myelopoiesis is an intriguing biological process during which multipotent stem cells limit their differentiation potential generating precursors that evolve into terminally differentiated cells. The differentiation process is correlated with differential gene expression and changes in nuclear architecture. In interphase, chromosomes are distinct entities known as chromosome territories and they show a radial localization that could result in a constrain of inter-homologous distance. This element plays a role in genome stability and gene expression. Here, we provide the first experimental evidence of 3D chromosomal arrangement considering two steps of human normal myelopoiesis. Specifically, multicolor 3D-FISH and 3D image analysis revealed that, in both normal human hematopoietic stem cells and myelod precursors CD14-, chromosomal position is correlated with gene density. However, we observed that inter-homologue distances are totally different during differentiation. This could be associated with differential gene expression that we found comparing the two cell types. Our results disclose an unprecedented framework relevant for deciphering the genomic mechanisms at the base of normal human myelopoiesis.

2018 - Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients [Articolo su rivista]
Ruberti, S; Bianchi, E; Guglielmelli, P; Rontauroli, S; Barbieri, G; Tavernari, L; Fanelli, T; Norfo, R; Pennucci, V; Fattori, G. Corbizi; Mannarelli, C; Bartalucci, N; Mora, B; Elli, L; Avanzini, M. A; Rossi, C; Salmoiraghi, S; Zini, R; Salati, S; Prudente, Z; Rosti, V; Passamonti, F; Rambaldi, A; Ferrari, S; Tagliafico, E; Vannucchi, A. M; Manfredini, R.
abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

2018 - KLF4 mediates the effect of 5-ASA on the b-catenin pathway in colon cancer cells [Articolo su rivista]
Parenti, Sandra; Montorsi, Lucia; Fantini, Sebastian; Mammoli, Fabiana; Gemelli, Claudia; Atene, Claudio Giacinto; Losi, Lorena; Frassineti, Chiara; Calabretta, Bruno; Tagliafico, Enrico; Ferrari, Sergio; Zanocco-Marani, Tommaso; Grande, Alexis
abstract

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing m-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores m-protocadherin expression and promotes the sequestration of b-catenin to the plasma membrane. Here, we show that 5-ASA–induced m-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated b-catenin inhibition is caused by m-protocadherin–dependent sequestration of b-catenin to the plasma membrane and by the direct binding of KLF4 to b-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA.

2017 - Epigenetically induced ectopic expression of uncx impairs the proliferation and differentiation of myeloid cells [Articolo su rivista]
Daniele, G.; Simonetti, G.; Fusilli, C.; Iacobucci, I.; Lonoce, A.; Palazzo, A.; Lomiento, M.; Mammoli, F.; Marsano, R. M.; Marasco, E.; Mantovani, V.; Quentmeier, H.; Drexler, H. G.; Ding, J.; Palumbo, O.; Carella, M.; Nadarajah, N.; Perricone, M.; Ottaviani, E.; Baldazzi, C.; Testoni, N.; Papayannidis, C.; Ferrari, S.; Mazza, T.; Martinelli, G.; Storlazzi, C. T.
abstract

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a lowmethylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1. We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.

2017 - Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells) [Articolo su rivista]
Perucca, Simone; Di Palma, Andrea; Piccaluga, Pier Paolo; Gemelli, Claudia; Zoratti, Elisa; Bassi, Giulio; Giacopuzzi, Edoardo; Lojacono, Andrea; Borsani, Giuseppe; Tagliafico, Enrico; Scupoli, Maria Teresa; Bernardi, Simona; Zanaglio, Camilla; Cattina, Federica; Cancelli, Valeria; Malagola, Michele; Krampera, Mauro; Marini, Mirella; Almici, Camillo; Ferrari, Sergio; Russo, Domenico
abstract

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygenlevel response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

2016 - Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes [Articolo su rivista]
Montorsi, Lucia; Parenti, Sandra; Losi, Lorena; Ferrarini, F; Gemelli, Claudia; Rossi, A; Manco, Gianrocco; Ferrari, Sergio; Calabretta, Bruno; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Grande, Alexis
abstract

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.

2016 - Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis [Articolo su rivista]
Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella
abstract

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.

2016 - Loss of zfp36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of zeb1, sox9 and macc1 [Articolo su rivista]
Montorsi, Lucia; Guizzetti, Filippo; Alecci, Claudia; Caporali, Andrea; Martello, Andrea; Giacinto Atene, Claudio; Parenti, Sandra; Pizzini, Silvia; Zanovello, Paola; Bortoluzzi, Stefania; Ferrari, Sergio; Grande, Alexis; ZANOCCO MARANI, Tommaso
abstract

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/β-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

2015 - Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations [Articolo su rivista]
Pennucci, Valentina; Zini, Roberta; Norfo, Ruggiero; Guglielmelli, Paola; Bianchi, Elisa; Salati, Simona; Sacchi, Giorgia; Prudente, Zelia; Tenedini, Elena; Ruberti, Samantha; Paoli, Chiara; Fanelli, Tiziana; Mannarelli, Carmela; Tagliafico, Enrico; Ferrari, Sergio; Vannucchi, ALESSANDRO MARIA; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract

Abnormal expression patterns of WT1-as, MEG3 and ANRIL long non-coding RNAs in CD34+ cells from patients with primary myelofibrosis and their clinical correlations.

2015 - MYB controls erythroid versus megakaryocyte lineage fate decision through the miR-486-3p-mediated downregulation of MAF [Articolo su rivista]
Bianchi, E.; Bulgarelli, J.; Ruberti, S.; Rontauroli, S.; Sacchi, G.; Norfo, R.; Pennucci, V.; Zini, R.; Salati, S.; Prudente, Z.; Ferrari, S.; Manfredini, R.
abstract

The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3′UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.

2015 - Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments [Articolo su rivista]
Hübner, Barbara; Lomiento, Mariana; Mammoli, Fabiana; Illner, Doris; Markaki, Yolanda; Ferrari, Sergio; Cremer, Marion; Cremer, Thomas
abstract

Background Previous studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization. Results Common human hematopoietic progenitors, myeloid precursor cells, differentiated monocytes and granulocytes analyzed by super-resolution fluorescence microscopy and electron microscopy revealed profound differences with respect to global chromatin arrangements, the nuclear space occupied by the interchromatin compartment and the distribution of nuclear pores. In contrast, we noted a consistent organization in all cell types with regard to two co-aligned networks, an active (ANC) and an inactive (INC) nuclear compartment delineated by functionally relevant hallmarks. The ANC is enriched in active RNA polymerase II, splicing speckles and histone signatures for transcriptionally competent chromatin (H3K4me3), whereas the INC carries marks for repressed chromatin (H3K9me3). Conclusions Our findings substantiate the conservation of the recently published ANC-INC network model of mammalian nuclear organization during human myelopoiesis irrespective of profound changes of the global nuclear architecture observed during this differentiation process. According to this model, two spatially co-aligned and functionally interacting active and inactive nuclear compartments (ANC and INC) pervade the nuclear space.

2015 - ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly [Articolo su rivista]
Selmi, Tommaso; Alecci, Claudia; Dell' Aquila, Miriam; Montorsi, Lucia; Martello, Andrea; Guizzetti, Filippo; Volpi, Nicola; Parenti, Sandra; Ferrari, Sergio; Salomoni, Paolo; Grande, Alexis; ZANOCCO MARANI, Tommaso
abstract

BACKGROUND: ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. METHODS: We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. RESULTS: Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. CONCLUSIONS: We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.

2014 - C-Myb Restrains Megakaryopoiesis through the Hsa-MiR-486-3p-Driven Down-Regulation of C-Maf [Abstract in Rivista]
Bianchi, Elisa; Bulgarelli, Jenny; Sacchi, Giorgia; Ruberti, Samantha; Norfo, Ruggiero; Rontauroli, Sebastiano; Pennucci, Valentina; Zini, Roberta; Salati, Simona; Prudente, Zelia; Ferrari, Sergio; Vannucchi, Alessandro M.; Manfredini, Rossella
abstract

INTRODUCTION The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. METHODS RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments. mRNA and miRNA expression for each sample were profiled by Affymetrix U219 GeneAtlas and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The effects of hsa-miR-486-3p overexpression and c-Maf silencing on CD34+ cells differentiation ability were studied by morphological and immunophenotypic analyses after liquid culture and by collagen-based clonogenic assay. Furthermore, gene expression changes in CD34+ cells upon hsa-miR-486-3p overexpression were profiled by Affymetrix U219. RESULTS The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs down-regulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p-overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes down-regulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as up-regulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. CONCLUSIONS Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p up-regulation and subsequent c-maf down-regulation as a new molecular mechanism through which c-Myb favours erythropoiesis while restraining megakaryopoiesis.

2014 - MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation [Articolo su rivista]
Gemelli, C.; Zanocco Marani, T.; Bicciato, S.; Mazza, E. M. C.; Boraschi, D.; Salsi, V.; Zappavigna, V.; Parenti, S.; Selmi, T.; Tagliafico, E.; Ferrari, S.; Grande, A.
abstract

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-. 7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-. 7R genes. © 2014 Elsevier B.V.

2014 - miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis [Articolo su rivista]
Norfo, Ruggiero; Zini, Roberta; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Guglielmelli, Paola; Bogani, Costanza; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Pietra, Daniela; Salmoiraghi, Silvia; Bisognin, Andrea; Ruberti, Samantha; Rontauroli, Sebastiano; Sacchi, Giorgia; Prudente, Zelia; Barosi, Giovanni; Cazzola, Mario; Rambaldi, Alessandro; Bortoluzzi, Stefania; Ferrari, Sergio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Investigators
abstract

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.

2014 - Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms [Articolo su rivista]
Tenedini, Elena; Bernardis, Isabella; Artusi, Valentina; Artuso, Lucia; Roncaglia, E.; Guglielmelli, P.; Pieri, L.; Bogani, C.; Biamonte, F.; Rotunno, G.; Mannarelli, C.; Bianchi, Elisa; Pancrazzi, A.; Fanelli, T.; MALAGOLI TAGLIAZUCCHI, Guidantonio; Ferrari, Sergio; Manfredini, Rossella; Vannucchi, A. M.; Tagliafico, Enrico
abstract

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2,000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in-vitro-expanded CD3+ T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG, and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest DIPSS-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing an NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

2013 - Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage [Articolo su rivista]
Salati, Simona; Lisignoli, G; Manfredini, C; Pennucci, Valentina; Zini, Roberta; Bianchi, Elisa; Norfo, Ruggiero; Facchini, A; Ferrari, Sergio; Manfredini, Rossella
abstract

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage.

2013 - Integrative Analysis Of mRNA/miRNA Expression Profiles Identified JARID2 As a Shared Target Of Deregulated Mirnas In Primary Myelofibrosis [Abstract in Rivista]
Zini, Roberta; Norfo, Ruggiero; Pennucci, Valentina; Bianchi, Elisa; Salati, Simona; Paola, Guglielmelli; Andrea, Bisognin; Vittorio, Rosti; Daniela, Pietra; Silvia, Salmoiraghi; Costanza, Bogani; Tiziana, Fanelli; Ruberti, Samantha; Sacchi, Giorgia; Prudente, Zelia; Giovanni, Barosi; Mario, Cazzola; Alessandro, Rambaldi; Stefania, Bortoluzzi; Ferrari, Sergio; Tagliafico, Enrico; Alessandro M., Vannucchi; Manfredini, Rossella
abstract

Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients [A.M. Vannucchi et al., Leukemia, 2013]. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported [P. Guglielmelli et al., Exp Hematol, 2007]. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Among 726 differentially expressed genes (DEG) we found that several putative cancer markers (WT1, ANGPT1) and several genes related to PMF progression, i.e. involved in megakaryocyte (MK) differentiation (NFE2, CD9), and fibrosis development (DLK1, LEPR1), were significantly more expressed in PMF samples than in the normal counterpart. Similarly, as regards the miEP, among 74 human differentially expressed miRNAs (DEM) in PMF compared to controls we found the upregulation of several miRNAs associated with hematological malignancies or known as oncomiRs (i.e. hsa-miR-155-5p [S. Jiang et al., Cancer Res, 2010], miRNAs belonging to the miR-17-92 cluster [L. Venturini et al., Blood, 2007]), and other aberrantly expressed miRNAs never described in hematopoiesis (i.e. hsa-miR-335-5p). Then, in order to construct regulatory networks of the functional human miRNA-target interactions, we performed an integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the miRNA expression profile with computational predicted targets and with the gene expression data. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. Of note, IA identified an interaction network (see Figure) in which the upregulated oncomirs miR-155-5p [R.M. O'Connel et al., J Exp Med, 2008], miR29a-3p [Y.C. Han et al., J Exp Med, 2010] and miR-19b-3p [K.J. Mavrakis et al., Nat Cell Biol, 2010] could explain the downregulation of targets whose lower expression was already described as involved in myeloproliferative phenotypes, such as NR4A3, CDC42, HMGB3. Additionally, IA disclosed the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of chronic myeloid malignancies, as a shared target of several upregulated miRNAs in PMF samples (i.e. miR-155-5p, miR-152-3p). Noteworthy, these miRNA-mRNA interactions were functionally confirmed by 3' UTR luciferase reporter assays. Next, in order to characterize the role of JARID2 in PMF pathogenesis, we performed RNAi-mediated gene silencing experiments on CD34+ cells of healthy donor. Interestingly, inhibition of JARID2 expression produces in silenced cells a significant increase of CD41 expression when compared with control (28.6±3.1% vs 15.3±1.8% at day 8, 52.6±7.6% vs 35.4±4.9% at day 12 of serum free liquid culture) and a remarkable increase in CFU-MK colonies (59.6±6.5% vs 39.8±5.9%). The values are reported as mean ± 2S.E.M from five independent experiments. Moreover, morphological analysis after May-Grunwald-Giemsa staining showed that JARID2 silencing induces in normal CD34+ cells a considerable enrichment in MK precursors at different stages of maturation. This study allowed the identification of different networks possibly involved in PMF onset, highlighting the potential contribution of miRNAs to PMF pathogenesis. Furthermore, for the first time, we demonstrated that the JARID2 downregulation in CD34+ cells might contribute to the abnormal megakaryopoiesis typical of PMF.

2013 - The Orosomucoid1 protein is involved in the vitamin D – mediated macrophage de-activation process [Articolo su rivista]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; ZANOCCO MARANI, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 - VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2012 - Hmgb3 Is Regulated by MicroRNA-206 during Muscle Regeneration [Articolo su rivista]
Maciotta, S; Meregalli, M; Cassinelli, L; Parolini, D; Farini, A; Fraro, Gd; Gandolfi, F; Forcato, Mattia; Ferrari, Sergio; Gabellini, D; Bicciato, Silvio; Cossu, G; Torrente, Y.
abstract

BACKGROUND: MicroRNAs (miRNAs) have been recently involved in most of human diseases as targets for potential strategies to rescue the pathological phenotype. Since the skeletal muscle is a spread-wide highly differentiated and organized tissue, rescue of severely compromised muscle still remains distant from nowadays. For this reason, we aimed to identify a subset of miRNAs major involved in muscle remodelling and regeneration by analysing the miRNA-profile of single fibres isolated from dystrophic muscle, which was here considered as a model of chronic damage. METHODOLOGY/PRINCIPAL FINDINGS: The miRNA-signature associated to regenerating (newly formed) and remodelling (resting) fibres was investigated in animal models of muscular dystrophies and acute damage, in order to distinguish which miRNAs are primary related to muscle regeneration. In this study we identify fourteen miRNAs associated to dystrophic fibres responsible for muscle regeneration and remodelling, and confirm over-expression of the previously identified regeneration-associated myomiR-206. In particular, a functional binding site for myomiR-206 was identified and validated in the 3'untranslated region (3'UTR) of an X-linked member of a family of sequence independent chromatin-binding proteins (Hmgb3) that is preferentially expressed in hematopoietic stem cells. During regeneration of single muscle fibres, Hmgb3 messenger RNA (mRNA) and protein expression was gradually reduced, concurrent with the up-regulation of miR-206. CONCLUSION/SIGNIFICANCE: Our results elucidate a negative feedback circuit in which myomiR-206 represses Hmgb3 expression to modulate the regeneration of single muscle fibres after acute and chronic muscle damage. These findings suggest that myomiR-206 may be a potential therapeutic target in muscle diseases

2012 - Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice [Articolo su rivista]
Salvestrini, V; Zini, Roberta; Rossi, L; Gulinelli, S; Manfredini, Rossella; Bianchi, Elisa; Piacibello, W; Caione, L; Migliardi, G; Ricciardi, Mr; Tafuri, A; Romano, M; Salati, Simona; Di Virgilio, F; Ferrari, Sergio; Baccarani, M; Ferrari, D; Lemoli, R. M.
abstract

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.

2012 - Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression [Articolo su rivista]
Zini, Roberta; Norfo, Ruggiero; Ferrari, Francesco; Bianchi, Elisa; Salati, Simona; Pennucci, Valentina; Sacchi, Giorgia; Carboni, Chiara; Ceccherelli, Gb; Tagliafico, Enrico; Ferrari, Sergio; Manfredini, Rossella
abstract

Histone deacetylase inhibitors represent a family of targeted anticancer compounds that are widely used against hematological malignancies. So far little is known about their effects on normal myelopoiesis. Therefore, in order to investigate the effect of histone deacetylase inhibitors on the myeloid commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with valproic acid (VPA). Our results demonstrate that VPA treatment induces H4 histone acetylation and hampers cell cycle progression in CD34(+) cells sustaining high levels of CD34 protein expression. In addition, our data show that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In fact, we demonstrate that VPA treatment is able to induce the expression of growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), which are crucial regulators of erythrocyte and megakaryocyte differentiation, and that the up-regulation of these genes is mediated by the histone hyperacetylation at their promoter sites. Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte commitment only. As a whole, our data suggest that VPA sustains the expression of stemness-related markers in hematopoietic stem/progenitor cells and is able to interfere with hematopoietic lineage commitment by enhancing erythrocyte and megakaryocyte differentiation and by inhibiting the granulocyte and mono-macrophage maturation.

2012 - ZFP36 expression impairs glioblastoma cell lines viability and invasiveness by targeting multiple signal transduction pathways. [Articolo su rivista]
Selmi, Tommaso; Martello, Andrea; Vignudelli, Tatiana; Ferrari, Erika; Grande, Alexis; Gemelli, Claudia; Salomoni, P; Ferrari, Sergio; ZANOCCO MARANI, Tommaso
abstract

RNA binding proteins belonging to the TIS11/TTP gene family regulate the stability of multiple targets. Their inactivation or deregulated expression has recently been related to cancer, and it has been suggested that they are capable of displaying tumor suppressor activities. Here we describe three new targets of ZFP36 (PIM-1, PIM-3 and XIAP) and show by different approaches that its ectopic expression is capable of impairing glioblastoma cell lines viability and invasiveness by interfering with different transduction pathways. Moreover, we provide evidence that compounds capable of inducing the expression of TIS11/TTP genes determine a comparable biological effect on the same cell contexts.

2011 - Alpha – 1 – acid glycoprotein-A is a new VDR transcriptional target involved in monocyte differentiation and activation processes. [Poster]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; ZANOCCO MARANI, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2011 - Down-regulation of μ-protocadherin expression is a common event in colorectal carcinogenesis [Articolo su rivista]
Losi, Lorena; Parenti, Sandra; Fabrizio, Ferrarini; Rivasi, Francesco; Margherita, Gavioli; Gianni, Natalini; Ferrari, Sergio; Grande, Alexis
abstract

We have previously reported that treatment of colorectal cancer cells with mesalazine results in the up-regulated expression of a novel member of the cadherin protein superfamily, named μ-protocadherin, which is able to sequester β-catenin on plasmatic membrane of treated cells inhibiting its proliferation signalling pathway. This finding suggests that μ-protocadherin could exert an oncosuppressive effect on colorectal epithelium. The purpose of our study was to assess whether μ-protocadherin expression is down-regulated during colorectal carcinogenesis. This issue was addressed by analyzing the messenger RNA and protein expression of μ-protocadherin in normal and tumor colorectal cell samples using a combination of quantitative real-time polymerase chain reaction, microarray analysis, and immunohistochemical examination. To better contextualize the role played by μ-protocadherin in the pathogenesis of colorectal cancer, this last assay was also extended to β-catenin, E-cadherin, and Ki-67 proteins. The results obtained evidenced that (1) levels of μ-protocadherin transcript were down-regulated in all the analyzed colorectal cancer samples as compared with normal mucosa; (2) expression of μ-protocadherin protein was completely lost in most analyzed colorectal cancer samples (71%); (3) μ-protocadherin retains β-catenin on the plasmatic membrane of normal colon enterocytes, which implies that β-catenin is released from this site and translocated to the nucleus in colorectal cancer cells. Our data consequently suggest that down-regulation of μ-protocadherin expression is a common event in colorectal carcinogenesis and might therefore play an important role in this pathologic process.

2011 - Expression profiling of FSHD-1 and FSHD-2 cells during myogenic differentiation evidences common and distinctive gene dysregulation patterns [Articolo su rivista]
S., Cheli; S., François; B., Bodega; F., Ferrari; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Sergio; E., Ginelli; R., Meneveri
abstract

BackgroundDetermine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis.Methodology/Principal FindingsBy in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.Conclusions/SignificanceFSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

2011 - Gene expression profiling in MDS and AML: potential and future avenues [Articolo su rivista]
K., Theilgaard Mönch; J., Boultwood; Ferrari, Sergio; K., Giannopoulos; J. M., Hernandez Rivas; A., Kohlmann; M., Morgan; B., Porse; Tagliafico, Enrico; C. M., Zwaan; J., Wainscoat; M. M., Van den Heuvel Eibrink; K., Mills; L., Bullinger
abstract

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients.

2011 - Liver Aging in Transplantation: Future Perspective on Donor-Recipient Age-Mismatch [Abstract in Rivista]
Grazi, Gl; Cescon, M; Olivieri, F; Capri, M; Lanzarini, C; Bellavista, E; Santoro, A; Martucci, M; Tenedini, Elena; Tagliafico, Enrico; Lazzarini, R; Remondini, D; Castellani, G; Ferrari, Sergio; Vasuri, F; D'Errico Grigioni, A; Procopio, A; Franceschi, C.
abstract

Research Areas:Gastroenterology & Hepatology; Surgery; Transplantation Web of Science Categories:Gastroenterology & Hepatology; Surgery; Transplantation

2011 - Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines [Articolo su rivista]
Ferrari, D; Gulinelli, S; Salvestrini, V; Lucchetti, G; Zini, Roberta; Manfredini, Rossella; Caione, L; Piacibello, W; Ciciarello, M; Rossi, L; Idzko, M; Ferrari, Sergio; Di Virgilio, F; Lemoli, Rm
abstract

Objective: Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions. Materials and Methods: ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology. Clonogenic and migration assays in vitro, as well as xenotransplant experiments in vivo, were performed to evaluate the effects of ATP on hMSCs proliferation and BM homing. Enzyme-linked immunosorbent assays were used to assess hMSCs cytokines production, whereas T-cell cultures demonstrated the immunoregulatory activity of ATP-treated hMSCs. Results: hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP-stimulated hMSCs underwent a downregulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly upregulated. The inhibitory activity of ATP on hMSCs proliferation was confirmed by assessing clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, the homing capacity of hMSCs to the BM of immunodeficient mice was significantly increased by pretreatment with ATP. Moreover, ATP increased the production of the proinflammatory cytokines interleukin-2, interferon-γ, and interleukin-12p70, while decreasing the anti-inflammatory cytokine interleukin-10, and this finding was associated with the reduced ability of MSCs to inhibit T-cell proliferation. Conclusions: Our data show that purinergic signaling modulates hMSCs functions and highlights a role for extracellular nucleotides in hMSCs biology.

2011 - TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources [Articolo su rivista]
Luca, Lenzi; Federica, Facchin; Francesco, Piva; Matteo, Giulietti; Maria, Pelleri; Flavia, Frabetti; Lorenza, Vitale; Raffaella, Casadei; Silvia, Canaider; Stefania, Bortoluzzi; Alessandro, Coppe; Gian, Danieli; Giovanni, Principato; Ferrari, Sergio; Pierluigi, Strippoli
abstract

BACKGROUND:Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input.RESULTS:TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified.CONCLUSIONS:TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes.

2010 - ANALISYS OF GENE EXPRESSION PROFILE DURING MYOGENESIS EVIDENCES DIFFERENT MOLECULAR DEFECTS AT THE BASIS OF FSHD-1 AND FSHD-2 PATHOGENESIS [Abstract in Atti di Convegno]
Cheli, Stefania; Francois, Stéphanie; Bodega, Beatrice; Ferrari, Francesco; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Sergio; Ginelli, Enrico; Meneveri, Raffaella
abstract

Germ line cell-derived pluripotent stem cells (GPSCs) are derived from spermatogonial stem cells and are similar to embryonic stem (ES) cells in that they can proliferate intensively and differentiate into a variety of cell types. Previous studies have revealed some inherent differences in gene expression between undifferentiated mouse ES cells and GPSCs. Our aims were to generate functional hepatocytes from mouse GPSCs in vitro and to investigate whether the differences in gene expression may impact on the hepatocyte differentiation capacity of the GPSCs compared with ES cells. Mouse GPSCs and ES cells were induced to differentiate into hepatocytes through embryoid body formation, with very high efficiency. These hepatocytes were characterized at cellular, molecular, and functional levels. The GPSCderived hepatocytes expressed hepatic markers and were metabolically active as shown by albumin and haptoglobin secretion, urea synthesis, glycogen storage, and indocyanine green uptake. We also performed an unprecedented DNA microarray analysis comparing different stages of hepatocyte differentiation. Gene expression profiling demonstrated a strong similarity between GPSC and ES cells at different stages of induced hepatic differentiation. Moreover, Pearson correlation analysis of the microarray datasets suggested that, at late hepatic differentiation stages, the in vitro-derived cells were closer to fetal mouse primary hepatocytes than to those obtained from neonates. We have shown for the first time that adult GPSCs can be induced to differentiate into functional hepatocytes in vitro. Moreover, our ongoing in vivo work shows that GPSC-derived hepatocytes can colonize the liver of monocrotaline-treated, partially hepatectomised mice. These GPSC-derived hepatocytes thus offer great potential for cell replacement therapy for a wide variety of liver diseases.

2010 - c-Myb supports erythropoiesis by transactivating KLF1 and LMO2 expression [Abstract in Atti di Convegno]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Ferrari, Sergio; Manfredini, Rossella
abstract

The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+ cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day. Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced cells, while the erythroid population was strongly decreased. Morphological evaluation of May Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression profiling of c-Myb silenced CD34+ cells enabled us to identify new putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Functional rescue experiments showed that the retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to rescue, at least in part, the impaired erythroid differentiation. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision.

2010 - c-Myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression. [Articolo su rivista]
Bianchi, Elisa; Zini, Roberta; Salati, Simona; Tenedini, Elena; Norfo, Ruggiero; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Sergio
abstract

The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-Myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity towards the macrophage and megakaryocyte lineages, while erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphological and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-Myb-silenced CD34+ cells identified the transcription factors KLF1 and LMO2 as putative targets which can account for c-Myb knockdown effects. Indeed, Chromatin Immunoprecipitation and Luciferase reporter assay demonstrated that c-Myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-Myb silencing, while only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-Myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

2010 - HGM 2010 Programme / Abstract [Abstract in Rivista]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning-on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNAs profiles from human CD34+ hematopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors, that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically up-regulated in one single cell progeny and their putative target genes, which resulted down-regulated. Among the up-regulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitors fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates the regulation of hematopoietic progenitors fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-miR-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

Cell fate decisions in the hematopoietic system appear to be directed by an antagonistic or synergistic interplay of transcription factors that pivot immature blood progenitors for cell specification. Multipotent progenitors initially trigger a promiscuous transcriptional program and, as soon as they commit to a restricted fate, they reinforce unilineage gene expression and withdraw transcripts affiliated with alternative blood cell types. MicroRNAs appear to be especially pertinent in driving this particular behavior representing a new component of the hematopoietic gene regulatory network. In fact, the archetypal microRNA can potentially regulate hundreds of genes even if most targets contain isolated microRNA recognition sites that may be inadequate for complete gene silencing. According to Bartel’s theory, microRNAs mediated post-transcriptional control offers a more flexible and rapid way of tuning genes compared to transcriptional control (Bartel DP and Chen CZ, Nat Rev Genet 2004). These issues encouraged some investigators to explore the association of microRNAs and genes expression profiles obtained from the same cell type and advocated that microRNAs evolved to regulate gene expression programs and remove gene products unnecessary or potentially dangerous more rapidly than might occur by natural decay. Although many studies addressed the role of microRNAs during the normal myeloid differentiation process, only Georgantas and co-workers focused onto the impact of microRNAs on mRNA expression levels but limited the analyses to data obtained from human CD34+ stem/progenitor cells (Georgantas RW 3rd et al, PNAS 2007). In order to shed light onto the interplay of mRNAs and microRNAs during the normal myeloid commitment and verify that increased expression of a microRNA is skillful to modulate the levels of corresponding target mRNAs, we obtained microRNAs profiles from CD34+ hematopoietic progenitor cells (CD34 HPCs) and in-vitro differentiated precursors: erythroblasts, megakaryoblasts, monoblasts and myeloblasts (ERY, MKC, MONO and MYELO). We therefore analyzed these microRNA expression profiles together with the gene expression profiles of the same populations and observed that for the most part of the microRNAs specifically up-regulated in one single progeny an inverse correlation between microRNAs and down-regulated putative targets expression levels occurs, i.e. down-regulated genes showed an enrichment for the conserved putative targets of up-regulated microRNA. Among these microRNAs, hsa-miR-299-5p emerged as an interesting candidate to demonstrate how the integrated analysis of microRNA and mRNA expression data can help shedding light on the regulatory mechanisms governing cell differentiation. In particular, we used hsa-miR-299-5p to prove that the forced expression of a single lineage-specific microRNA is able to control the cell fate of CD34 HPCs grown in multilineage culture conditions. Clonogenic and liquid culture differentiation assays after gain- and loss-of-function experiments revealed that indeed hsa-miR-299-5p regulates hematopoietic progenitors fate modulating megakaryocytic-granulocytic versus erythroid-monocytic development.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Articolo su rivista]
Tenedini, Elena; Roncaglia, Enrica; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio; Ferrari, Francesco
abstract

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort, which, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes, which resulted in downregulation. Among the upregulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, F; Orlandi, C; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio
abstract

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning-on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNAs profiles from human CD34+ hematopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors, that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically up-regulated in one single cell progeny and their putative target genes, which resulted down-regulated. Among the up-regulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitors fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates the regulation of hematopoietic progenitors fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Mesalazine inhibits the beta-catenin signalling pathway acting through the upregulation of mu-protocadherin gene in colo-rectal cancer cells [Articolo su rivista]
Parenti, Sandra; Ferrarini, F; Zini, Roberta; Montanari, Monica; Losi, Lorena; Canovi, B; Ferrari, Sergio; Grande, Alexis
abstract

BACKGROUND: Several reports indicate that mesalazine (5-aminosalicylic acid, 5-ASA) is a promising candidate for the chemoprevention of colo-rectal cancer because of its ability to reach the purpose avoiding the unwanted side effects usually associated with prolonged administration of nonsteroidal anti-inflammatory drugs. This activity of 5-ASA is probably the consequence of a number of effects determined on colo-rectal cancer cells, consisting of reduced proliferation, increased apoptosis and activation of cell cycle checkpoints and DNA repair processes. A recent observation has suggested that inhibition of beta-catenin signalling could induce these cellular effects. AIM: To characterize better the capacity of 5-ASA to inhibit the beta-catenin signalling pathway. METHODS: Genes belonging to the beta-catenin signalling pathway were analysed in colo-rectal cancer cell lines treated with 5-ASA using a combination of laboratory assays that are able to detect their phenotypic expression and functional activity. RESULTS: The results obtained indicated that 5-ASA induces the expression of a protein called mu-protocadherin that belongs to the cadherin superfamily and is able to sequester beta-catenin on the plasmatic membrane of treated cells hampering its function. CONCLUSION: These findings suggest that mu-protocadherin might be employed as a biological marker to monitor the chemopreventive efficacy of 5-ASA.

2010 - ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with Stat5b Pathway. [Articolo su rivista]
Vignudelli, Tatiana; Selmi, Tommaso; A., Martello; Parenti, Sandra; Grande, Alexis; Gemelli, Claudia; ZANOCCO MARANI, Tommaso; Ferrari, Sergio
abstract

ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3'-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by post-transcriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3' untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, since cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

2009 - Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells. the crucial role of PTEN. [Articolo su rivista]
Cellai, C; Laurenzana, A; Bianchi, Elisa; Sdelci, S; Manfredini, Rossella; Vannucchi, Am; Caporale, R; Balliu, M; Mannelli, F; Ferrari, Sergio; Bosi, A; Miniati, D; Cocco, Pl; Veronneau, S; Stankova, J; Paoletti, F.
abstract

OBJECTIVE:This study aimed to investigate the mechanisms of action of WEB-2170, an inverse agonist of platelet-activating factor receptor, capable of inducing apoptosis in human acute myelogenous leukemia (AML) cells.MATERIAL AND METHODS:Gene expression profiling followed by cytofluorimetric, morphologic, and biologic analyses were used to monitor WEB-2170 effects in AML cell lines (ie, NB4, KG1, NB4-MR4, THP1, and U937) and blasts from patients with different AML (M0-M5) subtypes. PTEN silencing with small interfering RNA was also performed.RESULTS:We have demonstrated that drug-mediated cytostasis/apoptosis in NB4 cells is characterized by upregulation of cyclin G2, p21/WAF1, NIX, TNF-alpha, and PTEN expression, and downregulation of cyclin D2 and BCL2 expression. We observed an increase in PTEN protein accompanied by a decrease in phospho-extracellular signal-regulated kinase 2 (ERK2) and phospho-AKT, and by forkhead box O3a (FOXO3a) cytoplasmic-nuclear translocation; the mitochondrial cytochrome C release and PARP cleavage marked the late apoptotic steps. We have found that WEB-2170 triggered apoptosis in NB4, KG1, and NB4-MR4 cells where PTEN was expressed, but not in THP1 and U937 cells where PTEN was absent. Finally, we show that PTEN silencing in NB4 cells by PTEN-specific small interfering RNA resulted in a significant reduction of drug-induced apoptosis.CONCLUSION:We demonstrated that WEB-2170 is a powerful antileukemic agent with interesting translational opportunities to treat AML and described mechanisms of drug-induced intrinsic and extrinsic apoptosis both in AML cell lines and blasts from AML patients by addressing PTEN as the master regulator of the whole process.

2009 - Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib [Articolo su rivista]
Lemoli, Rm; Salvestrini, V; Bianchi, Elisa; Bertolini, F; Fogli, M; Amabile, M; Tafuri, A; Salati, Simona; Zini, Roberta; Testoni, N; Rabascio, C; Rossi, L; Martin Padura, I; Castagnetti, F; Marighetti, P; Martinelli, G; Baccarani, M; Ferrari, Sergio; Manfredini, Rossella
abstract

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin–CD34–) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34– cells are leukemic. CML Lin–CD34– cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin–CD34– cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34+ cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin–CD34– cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin–CD34– cells in vitro. Moreover, leukemic CD34– cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34– leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

2009 - Molecular profile of CD34+ stem/progenitor cells according to JAK2V617F mutation status in essential thrombocythemia. [Articolo su rivista]
Catani, L; Zini, Roberta; Sollazzo, D; Ottaviani, E; Vannucchi, Am; Ferrari, Sergio; Baccarani, M; Vianelli, N; Lemoli, Rm; Manfredini, Rossella
abstract

Essential thrombocythemia (ET) is mainly characterized by the abnormal proliferation of a malignant megakaryocytic clone and by persistent thrombocytosis. Recently, a JAK2 mutation (JAK2V617F) has been reported in ET and other myeloproliferative neoplasms.

2009 - Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation [Articolo su rivista]
Coppe, A; Ferrari, F; Bisognin, A; Danieli, G. A.; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, S.
abstract

Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation.

2009 - Regeneration and repair in multiple sclerosis: The role of cell transplantation [Articolo su rivista]
Pluchino, S; Zanotti, L; Brini, E; Ferrari, Sergio; Martino, G.
abstract

Physiological (spontaneous) and reactive (reparative) regenerative processes are fundamental part of life and greatly differ among the different animals and tissues. While spontaneous regeneration naturally occurs upon cell attrition, reparative regeneration occurs as a consequence of tissue damage. Both spontaneous and reparative regeneration play an important role in maintaining the normal equilibrium of the central nervous system (CNS) as well as in promoting its repair upon injury. Cells play a critical role in reparative regeneration as regenerating structures (cells or tissues) depend on the proliferation without (de)differentiation of parenchymal cells surviving to the injury, proliferation of stem (progenitor) cells resident in the injured tissue, dedifferentiation of mature cells in the remaining tissue, or by the influx of stem cells originating outside the damaged tissue. Considering the central role of stem and progenitor cells in regeneration, a spur of experimental stem cell-based transplantation approaches for tissue (e.g. CNS) repair has been recently generated. This review will focus on the therapeutic efficacy of different sources of somatic stem cells – and in particular on those of neural origin – in promoting CNS repair in a chronic (auto)immune-mediated inflammatory disorder such as multiple sclerosis.

2009 - TFE3 transcription factor regulates the expression of MAFB during macrophage differentiation [Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Parenti, Sandra; Gemelli, Claudia; Condorelli, F; Martello, A; Selmi, Tommaso; Grande, Alexis; Ferrari, Sergio
abstract

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

2008 - Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells [Articolo su rivista]
Salati, Simona; Zini, Roberta; Bianchi, Elisa; Testa, Anna; Mavilio, Fulvio; Manfredini, Rossella; Ferrari, Sergio
abstract

CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.

2008 - Silencing CD34 antigen in human hematopoietic stem cells [Articolo su rivista]
Zini, Roberta; Salati, Simona; Bianchi, Elisa; Ferrari, Sergio; Manfredini, Rossella
abstract

CD34 is a highly glycosylated transmembrane protein strongly expressed on hematopoietic stem/progenitor cells (HSPCs); despite its importance as a marker of HSPCs, its function is still poorly understood, even if a role in cell adhesion has been demonstrated. In order to characterize the function of CD34 antigen in human HSPCs, we evaluated by small interfering RNAs (siRNAs) mediated gene silencing the role of CD34 antigen in HSPCs differentiation. By using the Nucleofection Amaxa technology for siRNA transfection in HSPCs, we obtained a rapid and effective down-regulation of the CD34 antigen. In this paper, we have demonstrated that CD34 silencing in HSPCs enhances their granulocyte and megakaryocyte differentiation and reduces erythroid maturation as shown by clonogenic assay, morphological analysis and expression of differentiation markers. In agreement with these results, the gene expression profile of HSPCs-silenced cells reveals the up-regulation of genes involved in granulocyte and megakaryocyte commitment and the down-regulation of erythroid genes. These data indicate that CD34 transmembrane protein promotes the differentiation of CD34+ hematopoietic progenitors towards the erythroid lineage at the expense of granulocyte and megakaryocyte ones.

2008 - The vitamin D3/Hox-A10 pathway supports MafB function during the monocyte differentiation of human CD34+ hemopoietic progenitors [Articolo su rivista]
Gemelli, Claudia; Orlandi, Claudia; ZANOCCO MARANI, Tommaso; Martello, A; Vignudelli, Tatiana; Ferrari, Francesco; Montanari, Monica; Parenti, Sandra; Testa, Anna; Grande, Alexis; Ferrari, Sergio
abstract

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

2007 - Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity [Articolo su rivista]
Salati, Simona; Bianchi, Elisa; Zini, Roberta; Tenedini, Elena; Quaglino, Daniela; Manfredini, Rossella; Ferrari, Sergio
abstract

BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils.

2007 - Gene expression analysis of angioimmunoblastic lymphoma indicates derivation from T follicular helper cells and vascular endothelial growth factor deregulation [Articolo su rivista]
PICCALUGA P., P; Agostinelli, C; Califano, A; Carbone, A; Fantoni, L; Ferrari, Sergio; Gazzola, A; Gloghini, A; Righi, S; Rossi, M; Tagliafico, Enrico; ZINZANI P., G; Zupo, S; Baccarani, M; Pileri, S. A.
abstract

Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4+, CD8+, and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4+, rather than to resting or CD8+ lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-B (NF-B) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor ) and VEGF. [Cancer Res 2007;67(22):10703–10]

2007 - Genomic expression during human myelopoiesis [Articolo su rivista]
Ferrari, F; Bortoluzzi, S; Coppe, A; Basso, D; Bicciato, Silvio; Zini, Roberta; Gemelli, Claudia; Danieli, Ga; Ferrari, Sergio
abstract

BACKGROUND: Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their differentiation potential, generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization. RESULTS: Gene expression data from 24 experiments for 8 different cell types of the human myelopoietic lineage were used to generate an integrated myelopoiesis dataset of 9,425 genes, each reliably associated to a unique genomic position and chromosomal coordinate. Lists of genes constitutively expressed or silent during myelopoiesis and of genes differentially expressed in commitment phase of myelopoiesis were first identified using a classical data analysis procedure. Then, the genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. This approach allowed identifying specific chromosomal regions significantly highly or weakly expressed, and clusters of differentially expressed genes and of transcripts related to specific functional modules. CONCLUSION: The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions.

2007 - Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells [Articolo su rivista]
S., Biressi; Tagliafico, Enrico; G., Lamorte; S., Monteverde; Tenedini, Elena; Roncaglia, Enrica; Ferrari, Stefano; Ferrari, Sergio; MG CUSELLA DE, Angelis; S., Tajbakhsh; G., Cossu
abstract

Skeletal muscle development occurs asynchronously and it has been proposed to be dependent upon the generation of temporally distinct populations of myogenic cells. This long-held hypothesis has not been tested directly due to the inability to isolate and analyze purified populations of myoblasts derived from specific stages of prenatal development. Using a mouse strain with the GFP reporter gene targeted into the Myf5 locus, a cell-sorting method was developed for isolating embryonic and fetal myoblasts. The two types of myoblasts show an intrinsic difference in fusion ability, proliferation, differentiation and response to TGFβ, TPA and BMP-4 in vitro. Microarray and quantitative PCR were used to identify differentially expressed genes both before and after differentiation, thus allowing a precise phenotypic analysis of the two populations. Embryonic and fetal myoblasts differ in the expression of a number of transcription factors and surface molecules, which may control different developmental programs. For example, only embryonic myoblasts express a Hox code along the antero-posterior axis, indicating that they possess direct positional information. Taken together, the data presented here demonstrate that embryonic and fetal myoblasts represent intrinsically different myogenic lineages and provide important information for the understanding of the molecular mechanisms governing skeletal muscle development.

2007 - MicroRNA expression profile in granulocytes from primary myelofibrosis patients [Articolo su rivista]
Guglielmelli, P; Tozzi, L; Pancrazzi, A; Bogani, C; Antonioli, E; Ponziani, V; Poli, G; Zini, Roberta; Ferrari, Sergio; Manfredini, Rossella; Bosi, A; Vannucchi, Am
abstract

Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET).PATIENTS AND METHODS:Using stem loop-primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2(617V>F) mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes.RESULTS:There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present, displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31, -150, and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a, -342, -326, -105, -149, and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2(617V>F) allele burden. Three in silico-predicted putative target genes (DTR, HMGA2, and MYB), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA.CONCLUSIONS:A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients

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) [Articolo su rivista]
Guglielmelli, P; Zini, Roberta; Bogani, C; Salati, Simona; Pancrazzi, A; Bianchi, Elisa; Mannelli, F; Ferrari, Sergio; Le Bousse Kerdilès, Mc; Bosi, A; Barosi, G; Migliaccio, Ar; Manfredini, Rossella; Vannucchi, A. M.
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.

2007 - Novel definition files for human GeneChips based on GeneAnnot [Articolo su rivista]
Ferrari, F; Bortoluzzi, S; Coppe, A; Sirota, A; Safran, M; Shmoish, M; Ferrari, Sergio; Lancet, D; Danieli, Ga; Bicciato, Silvio
abstract

BACKGROUND: Improvements in genome sequence annotation revealed discrepancies in the original probeset/gene assignment in Affymetrix microarray and the existence of differences between annotations and effective alignments of probes and transcription products. In the current generation of Affymetrix human GeneChips, most probesets include probes matching transcripts from more than one gene and probes which do not match any transcribed sequence. RESULTS: We developed a novel set of custom Chip Definition Files (CDF) and the corresponding Bioconductor libraries for Affymetrix human GeneChips, based on the information contained in the GeneAnnot database. GeneAnnot-based CDFs are composed of unique custom-probesets, including only probes matching a single gene. CONCLUSION: GeneAnnot-based custom CDFs solve the problem of a reliable reconstruction of expression levels and eliminate the existence of more than one probeset per gene, which often leads to discordant expression signals for the same transcript when gene differential expression is the focus of the analysis. GeneAnnot CDFs are freely distributed and fully compliant with Affymetrix standards and all available software for gene expression analysis. The CDF libraries are available from http://www.xlab.unimo.it/GA_CDF, along with supplementary information (CDF libraries, installation guidelines and R code, CDF statistics, and analysis results).

2007 - The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration [Articolo su rivista]
Rossi, L; Manfredini, Rossella; Bertolini, F; Ferrari, D; Fogli, M; Zini, Roberta; Salati, Simona; Salvestrini, V; Gulinelli, S; Adinolfi, E; Ferrari, Sergio; Di Virgilio, F; Baccarani, M; Lemoli, R. M.
abstract

Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Galphai) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5'-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase-activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Galphai proteins and RhoGTPases

2007 - Transcriptional profiles in melanocytes from clinically unaffected skin distinguish the neoplastic growth pattern in patients with melanoma [Articolo su rivista]
Magnoni, Cristina; Tenedini, Elena; Ferrari, Francesco; Benassi, Luisa; Bernardi, Chiara; Gualdi, Giulio; Bertazzoni, Giorgia; Roncaglia, Enrica; Fantoni, Luca Isaia; Manfredini, Rossella; Bicciato, Silvio; Ferrari, Sergio; Giannetti, Alberto; Tagliafico, Enrico
abstract

Background It is generally accepted that sunlight may contribute to the development of melanoma. Objectives To analyse gene expression of melanocytes obtained from clinically unaffected skin of patients with melanoma and healthy controls before and after exposure to ultraviolet B radiation. Methods Using GeneChip array technology, the gene expression of melanocytes obtained from the two donor groups was profiled, in order to identify transcriptional differences affecting susceptibility to melanoma. Results The data collected did not show any difference between the expression profiles of melanocytes purified from normal donors and from patients with melanoma that was able to give a statistically significant class separation. However, by means of unsupervised clustering our data could be divided into two main classes. The first class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a vertical growth phase (VGP) melanoma, while the second class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a radial growth phase (RGP) melanoma. Conclusions These data suggest that melanocytes in patients with VGP and RGP melanomas show significant differences in gene expression profiles, which allow us to classify patients with melanoma also from clinically unaffected skin.

2006 - Embryonic stem-derived versus somatic neural stem cells: A comparative analysis of their developmental potential and molecular phenotype [Articolo su rivista]
Colombo, Elena; Giannelli, Serena G.; Galli, Rossella; Tagliafico, Enrico; Foroni, Chiara; Tenedini, Elena; Ferrari, Sergio; Ferrari, Stefano; Corte, Giorgio; Vescovi, Angelo; Cossu, Giulio; Broccoli, Vania
abstract

Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES-induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES-derived neural proliferating cells are endowed with stem cell properties such as extensive self-renewal capacity and single-cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES-derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES-derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen-activated protein kinase) signaling is acting in ES-induced NSCs, probably triggered by insulin-like growth factor-H. This may contribute to the high proliferation rate exhibited by these cells in culture.

2006 - Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features [Articolo su rivista]
Rutella, S; Bonanno, G; Procoli, A; Mariotti, A; de Ritis, Dg; Curti, A; Danese, S; Pessina, G; Pandolfi, S; Natoni, F; Di Febo, A; Scambia, G; Manfredini, Rossella; Salati, Simona; Ferrari, Sergio; Pierelli, L; Leone, G; Lemoli, R. M.
abstract

Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.

2006 - Identification of a molecular signature for leukemic promyelocytes and their normal counterparts: focus on DNA repair genes [Articolo su rivista]
I., Casorelli; Tenedini, Elena; Tagliafico, Enrico; Mf, Blasi; A., Giuliani; M., Crescenzi; E., Pelosi; U., Testa; C., Peschle; L., Mele; D., Diverio; M., Breccia; F., Lo Coco; Ferrari, Sergio; M., Bignami
abstract

Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34(+)-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.

2006 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.

2006 - Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Grande, Alexis; Ferrari, F.; Roncaglia, Enrica; Bicciato, Silvio; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Gemelli, Claudia; Montanari, Monica; Vignudelli, Tatiana; ZANOCCO MARANI, Tommaso; Parenti, Sandra; Paolucci, Paolo; Martinelli, G.; Piccaluga, P. P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, Sergio
abstract

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

2006 - Identification of new p63 targets in human keratinocytes [Articolo su rivista]
B., Testoni; S., Borrelli; Tenedini, Elena; D., Alotto; C., Castagnoli; S., Piccolo; Tagliafico, Enrico; Ferrari, Sergio; M. A., Vigano; Mantovani, Roberto
abstract

P63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63 depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.

2006 - Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors. [Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio
abstract

The MItf-Tfe family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

2006 - Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells [Articolo su rivista]
Gemelli, Claudia; Montanari, Monica; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Siena, Michela; Zini, Roberta; Salati, Simona; Tagliafico, Enrico; Manfredini, Rossella; Specchia, G; Grande, Alexis; Ferrari, Sergio
abstract

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

2005 - Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors [Articolo su rivista]
Montanari, Monica; Gemelli, Claudia; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, T; Siena, M; Zini, Roberta; Salati, Simona; Chiossi, G; Tagliafico, Enrico; Manfredini, Rossella; Grande, Alexis; Ferrari, Sergio
abstract

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

2005 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.

2005 - Stem cell plasticity: time for a reappraisal? [Articolo su rivista]
Rm, Lemoli; F., Bertolini; R., Cancedda; DE LUCA, Michele; A., DEL SANTO; G., Ferrari; Ferrari, Sergio; G., Martino; Mavilio, Fulvio; S., Tura
abstract

n recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline.

2005 - The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment. [Articolo su rivista]
Manfredini, Rossella; Zini, Roberta; Salati, Simona; Siena, M; Tenedini, Elena; Tagliafico, Enrico; Montanari, Monica; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, T; Grande, A; Fogli, M; Rossi, L; Fagioli, Me; Catani, L; Lemoli, Rm; Ferrari, Sergio
abstract

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior

2004 - Evaluation of widely used models for predicting BRCA1 and BRCA2 mutations [Articolo su rivista]
F., Marroni; P., Aretini; E., D'Andrea; M. A., Caligo; L., Cortesi; A., Viel; E., Ricevuto; M., Montagna; G., Cipollini; Ferrari, Sergio; M., Santarosa; R., Bisegna; J. E., Bailey Wilson; G., Bevilacqua; G., Parmigiani; S., Presciuttini
abstract

Deleterious mutations of the BRCA1 and BRCA2 genes are a major risk factor for the development of breast and ovarian cancers.1–4 Mutation tests for these two genes commonly are now offered in specialised clinics.5,6 As a result, a large number of women with personal or family histories of breast or ovarian cancer seek genetic counselling. Accurate evaluation of the probability that a woman carries a germline pathogenic mutation at BRCA1 or BRCA2 therefore is essential to help counsellors and those being counselled to decide whether testing is appropriate. In this context, the questions of practical interest are: Given the pedigree, what is the chance of a mutation being present? and What is the chance of the DNA laboratory finding a mutation?

2004 - Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells [Articolo su rivista]
Tenedini, Elena; Fagioli, Me; Vianelli, N; Tazzari, Pl; Ricci, F; Tagliafico, Enrico; Ricci, P; Gugliotta, L; Martinelli, G; Tura, S; Baccarani, M; Ferrari, Sergio; Catani, L.
abstract

Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V-positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.

2004 - Msx2 and Necdin regulate smooth muscle determination in mesoangioblasts. [Articolo su rivista]
Brunelli, S.; Tagliafico, Enrico; De Angelis, F. G.; Tonlorenzi, R.; Baesso, S.; Ferrari, Sergio; Niinobe, M.; Yoshikawa, K.; Schwartz, R. J.; Bozzoni, I.; Ferrari, Stefano; Cossu, G.
abstract

Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for “stem” cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor . Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor , and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.

2004 - TGFβ/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts [Articolo su rivista]
Tagliafico, Enrico; S., Brunelli; A., Bergamaschi; L., De Angelis; R., Scardigli; D., Galli; Battini, Renata; P., Bianco; Ferrari, Sergio; G., Cossu; Ferrari, Stefano
abstract

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Writ or TGFbeta/BMP Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Writs and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, evens after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

2003 - Development of an IL-6 antagonist peptide that induces apoptosis in IL-6 dependent 7TD1 cells. [Articolo su rivista]
Manfredini, Rossella; Tenedini, Elena; M., Siena; Tagliafico, Enrico; Montanari, Monica; Grande, Alexis; ZANOCCO MARANI, Tommaso; C., Poligani; Zini, Roberta; A., Bergamaschi; DE RIENZO, Francesca; DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Ferrari, Sergio
abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.

2003 - Functional and kinetic characterization of granulocyte colony-stimulating factor-primed CD34) human stem cells [Articolo su rivista]
Rm, Lemoli; F., Bertolini; Mt, Petrucci; C., Gregorj; Mr, Ricciardi; M., Fogli; A., Curti; C., Rabascio; S., Pandolfi; Ferrari, Sergio; R., Fo; M., Baccarani; A., Tafuri
abstract

We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin(-)CD34(-) and Lin(-)CD34(+) cells were isolated from granulocyte colony-stimulating factor-primed peripheral blood (PB) cells of healthy donors. The CD34(-) cell fraction did not contain either clonogenic cells in semisolid culture or long-term culture initiating cells (LTC-IC). However, stroma-dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC-IC. Significantly higher percentages of quiescent G(0) cells and lower percentages of cycling G(1) cells were found in Lin(-)CD34(-) cells when compared with Lin(-)CD34(+) cells. Kinetic quiescence of Lin(-)CD34(-) cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27(Kip1) and p21(cip1/waf1). Cytokine-mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long-term engraftment in immunocompromised non-obese diabetic (NOD)/recombination activating gene 1(null) and NOD/severe combined immunodeficient-beta(2)microglobulin(null) mice injected with CD34(+) cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell-cycle recruitment and downregulation of p27(Kip1)in vitro and higher engraftment capacity in vivo.

2003 - Italian family with two independent mutations:3358T/A in BRCA1 and 8756delA in BRCA2 genes. [Articolo su rivista]
Cortesi, L.; Turchetti, D.; Bertoni, Carlo Maria; ZANOCCO MARANI, Tommaso; Silvestri, C.; Vinceti, Marco; Federico, Massimo; Silingardi, Vittorio; Ferrari, Sergio
abstract

Hereditary breast/ovarian cancer is a well-characterized clinical entity, largely attributed to the inheritance of BRCA1 or BRCA2 mutations. Among general population, the mutation's frequency of these genes is very low; therefore, the identification of two independent mutations in the same family is a rare event. This study reports the presence of two mutations, one in the BRCA1 and the second in the BRCA2 gene in an Italian Caucasian kindred. This family is composed of more than 250 individuals, spanning through five generations, among which endogamy was a common phenomenon. Considering the tumor spectrum, this family is characterized by a high incidence of different types of cancer. In our study, we considered only three out of seven family units for BRCA1 and BRCA2 analysis. In one of the family units, we found independent mutations of both BRCA genes. The BRCA1 mutation on exon 11 (3358TA) was identified originally in the index case and subsequently in 18 members of this family, whereas the same mutation was not detected in a related family member with male breast cancer. The male breast cancer patient led to the identification, through mutational analysis, of a new BRCA2 mutation (8756delA). This BRCA2 mutation was also found in the male breast cancer patient's daughter. The discovery of the BRCA2 mutation allowed us to alert the patient's daughter who, otherwise, could be falsely reassured since she had a negative BRCA1 test.

2003 - Requirement of the coiled-coil domains of p92(c-Fes) for nuclear localization in myeloid cells upon induction of differentiation [Articolo su rivista]
Tagliafico, Enrico; M., Siena; ZANOCCO MARANI, Tommaso; Manfredini, Rossella; Tenedini, Elena; M., Montanari; Grande, Alexis; Ferrari, Sergio
abstract

The nonreceptor tyrosine kinase Fes is implicated in myeloid cells differentiation. It has been observed that its localization can be cytoplasmic, perinuclear, or nuclear. To further characterize this point, we studied Fes subcellular localization in myeloid cell lines (HL60 and K562) and in COS1 cells. Fes was observed in both the nucleus and the cytoplasm of HL60, K562 cells over-expressing Fes and only in the cytoplasm of COS1 cells, suggesting that nuclear localization is cell context dependent. Moreover, in myeloid cells, the treatment with differentiation-inducing agents such as retinoic acid, phorbol esters and vitamin D, is followed by an increase of the oligomeric form of Fes in the nucleus. In fact, oligomerization seems to be necessary for translocation to occur, since Fes mutants missing the coiled-coil domains are not able to form oligomers and fail to localize in the nucleus. The active form of Fes is tyrosine phosphorylated; however, phosphorylation is not required for Fes to localize in the nucleus, since tyrosine kinase inhibitors do not block the translocation process.

2002 - Gene expression Profile of Human Myeloid Cells [Abstract in Atti di Convegno]
Siena, M; Manfredini, R; Bergamaschi, A; Tenedini, Elena; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Montanari, Monica; Gemelli, Claudia; Grande, Alexis; Ferrari, Sergio
abstract

Array technologies have made it possible to monitor simultaneously the expression pattern of thousands of genes. Working on normal human hempoietic stem cells it is possible to evaluate their gene expression profile, changes in gene expression occurring in their early commitment phase and to compare the gene expression profiling with normallly differentiated myeloid cells, i.e. granulocytes and monocytes.

2002 - Gene Expression profile of Vitamin D3 treated HL60 cells shows a phenotypic but not a complete functional conversion to monocytes [Abstract in Atti di Convegno]
Tenedini, Elena; Bergamaschi, A; Manfredini, Rossella; Percudani, R; Siena, M; ZANOCCO MARANI, Tommaso; Grande, Alexis; Montanari, Monica; Gemelli, Claudia; Torelli, U; Ferrari, Sergio; Tagliafico, Enrico
abstract

Acute Myeloid leukemia blast cells are characterized by their inability to proceed spontaneously toward terminal differentiation. To tackle this problem we have studied the changes occurring in the gene expression profile during the differentiation of HL60 cells treated with VD using the Affymetrix GeneChip technology and we have compared the molecular phenotype of VD induced cells to that of CD14+ pheripheral monocytes.

2002 - Gene expression profile of vitamin D3 treated HL60 cells shows an incomplete molecular phenotypic conversion to monocytes [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; A., Bergamaschi; ZANOCCO MARANI, Tommaso; R., Percudani; M., Siena; Manfredini, Rossella; Grande, Alexis; M., Montanari; C., Gemelli; U., Torelli; Ferrari, Sergio
abstract

By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.

2002 - In vivo effects of low-frequency low energy pulsing electromagnetic fields (PEMFs) on gene expression during the inflammation phase of bone repair [Articolo su rivista]
Zucchini, P; Zaffe, Davide; Botti, P; Grande, Alexis; Cavani, Francesco; Cadossi, M; Ferrari, Sergio; Cadossi, Ruggero; Fini, M; Canè, Valerio
abstract

It has been widely demonstrated that pulsed low-frequency electromagnetic fields (PEMFs) positively affect bone repair. The aim of this study is to highlight if PEMFs influence cell metabolic activity during the replacement of the blood clot with granulation tissue in the inflammation phase of bone repair. Four equal transcortical holes were made, at the same diaphyseal level, in both metacarpals (McIII) of five mate adult horses. The left McIII were exposed to PEMFs 24 hr/day; the right untreated McIII were used as controls. Eight days after surgery, the horses were sacrificed. We investigated the effect of PEMFs on 1) histological aspects of the lesion, 2) histochemical detection of the bone marker alkaline phosphatase, and 3) molecular markers as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-beta) by reverse transcriptase-polymerase chain reaction amplification (RT-PCR). The histological analysis indicates that the blood clot, in both PEMF treated and control holes, is being replaced by granulation tissue extending from the endosteal towards the periosteal side of the lesion. TALP positive areas do not exactly correspond to the areas where fibroblasts are present, these being wider than the former. The study of the expression of the mRNA of TGF-beta1 shows no differences between treated holes and control ones. The expression of TNF-alpha and IL-6 however, is not univocal, being sometimes more expressed, sometimes less in treated or control holes. These data show that PEMFs exposure affects the expression of inflammatory cytokines (TNF-alpha and IL-6) during the very early stages of bone repair. On the contrary TFG beta expression and histological findings are not modified by PEMF exposure at least in this experimental condition.

2002 - Physiological levels of 1 alpha, 25 di-hydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors [Articolo su rivista]
Grande, Alexis; M., Montanari; Tagliafico, Enrico; Manfredini, Rossella; ZANOCCO MARANI, Tommaso; M., Siena; Tenedini, Elena; A., Gallinelli; Ferrari, Sergio
abstract

Although supraphysiological levels of 1alpha, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38-hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

2002 - Requirement of the coiled coil domains of p92c-Fes for nuclear translocation in myeloid cells upon induction of differentiation [Abstract in Atti di Convegno]
ZANOCCO MARANI, Tommaso; Siena, M; Tagliafico, Enrico; Manfredini, Rossella; Tenedini, Elena; Montanari, Monica; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio
abstract

The non-receptor tyrosine kinase Fes is expressed in hematopoietic progenitors, differentiated myeloid cells and other cell types, such as vascular endothelial cells and neuroblastoma cell lines. To further clarify this point we performed confocal microscopy and western blot experiments on myeloid cell lines and COS1 cells. In myeloid cells the treatment with differentiation inducing agents such as ATRA, PMA and VD is followed by an increase of Fes abundance in the nuclear compartment. The active form of Fes is phosphorylated on residue 713 and is present into the nucleus while treated cells with the tyrosine kinase inhibitor Genistein clearly showed that phosphorylation is not a required event in order to Fes to translocate to the nucleus.

2002 - Usefulness of breast MRI in a patient with genetic risk [Articolo su rivista]
L., Cortesi; B., Canossi; M., De Santis; P., Panizza; G., Rossi; D., Turchetti; A., Del Maschio; Ferrari, Sergio; Romagnoli, Renato; Federico, Massimo; Silingardi, Vittorio
abstract

We describe an interesting case-report represented by a patient carrying BRCA1 mutation, recruited for the study Multicenter evaluation of Magnetic Resonance Imaging (MRI) in early diagnosis and prevention of breast cancer in high risk population, diagnosed with breast cancer on the basis of MRI findings but not with conventional mammography and ultrasound (US). She was already affected at 53 years of age by a multifocal Ductal Infiltrating Carcinoma (DIC) in the left breast; then, she had an axillary and sovraclavear nodal recurrence of the disease, three years after the initial diagnosis. Since other relatives were affected by breast cancer (mother, sister and niece) and two arose at early age (<40 years), BRCA1 mutational analysis was offered to the patient, identifying a nonsense mutation on the exon 13. Furthermore, this patient was recruited to study contralateral breast and at the second round, two little foci, suspicious of malignancy, were identified only with MRI, but not with mammography and ultrasonography. The final diagnosis was multifocal Ductal Carcinoma in situ (DCIS); the major focus measured 3 mm. In our patient MRI has shown a major sensitivity with respect to conventional radiology and US and has provided a very early diagnosis in this woman at genetic risk.

2001 - A functionally active RAR alpha nuclear receptor is expressed in retinoic acid non responsive early myeloblastic cell lines [Articolo su rivista]
Grande, Alexis; M., Montanari; Manfredini, Rossella; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; F., Trevisan; Ligabue, Giulia; M., Siena; Ferrari, Stefano; Ferrari, Sergio
abstract

Although ail-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RAR alpha), we performed an analysis of RAR alpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RAR alpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myetoblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RAR alpha, play a role in the resistance of these cells to ATRA treatment.

2000 - Comparison between genotype and phenotype identifies a high-risk population carryingBRCA1 mutations [Articolo su rivista]
Laura, Cortesi; Daniela, Turchetti; Chiara, Bertoni; Roberta, Bellei; Lucia, Mangone; Vinceti, Marco; Federico, Massimo; Vittorio, Silingardi; Ferrari, Sergio
abstract

Hereditary breast carcinomas constitute about 10% of all malignant mammary tumors, but the selection criteria to identify a high-risk population carrying BRCAI mutations are not yet well-defined. We have collected $1 pedigrees of familial breast cancer, 16 pedigrees of familial breast and ovarian cancer, and 30 cases of early-onset breast cancer (<35 years of age) without any family history of breast cancer. The index cases of the 97 selected families were further subdivided into three groups based on histopathological parameters: group A (n = 19) was characterized by tumor grade III, negative estrogen and progesterone receptors, and high proliferative rate; group B (n = 20) was characterized by grade I-II tumors, positive hormonal receptors, and low proliferative rate; and group C (n = 58) was not homogeneous for the histopathological criteria. The aim of our study was to evaluate, in patients with a family history of breast cancer or with early diagnosis of breast cancer, the incidence of BRCAI mutation on the basis of tumor phenotype. We found the highest rate of BRCAI mutations in group A (53%), and low frequencies in groups B (5%) and C (0%). Our data strongly indicate that an aggressive tumor phenotype in patients with a positive family history or early diagnosis identifies a population with high probability of carrying BRCAI mutations.

2000 - Il rene policistico autosomico dominante (ADPKD): aspetti genetico-molecolari, clinici ed epidemiologici nella provincia di Modena. [Articolo su rivista]
Magistroni, Riccardo; Furci, L.; Ligabue, Giulia; Baraldi, A.; Medici, G.; Olmeda, F.; Ballestri, M.; Miscia, M. C.; Ferrari, Sergio; Albertazzi, Alberto
abstract

non disponibile

1999 - Antisense oligonucleotides and PNAs for sequence-specific regulation of gene expression [Articolo su rivista]
Ferrari, Sergio
abstract

It has become increasingly clear in recent years that there are many possible regulatory steps in the pathway from gene to protein: chromatin conformation and transcriptional control; post-transcriptional regulation, particularly splicing, mRNA stability, nuclear cytoplasm transport of mRNAs, natural antisense transcripts; translational and post-translational control. Using antisense strategies it is possible to interfere with most of the regulatory mechanisms underlying gene expression both in embryonic and somatic cells. The specific gene inactivation in a particular cellular context is the necessary step to evaluate its possible biological function. Several different technical approaches based on different methodologies underlie antisense strategies. Among these, antisense oligonucleotides and peptide nucleic acids (PNAs) are considered in this short review. In these experimental approaches it is essential to identify the target genes and the most suitable cellular system. In fact, gene inactivation in somatic cells allows us to study basic questions in cell biology, such as cell cycle regulation, differentiation apoptosis, cell activation, cellular transformation, without having to struggle with the complexities of animal models. We have studied the biological consequences of c-myb, c-fes and Bax genes inactivation in normal and leukemic myeloid cells and their possible therapeutic applications. New delivery systems, based on translocating peptides to efficiently penetrate the cells with antisense oligonucleotides, PNAs and biologically active peptides are also discussed.

1999 - Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia [Articolo su rivista]
F., Parise; L., Simone; M. A., Croce; Ghisellini, Margherita; Battini, Renata; S., Borghi; Tiozzo, Roberta; Ferrari, Sergio; CALANDRA BUONAURA, Sebastiano; Ferrari, Stefano
abstract

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

1999 - Induction of a functional vitamin D receptor in all-trans-retinoic acid- induced monocytic differentiation of M2-type leukemic blast cells [Articolo su rivista]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Lemoli, R.; Visani, G.; Tura, S.; Ferrari, S.; Ferrari, S.
abstract

Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2- type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D- responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD- responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.

1998 - Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells [Articolo su rivista]
Manfredini, R.; Capobianco, M. L.; Trevisan, F.; Rauzi, F.; Barbieri, D.; Citro, G.; Tagliafico, E.; Ferrari, S.
abstract

Cell sensitivity to programmed cell death is primarily modulated by members of the Bcl-2 family, as the balance of homodimer or heterodimer formation between proapoptotic and antiapoptotic members defines apoptosis susceptibility in the great majority of cellular contexts. It is, therefore, important to clarify if the Bax protein is limiting for activation of the genetic program of programmed cell death or can be complemented by different Bcl-2 family members, such as Bah or Bad. To gain some insight into the role of Bax in the molecular mechanisms of apoptosis of myeloid cells, we inhibited this gene in all-trans-retinoic acid (ATRA)-treated HL60 cells using the methodology of antisense oligodeoxynucleotides (AS-ODN). Our results indicate that Bax inhibition has no effect on the proliferation and differentiation capacity of HL60 cells. Instead, the survival rate of terminally differentiated Bax-inactivated HL60 (Bax((-)) HL60) cells is almost three times higher in respect to control cultures, indicating that in mature granulocytes Bax is not efficiently complemented by others members of the Bcl-2 family proteins.

1998 - Wt-p53 action in human leukaemia cell lines corresponding to different stages of differentiation [Articolo su rivista]
Rizzo, Mg; Zepparoni, A; Cristofanelli, B; Scardigli, R; Crescenzi, M; Blandino, G; Giuliacci, S; Ferrari, Sergio; Soddu, S; Sacchi, A.
abstract

Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53(Val135) mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including M1 (KG1), M2 (HL-60), M3 (NB4), M5 (U937) and M6 (HEL 92.1.7), as well as blast crisis of chronic myelogenous leukaemia (BC-CML: K562, BV173) showing diverse differentiation features. By morphological, molecular and biochemical analyses, we have shown that exogenous wt-p53 gene expression induces apoptosis only in cells corresponding to M1, M2 and M3 of the FAB classification and in BC-CML showing morphological and cytochemical features of undifferentiated blast cells. In contrast. it promotes differentiation in the others. Interestingly, cell responsiveness was independent of the vector used and the status of the endogenous p53 gene.

1997 - Antisense inhibition of c-fes proto-oncogene blocks PMA-induced macrophage differentiation in HL60 and in FDC-P1/MAC-11 cells [Articolo su rivista]
Manfredini, Rossella; R., Balestri; Tagliafico, Enrico; F., Trevisan; Grande, Alexis; M., Pizzanelli; D., Barbieri; P., Zucchini; G., Citro; C., Franceschi; Ferrari, Sergio
abstract

To gain some insight into the role of c-fes in macrophage differentiation, we have analyzed the ability of HL60 leukemic promyelocytic cells and FDC-P1/MAC-11 murine myeloid precursor cells to differentiate in response to phorbol esters after inhibition of c-fes function. Fes inactivation has been obtained by using oligodeoxynucleotides (ODN) complementary to the 5´ region of c-fes mRNA and to 5´ splice junctions of c-fes primary transcript. After 5 days (d) in culture, in several separate experiments performed with different ODN preparations, a complete inhibition of c-fes expression was observed in HL60 and in FDC-P1/MAC-11 cells. No perturbation of cell growth was evident in our experimental conditions in both cell lines after c-fes inhibition. Furthermore, in HL60 cells lacking c-fes product, an almost complete downregulation of the alpha 4 beta 1 fibronectin receptor occurred. However, in both cell lines, the induction of macrophage differentiation by phorbol esters resulted in an almost complete maturation arrest as evaluated by morphological, cytochemical, immunological criteria, and by the cytofluorimetric cell cycle analysis. A loss of the adhesion capacity of both myeloid cell lines, when compared to terminally differentated macrophages, was also observed. These results suggest that HL60 and FDC-P1/MAC-11 cells, when treated with phorbol 12-myristate 13-acetate, require c-fes protein expression to activate the genetic program underlying macrophage differentiation.

1997 - Correlations between the FAB phenotype and differentiation potential in leukemic myelopoiesis [Relazione in Atti di Convegno]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Pignatti, E.; Ligabue, G.; Barbieri, D.; Ferrari, Se.
abstract

1997 - Interleukin-9 in human myeloid leukemia cells [Articolo su rivista]
Lemoli, R; Fortuna, A; Tafuri, A; Grande, Alexis; Amabile, M; Martinelli, G; Ferrari, Sergio
abstract

Here we review our recent data addressing the role of recombinant human (rh) interleukin 9 (IL-9) in acute myeloblastic leukemia (AML). We first evaluated the proliferative response of 3 leukemic cell lines and 32 primary samples from AML patients to IL-9 alone and combined with rh-IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF, c-kit ligand). The colony forming ability of leukemic cells was assessed by a clonogenic assay in methylcellulose, whereas the cell cycle characteristics of the same samples were determined by the acridine-orange (AO) flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase Assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction or prevention of apoptosis by IL-9. IL-9, used as a single cytokine, at various concentrations stimulated the colony formation of the 3 myeloid cell lines under serum-containing and serum-free conditions and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in the increase of the blast colony formation in all the cases studied and was the most effective CSF for promoting leukemic cell growth among those tested in this study including SCF, IL-3, and GM-CSF. The addition of SCF to IL-9 demonstrated an additive or synergistic effect of the 2 cytokines in 5 out of 8 AML cases tested for their CFU-L growth (187 +/- 79 colonies in comparison with 107 +/- 32 CFU-L; p = 0.05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of the cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2 +/- 24% compared to 58.6 +/- 22% of control cultures; p < 0.05) and induced an increase of G1 and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. Furthermore, in this study, reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did not show the constitutive expression of IL-9 mRNA in the cell lines and the AML samples studied at diagnosis. In summary, IL-9 may play a role in the development of acute myeloid leukemia by stimulating the proliferation of leukemic cells perhaps through a paracrine growth loop.

1997 - Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation [Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; M., Pizzanelli; Tagliafico, Enrico; R., Balestri; F., Trevisan; D., Barbieri; C., Franceschi; Battini, Renata; Ferrari, Stefano; Ferrari, Sergio
abstract

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function,we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.

1996 - Interleukin-9 (IL-9) stimulates the proliferation of human myeloid leukemic cells [Articolo su rivista]
Rm, Lemoli; A., Fortuna; A., Tafuri; M., Fogli; M., Amabile; Grande, Alexis; Mr, Ricciardi; Mt, Petrucci; L., Bonsi; Gp, Bagnara; G., Visani; G., Martinelli; Ferrari, Sergio; S., Tura
abstract

Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh)-IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction or prevention of apoptosis by IL-9. IL-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU-L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9-supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (ie, SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G(0) cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P < .05) and induced an increase of G(1)- and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle.

1995 - All-trans-retinoic acid induces simultaneously granulocytic differentiation and expression of inflammatory cytokines in HL-60 cells [Articolo su rivista]
Grande, Alexis; Manfredini, Rossella; Tagliafico, Enrico; R., Balestri; M., Pizzanelli; S., Papa; P., Zucchini; L., Bonsi; G., Bagnara; U., Torelli; Ferrari, Sergio
abstract

All-trans-retinoic acid (ATRA) can induce granulocytic differentiation both in vitro and in vivo, and its activity is mediated by the retinoic acid receptor-alpha (RAR-alpha). In the present study, we evaluated the ability of this inducer in HL-60 cells, to stimulate simultaneously granulocytic differentiation and the expression of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-3, IL-6, tumor necrosis factor-a: (TNF-a), and stem cell factor (SCF). The level of expression of these cytokines in ATRA-treated HL-60 cells was compared with that observed in normal and lipopolysaccharide (LPS)-heated peripheral granulocytes. The results indicate that the expression of these cytokines is enhanced during differentiation so that the pattern observed in ATRA-treated HL-60 cells is close to that of LPS-stimulated normal granulocytes. In addition, tetra phorbol acetate (TPA)-treated HL-60 cells express several of the above listed cytokines. It is concluded that ATRA not only induces granulocytic differentiation of HL-60 cells, but also activation of these terminally differentiated cells. The activating cytokine expression in these cells appears related to the progress of the differentiation program induced by ATRA since normal granulocytes do not respond to this inducer by activation of the expression of these genes. Furthermore, the cytokine activation is a specific effect of ATRA, since DMSO does not have any stimulatory effect.

1995 - Interleukin-11 (IL-11) acts as synergistic factor for the proliferation of human myeloid leukemic cells [Articolo su rivista]
Rm, Lemoli; M., Fogli; A., Fortuna; M., Amabile; P., Zucchini; Grande, Alexis; G., Martinelli; G., Visani; Ferrari, Sergio; S., Tura
abstract

Interleukin-11 is a stromal cells derived cytokine which stimulates the proliferation of primitive haemopoietic progenitor cells. For this paper we have studied the constitutive expression of IL-11 mRNA in a panel of well-known leukaemic cell lines and samples from AML patients at diagnosis. Moreover, the same cellular populations were evaluated for their proliferative response to recombinant-human-(r-hu) IL-11 alone and combined with r-hu-IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCE, c-kit ligand). The colony-forming ability of HL60, K562, KG1 cells and eight fresh AML cell populations was assessed by a clonogenic assay in methylcellulose. In eight additional AML cases the number of S-phase leukaemic cells induced by IL-11 was determined by the bromodeoxyuridine (BRDU) incorporation assay after 3 d of liquid culture. IL-11, as single cytokine, did not stimulate the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions. In contrast, the proliferation of the leukaemic cells in response to IL-3, GM-CSF and SCF was enhanced by co-incubation with IL-11, and this effect was reversed in blocking experiments by the anti-IL-11 Moab. When tested on primary AML samples, IL-11 alone showed little, if any, proliferative activity. However, it increased the IL-3-dependent blast colony formation in eight out of eight cases and GM-CSF in seven cases. IL-11 also augmented synergistically the number of CFU-L stimulated by SCF in seven cases. A combination of three factors (1L-11 SCF and IL-3) yielded optimal colony formation. The BRDU studies showed the significant increase of AML cells in S-phase when IL-11 was combined with SCF, whereas the two CSF had no activity on their own. Positive interaction was also observed when IL-11 was added to IL-3 supplemented cultures in five out of eight cases tested. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) demonstrated the constitutive expression of IL-11 mRNA in all the cell lines and 11/12 AML samples studied at diagnosis. These results indicate that IL-11 is expressed in leukaemic myeloid cells and that their proliferation is regulated by the cytokine which acts as a synergistic factor.

1995 - ROLE OF C-FES PROTOONCOGENE IN MYELOID DIFFERENTIATION [Articolo su rivista]
FERRARI, Sergio; GRANDE, Alexis; MANFREDINI, Rossella; TAGLIAFICO, Enrico
abstract

The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes, The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.

1995 - Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Paola, Cocconcelli; Elisa, Merelli; Luca Ceccherini, Nelli; Ferrari, Sergio; Mario, Luppi; Barozzi, Patrizia
abstract

Out of 64 cases of non-Hodgkin's lymphomas (NHL), 55 cases of Hodgkin's disease (HD) and 31 cases of multiple sclerosis (MS), 2 NHL, 7 HD and 1 MS cases were found positive by polymerase chain reaction (PCR) for the presence of HHV-6 sequences in pathologic lymph nodes of the lymphomas and in peripheral blood mononuclear cells (PBMCs) of MS. A further analysis of the PBMCs of the PCR positive cases by standard Southern blot technique revealed only 2 NHL, 3 HD and 1 MS cases as positive, indicating that these six patients have an unusually high viral copy number in the PBMCs. Restriction analysis, carried out using probes representative of different regions of the virus, showed that three cases retain only a deleted portion of the viral genome. In the remaining three cases a complete viral genome was present, containing the right end sequences in which the rep-like gene, possibly crucial to the viral and cellular life cycle, is located. The analysis by pulsed field gel electrophoresis (PFGE) of the total DNA of the PBMCs obtained directly, without culture from PBMCs of these last three cases (1 NHL, 1 HD, and 1 MS), using the same probes, showed the absence of free viral molecules and the association of viral sequences with high molecular weight DNA. These results are consistent with in vivo integration of the entire virus in the cellular genome. A further study of the same patients with chromosome fluorescence in situ hybridization (FISH) showed in all the three cases the presence of a specific hybridization site, located at the telomeric extremity of the short arm of chromosome 17 (17p13), suggesting that this location is at least a preferred site of an infrequent, but possibly biologically important, integration phenomenon.

1994 - Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation [Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Tagliafico, Enrico; Grande, Alexis; D., Barbieri; R., Balestri; M., Pizzanelli; Zucchini, Patrizia; G., Citro; G., Zupi
abstract

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

1994 - ANTISENSE STRATEGIES IN LEUKEMIA [Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Torelli, U.
abstract

1994 - Expression And Function Of Nerve Growth-Factor And Nerve Growth-Factor Receptor On Cultured Keratinocytes. [Articolo su rivista]
Pincelli, Carlo; C., Sevignani; Manfredini, Rossella; Grande, Alexis; F., Fantini; L., Bracci Laudiero; L., Aloe; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, Alberto
abstract

Keratinocytes, a key cellular component both for homeostasis and pathophysiologic processes of the skin, secrete a number of cytokines and are stimulated by several growth factors. Nerve growth factor (NGF) is synthesized in the skin and basal keratinocytes express the low-affinity nerve growth factor receptor (NGF-R). We present evidence that normal human keratinocytes in culture express the low- and the high-affinity NGF-R both at the mRNA level, as determined by reverse-transcription polymerase chain reaction and at the protein level, as shown by cytofluorimetric analysis. NGF significantly stimulates the proliferation of normal human keratinocytes in culture in a dose-dependent manner. This effect can be prevented by the addition of both an anti-NGF neutralizing antibody and a high-affinity NGF-R (trk) specific inhibitor, the natural alkaloid K252a. By contrast, keratinocyte proliferation is not inhibited by an anti - low-affinity NGF-R monoclonal antibody, thus suggesting that NGF effect on human keratinocytes is mediated by the high-affinity NGF-R. Moreover, NGF mRNA is expressed in normal human keratinocytes and NGF is secreted by keratinocytes in increasing amounts during growth, as detected by enzyme-linked immunosorbent assay. These results suggest that NGF could act as a cytokine in human skin and take part in disorders of keratinocyte proliferation.

1994 - EXPRESSION AND FUNCTIONAL-ROLE OF C-KIT LIGAND (SCF) IN HUMAN MULTIPLE-MYELOMA CELLS [Articolo su rivista]
R. M., Lemoli; A., Fortuna; Grande, Alexis; B., Gamberi; L., Bonsi; M., Fogli; M., Amabile; M., Cavo; Ferrari, Sergio; S., Tura
abstract

In this study we investigated the proliferation of three well-documented MM lines and 10 bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3 and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (>90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 and 7 d of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. AU the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase (24.5 +/- 2% SEM v 14.5 +/- 1% SEM and 32 +/- 3% SEM v 21 +/- 4% SEM, respectively; P < 0.05). The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% v 3.4 +/- 1.3% in control cultures; P = 0.02). Significant proliferation was also induced by IL-6 (7 +/- 2.3% of BRDU(+) cells; P = 0.006), IL-3 (5.3 +/- 1.3%; P = 0.01) and PIXY-321 (5.4 +/- 1.6%; P = 0.02). The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. In summary, our results indicate that SCF is expressed in MM cells and stimulates the proliferation of neoplastic plasma cells.

1994 - Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level. [Articolo su rivista]
Girolomoni, G; Zambruno, G; Manfredini, Rossella; Zacchi, V; Ferrari, Sergio; Cossarizza, Andrea; Giannetti, A.
abstract

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37°C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (&gt; 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.

1994 - LOCAL PRODUCTION AND ACTION OF FOLLISTATIN IN HUMAN PLACENTA [Articolo su rivista]
Petraglia, F; Gallinelli, A; Grande, Alexis; Florio, P; Ferrari, Sergio; Genazzani, Ar; Ling, N; Depaolo, Lv
abstract

The aim of the present study was to investigate the possible production, localization, and action of follistatin in human placenta, fetal membranes (amnion, chorion), and maternal decidua. Four different experimental approaches were used: 1) Southern blot analysis following reverse polymerase chain reaction to identify follistatin messenger RNA (mRNA) in tissue homogenates; 2) immunohistochemistry to localize immunoreactive (ir-) follistatin in the various intrauterine tissues; 3) measurement by RIA of ir-follistatin levels in culture medium of placental cells; and 4) possible action of follistatin on human CG (hCG) and progesterone release from cultured placental cells. Placental and decidual cells collected during first trimester or at term gestation express follistatin mRNA; fetal membranes (amnion, chorion) at term also express follistatin mRNA. Immunoreactive follistatin is localized in syncytial cells of placental villi at term as well as in large decidual cells, in amnion epithelium, and in chorionic cells. The placental secretion of follistatin has been confirmed by the evidence of measurable levels of ir-follistatin in the medium of cultured placental cells at term; the release is time dependent and is not modified by the addition of forskolin or progesterone. The addition of increasing doses of recombinant human follistatin does not significantly influence the release of hCG or progesterone from cultured placental cells, whereas the activin A-induced hCG and progesterone release are completely reversed. The present data showed that 1) human placenta, fetal membranes, and decidua express follistatin mRNA; 2) ir-follistatin is localized and released from placental cells at term; and 3) follistatin has a functional role in the local control system regulating placental hormone production.

1993 - Expression of interleukins 1, 3, 6, stem cell factor and their receptors in acute leukemia blast cells and in normal peripheral lymphocytes and monocytes [Articolo su rivista]
Ferrari, S.; Grande, A.; Manfredini, R.; Tagliafico, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract

Abstract: Reverse transcriptase‐polymerase chain reaction amplification (RT‐PCR) and Southern blot analysis were used to evaluate ligand and receptor expression of interleukin 1α (IL‐1α), interleukin 3 (IL‐3), interleukin 6 (IL‐6) and stem cell factor (SCF) in peripheral blood lymphocytes and monocytes and in several acute leukemia blast cell populations. Resting peripheral lymphocytes and monocytes expressed both ligand and receptor of the four cytokines at considerable levels. The leukemic blast cells of the M1‐M4 phenotypes are characterized by almost complete lack of expression of IL‐1α, IL‐3 and IL‐6 and the constant and usually high expression of SCF. On the other hand, these myeloid blast cells express generally high levels of the four cytokine receptors. The data suggest that the regulation of the expression of IL‐1α, IL‐3 and IL‐6, at least in our limited number of leukemic cell populations studied, is independent of that of SCF. The results indicate that, at least in most of the leukemic myeloid blasts cells, the expression of SCF and its receptor, the c‐kit oncogene, may permit an autocrine regulation of cell cycling. © Munksgaard 1993

1993 - Inhibition of c-fes expression by an antisense oligomer causes apoptosis oh HL60 cells induced to granulocytic differentiation [Articolo su rivista]
Manfredini, Rossella; Grande, Alexis; Tagliafico, Enrico; D., Barbieri; P., Zucchini; G., Citro; G., Zupi; C., Franceschi; U., Torelli; Ferrari, Sergio
abstract

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

1993 - Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity [Articolo su rivista]
Ferrari, S.; Grande, A.; Zucchini, P.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Temperani, P.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1993 - 3 CASES OF HUMAN HERPESVIRUS-6 LATENT INFECTION - INTEGRATION OF VIRAL GENOME IN PERIPHERAL-BLOOD MONONUCLEAR CELL-DNA [Articolo su rivista]
Luppi, Mario; Marasca, Roberto; Barozzi, Patrizia; Ferrari, Sergio; Ceccherininelli, L; Batoni, G; Merelli, E; Torelli, Giuseppe
abstract

Saliva and peripheral blood mononuclear cells from three patients, two with lymphoproliferative disorders and one suffering from multiple sclerosis, were examined for the presence of human herpesvirus-6 (HHV-6) genome by using the polymerase chain reaction and Southern blot analysis. The search for anti-HHV-6 antibodies, carried out in the sera of the same cases by an immunofluorescence assay, was negative in two cases at the lowest dilution used (1:40). These three patients had a high number of HHV-6 specific sequences in uncultured peripheral blood mononuclear cells, which are thought to be a normal site of viral latency although, in healthy individuals, the infected cells are extremely rare. In order to gain some insight into the state of the viral genome in this latent HHV-6 infection, we used pulsed field gel electrophoresis to separate HHV-6 DNA directly from HHV-6 (strain GS) infected HSB-2 cells and from the peripheral blood mononuclear cells of these three patients. Our study showed the presence of intact viral genome, of the expected length of 170 kb, persisting as free extrachromosomal element in the HSB-2 cells but not in patients' peripheral blood mononuclear cells. On the other hand, in strong contrast with the results obtained in infected HSB-2 DNA, the restriction analysis of the three patients' peripheral blood mononuclear cell DNA showed fragments of molecular weight constantly higher than the 170 kb segment, indicating that the viral sequences are linked to high molecular weight cellular DNA. Our findings are consistent only with a latent infection in which HHV-6 is integrated in vivo and suggest that pulsed field gel electrophoresis analysis is well worth using to evaluate the presence of integrated, intact, or fragmented viral genomes in HHV-6 associated lymphoproliferative diseases and immune disorders.

1992 - ANTISENSE STRATEGIES TO CHARACTERIZE THE ROLE OF GENES AND ONCOGENES INVOLVED IN MYELOID DIFFERENTIATION [Articolo su rivista]
Ferrari, Sergio; Manfredini, Rossella; Grande, Alexis; Torelli, Umberto
abstract

Several genes that ara associated with cell proliferation and differentiation have been isolated. The definition of "cell cycle-related or differentiation-related genes" is simply based on the concept that the mRNA and/or protein abundance of these genes increases in these two functional states of the cell .....

1992 - Proliferation, Differentiation Arrest, and Survival in Leukemic Blast Cells [Articolo su rivista]
Ferrari, S.; Manfredini, R.; Grande, A.; Torelli, G.; Torelli, U.
abstract

1992 - Terminal differentiation [Articolo su rivista]
Ferrari, Sergio; Grande, Alexis; Manfredini, Rossella; Torelli, Umberto
abstract

Cell differentiation ultimately proceeds as a response to signals from the extracellu- lar microenvironment. If the cells are competent, that is, if they carry the correspond- ing receptors and signal transduction pathways, they can be triggered in the commit- ment state. In many cases the response includes complex gene expression programs whose progress and completion are relatively autonomous and whose outcome is determined by inherent regulatory factors of the cell type affected.' As a short introduction to the problems of differentiation and aging, we survey the main points that characterize the differentiation of hematopoietic cells. We can identify several main aspects.

1991 - HHV-6 INFECTION AND HUMAN HODGKIN AND NON-HODGKIN LYMPHOMAS [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Selleri, L; Luppi, Mario; Montorsi, M; Federico, Massimo; Narni, Franco; Ceccherininelli, L; Ferrari, Sergio
abstract

1991 - Human Herpes virus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction [Articolo su rivista]
Torelli, Giuseppe; Marasca, Roberto; Luppi, Mario; L., Selleri; Ferrari, Sergio; Narni, Franco; Mt, Mariano; Federico, Massimo; L., CECCHERINI NELLI; M., Bendinelli; G., Montagnani; M., Montorsi; Artusi, Tullio
abstract

In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV-6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.

1991 - TRANSCRIPTION OF N-MYC AND PROLIFERATION-RELATED GENES IS LINKED IN HUMAN NEUROBLASTOMA [Articolo su rivista]
Raschella, G; Negroni, A; Giubilei, C; Romeo, A; Ferrari, Sergio; Castello, Ma; Dominici, C.
abstract

A definite association between the transcription of N-myc oncogene and proliferation-related genes, histone H3, c-myc and p53, was found in a set of 12 primary untreated neuroblastomas and a metastasis of one of these at relapse. Molecular analysis allowed us to discriminate between actually proliferating and non-proliferating tumors, and suggested a link between N-myc and proliferation. Flow cytometric analysis of DNA distribution was less reliable for assessing tumor proliferative activity. Our data also seem to indicate a down-regulation of c-myc by N-myc in human neuroblastoma.

1990 - Detection of low abundance mRNA of myeloid specific genes in cells of acute and chronic lymphoid leukemias by cRNA hybridization [Articolo su rivista]
Ferrari, S.; Ceccherelli, G.; Tagliafico, E.; Zucchini, P.; Manfredini, R.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.

1990 - Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells [Articolo su rivista]
Ferrari, S.; Donelli, A.; Manfredini, R.; Sarti, M.; Roncaglia, R.; Tagliafico, E.; Rossi, E.; Torelli, G.; Torelli, U.
abstract

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.

1990 - Noncoordinated Expression of S6, S11, and S14 Ribosomal Protein Genes in Leukemic Blast Cells [Articolo su rivista]
Ferrari, S.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Donelli, A.; Torelli, G.; Torelli, U.
abstract

The steady state levels of mRNAs codying for the ribosomal proteins S6, Sil, and SI4 have been evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. It was found that the amounts of ribosomal protein mRN A are very similar and are not increased by serum or mitogen stimulation. The constitutive expression of these genes appears to be coordinately regulated and it is not modified after protein synthesis inhibition by cycloheximide. The ribosomal protein mRNA was also assayed in 15 different populations of human leukemic blast cells. In these populations the abundance of each ribosomal protein mRNA is remarkably different from the other. The results of our present experiments indicate that the expression of the three ribosomal protein genes undergoes independent noncoordinated changes in the large majority of the leukemic populations studied. © 1990, American Association for Cancer Research. All rights reserved.

1990 - Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics [Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Temperani, P.; Manfredini, R.; Ceccherelli, G.; Zucchini, P.; Tabilio, A.; Donelli, A.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression. © 1990.

1989 - "Biologia molecolare delle leucemie e dei linfomi: Biologia molecolare della cellula, citogenetica e genetica molecolare [Monografia/Trattato scientifico]
Torelli, Umberto; Emilia, Giovanni; Torelli, Giuseppe; Ferrari, Sergio; Narni, Franco
abstract

L'applicazione delle tecniche del DNA ricombinante allo studio delle leucemie e dei linfomi ha permesso di approfondire la conoscenza dei meccanismi molecolari dei processi mielo- e linfo-proliferativi, di valutarne il significato prognostico e di aprire nuove prospettive di terapia mirata. Gli autori raccolgono in questa rassegna i dati più significativi emersi degli studi condotti in questo in settore in costante aggiornamento.

1989 - Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes. [Articolo su rivista]
Ferrari, Sergio; Tagliafico, Enrico; Ceccherelli, G; Selleri, L; Calabretta, Bruno; Donelli, A; Temperani, Paola; Sarti, M; Sacchi, Stefano; Emilia, Giovanni; Torelli, Giuseppe; Torelli, Umberto
abstract

The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.

1988 - Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts. [Articolo su rivista]
Ferrari, Sergio; Calabretta, Bruno; Battini, Renata; Cosenza, Sc; Owen, Ta; Soprano, Kj; Baserga, R.
abstract

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

1988 - Expression of oncogenes and cell cycle related genes in acute and chronic leukemias. [Articolo su rivista]
Ferrari, Sergio; Calabretta, Bruno; Selleri, L; Ceccherelli, G; Torelli, Giuseppe; Torelli, Umberto
abstract

The authors have assayed the level of expression of several cell-cycle related genes in several populations of circulating myeloid leukemic blast cells. The genes explored included oncogenes such as c-myc, c-myb, p53, and cell-cycle-related genes such as vimentin, calcyclin, ornithine decarboxylase (ODC) and histone H3. Particular attention was given to analysis of the relationship existing between the mRNA levels of the histone H3 gene, which is expressed specifically in the S phase of the cell cycle, and the levels of other genes that are expressed in different stages of the G1 phase. Remarkable differences were observed among the different cases indicating that a differential expression of cell-cycle-related genes characterizes many acute leukemias. This differential expression is reflected in an altered ratio among G1-related genes and the H3 histone gene. The large fraction of leukemic cells which does not express histone H3 and therefore is functionally noncycling, shows a heterogeneous pattern of G1-related gene expression. This reflects the inability of most leukemic cells to progress through the G1 phase into the S phase of the cell cycle. This inability represents an abnormality of the cell cycle. It is concluded that the study of the expression of cell-cycle genes and protooncogenes in in understanding how leukemic cells enter a state of proliferation arrest, which appears to occur in a large fraction of leukemic cells.

1988 - Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia. [Articolo su rivista]
Ferrari, Sergio; M. T., Mariano; Tagliafico, Enrico; M., Sarti; G., Ceccherelli; L., Selleri; F., Merli; Narni, Franco; A., Donelli; Torelli, Giuseppe
abstract

By using a cDNA clone of the myeloperoxidase (MPO) gene, we have studied, by Northern blot analysis, the level of MPO mRNA in eight cases of acute lymphoblastic leukemia (ALL). The blast cell populations studied were characterized by morphologic, cytochemical, immunochemical, and molecular criteria. With all the methods used the populations were found to be highly homogeneous and showed a typical lymphoid phenotype. In particular, the Ig heavy-chain gene rearrangement was largely prevalent, and the germ line configuration was almost absent. However, in three of eight cases, high levels of MPO mRNA were detected. The remarkable homogeneity of the cell populations examined suggests that the MPO mRNA observed was present in cellular elements certainly identified as lymphoid. The absence of contamination by myeloid cells was confirmed by the results of Western blot analysis of the proteins of the cell population studied: no MPO protein was detectable. The levels of mRNA observed were high enough to be comparable to those observed in a promyelocytic cell population.

1987 - Cell-cycle-dependent expression of human ornithine decarboxylase. [Articolo su rivista]
Kaczmarek, L; Calabretta, Bruno; Ferrari, Sergio; de Riel, J. K.
abstract

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.

1987 - Molecular cloning of a cDNA for a human ADP/ATP carrier which is growth-regulated. [Articolo su rivista]
Battini, Renata; Ferrari, Sergio; Kaczmarek, L; Calabretta, Bruno; Chen, St; Baserga, R.
abstract

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.

1987 - Structural and functional analysis of a growth-regulated gene, the human calcyclin [Articolo su rivista]
Ferrari, Sergio; Calabretta, Bruno; Deriel, Jk; Battini, Renata; Ghezzo, F; Lauret, E; Griffin, C; Emanuel, Bs; Gurrieri, F; Baserga, R.
abstract

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

1986 - Coding sequence and growth regulation of the human vimentin gene. [Articolo su rivista]
Ferrari, Sergio; Battini, Renata; Kaczmarek, L; Rittling, S; Calabretta, Bruno; de Riel, Jk; Philiponis, V; Wei, Jf; Baserga, R.
abstract

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

1986 - Expression of growth regulated genes in human acute leukemias. [Articolo su rivista]
Ferrari, Sergio; Narni, Franco; Mars, W.; Kaczmarek, L.; Venturelli, D.; Anderson, B.; Calabretta, Bruno
abstract

We have investigated the expression of six growth-regulated genes (c-myc, c-myb, p53, 4F1, 2F1, and ornithine decarboxylase) and the S-phase-specific histone H3 gene in acute myeloid and lymphoid leukemic cells. We have purposely chosen three growth-regulated protooncogenes that share similar biological features and three gene sequences that have in common the cell cycle dependence of their expression in cells of different tissue and in different species. The level of expression was determined by measuring the amounts of specific RNA by Northern blot analysis. Levels of expression of the six growth-regulated genes were compared to the level of expression of the S-phase-specific H3 gene and among themselves. This method distinguishes the increased expression of a growth-regulated gene due to a true altered activation from over-expression which simply reflects an increase in the fraction of cycling cells. We have found that six of 14 patients with acute leukemias have markedly high ratios of c-myc/H3, c-myc/p53, and c-myc/c-myb expression. Two patients with altered c-myc expression have also a high ratio p53/H3. Within the group of cell cycle-dependent genes the ratios of expression seem in the overall much more regular with the clear exception of a patient with acute myelogenous leukemia in which the ratios 4F1/H3 and 2F1/H3 are significantly increased. A possible interpretation of these findings is that the fraction of noncycling leukemic cells that often constitute the majority of the entire leukemic population is in some cases in a true resting state, whereas in other cases heterogeneous degrees of growth arrest might occur. The altered expression of c-myc seems the feature most commonly associated with this putative growth arrest of leukemic cells suggesting that this gene may contribute to the impairment of proliferative control that is associated with the leukemic phenotype.

1985 - Study of the levels of expression of two oncogenes, c-myc and c-myb, in acute and chronic leukemias of both lymphoid and myeloid lineage.. [Articolo su rivista]
Ferrari, Sergio; Torelli, Umberto; Selleri, L; Donelli, A; Venturelli, D; Narni, Franco; Moretti, Luigi; Torelli, Giuseppe
abstract

Total cellular RNA from a series of leukemic cell populations, both myeloid and lymphoid, as well as from normal circulating lymphocytes was analysed for the expression of two cellular oncogenes, c-myc and c-myb, by Northern blot hybridization assay. Expression of c-myc but not of c-myb was observed in unstimulated normal lymphocytes. Stimulation by PHA was shown to activate the expression of both genes. Remarkably different levels of expression of c-myc were observed in ALL, whereas in CLL the expression of c-myc was uniformly low or absent. Differential expression of c-myc was detected in AML as well as in CML, c-myb was differentially expressed in AML and ALL, and absent in CLL and CML. Other single cases of hemopoietic disorders were studied, but the expression of the two oncogenes was low or absent. Neither evident genome amplification nor genome rearrangements were detected in the cell DNAs digested with restriction endonucleases.

1984 - Isolation of a cDNA clone containing a sequence complementary to the intestinal calcium-binding protein of the chick [Articolo su rivista]
Ferrari, S.; Battini, R.; Leone, A.; Ferrari, S.; Torelli, G.; Barbiroli, B.
abstract

The present work describes the construction of a cDNA library in pBR322 plasmid from an mRNA population enriched for the intestinal calcium-binding protein (CaBP) mRNA of the chick. We report the isolation of one recombinant clone containing a vitamin D-regulated sequence, which is complementary to part of the CaBP mRNA. Northern blot hybridization experiments allowed us to identify a 1900 nucleotide RNA species as the CaBP mRNA. © 1984.

1981 - Different frequency classes of sequences in heterogeneous nuclear RNA of normal promyelocytes and lymphoblasts and of leukemic blast cells of circulating blood and of the HL60 line. [Relazione in Atti di Convegno]
Torelli, U; Torelli, Giuseppe; Narni, Franco; Donelli, A; Ferrari, Sergio; Franchini, G; Calabretta, Bruno
abstract

No abstract available.

1980 - Characteristics of macromolecular RNA metabolism in leukemic promyelocytes of the HL 60 line [Articolo su rivista]
Torelli, Umberto; A., Donelli; G., Franchini; Calabretta, Bruno; Ferrari, Sergio; Narni, Franco; Torelli, Giuseppe; G. P., Bagnara; M. A., Brunelli
abstract

The macromolecular RNA metabolism of the HL-60 promyelocytic cell line was studied after labelling with 3H-5 uridine and sedimentation in a linear sucrose gradient. The pattern obtained in exponentially growing HL-60 cells is strictly similar to that observed in resting circulating leukemic cells, whereas cleavage of early transcripts is very rapid in HeLa cells.

1980 - Self complementary sequences in the heterogeneous nuclear RNA of leukemic cells of the HL 60 promyelocytic cell line. [Articolo su rivista]
Torelli, Umberto; A., Donelli; G., Franchini; Ferrari, Sergio; Calabretta, Bruno; Narni, Franco; Torelli, Giuseppe; G. P., Bagnara; M. A., Brunelli
abstract

After extensive self annealing up to 30% double stranded RNA can be obtaind from HL-60 cell line. The annealing kinetics are consistent with the presence of at least two components. The smallest component is presumably transcribed from repeated DNA sequences. The largest component includes sequences annealing at low rate, as expected for sequences transcribed from unique DNA sequences..

1979 - Immunological evidence for the presence of double-stranded RNA regions in intact nuclei of normal human lymphocytes [Articolo su rivista]
Torelli, Umberto; Ferrari, Sergio; Franchini, G; Donelli, A; Narni, Franco; Calabretta, Bruno; Torelli, Giuseppe
abstract

Nuclei isolated from unstimulated and PHA stimulated human lymphocytes were incubated with a labeled purified antibody to a double-stranded polyribonucleotide. A relatively high amount of double stranded RNA could be detected in intact lymphocyte nuclei.

1979 - Kinetics of hybridization to human DNA of heterogeneous nuclear RNA isolated from normal human lymphoblasts and acute leukemia blast cells. [Articolo su rivista]
Torelli, Giuseppe; Narni, Franco; Franchini, G; Donelli, A; Ferrari, Sergio; Calabretta, Bruno; Torelli, Umberto; Bosi, Paolo
abstract

Heterogeneous nuclear RNA was extracted from normal PHA-stimulated human lymphocytes and acute myeloid leukemia blast cells. Experiments were performed to determine the hybridization kinetics of these RNA's to human DNA. The best least squares solutions indicate in the hybridization reaction of both normal and leukemic RNA two main components. For leukemic cell RNA the rate constants of both components were significantly different from that of normal cell RNA. In particular, the difference between the rate constants of the second lower component suggests that the slowly hybridizing sequences in leukemic cell RNA have a degree of repetition higher than of the corresponding sequences of normal cell RNA.

1979 - Reassociation kinetics of the DNA of human acute leukemia cells. [Articolo su rivista]
Torelli, Giuseppe; Cadossi, Ruggero; Ferrari, Sergio; Narni, Franco; Ferrari, Stefano; Montagnani, Giuliano; Torelli, Umberto; Bosi, Paolo
abstract

Human DNA isolated from normal phytohaemagglutinin-stimulated human lymphocytes and from acute leukemia blast cells have been studied by renaturation techniques using hydroxyapatite binding and DNA hyperchromism. In the leukemic genome, the unique sequences account for 62% of the genome of leukemic DNA. Repetitive sequences may be subdivided into at least three fractions: (a) foldback sequences, which represent 5% of the genome; (b) sequences with high repetition frequency (3. 10(4) times on the average), which represent 12% of the genome; (c) sequences with low repetition frequency (10 times on the average), which represent 16% of the genome. The average length of the repetitive sequences is evaluated to be between 200 and 500 nucleotides. There are at least two patterns of interspersion of repetitive sequences with unique sequences of different length: short (about 2000 nucleotides on average) and long (not defined). The results of our experiments on DNA from normal phytohaemagglutinin-stimulated human lymphocytes are in close agreement with those reported by other authors studying different types of human cells. The human leukemic DNA, as far as the parameters that have been studied, does not significantly differ from normal human DNA.

1978 - The primary product of DNA transcription in the blast cell of acute leukemia. Its characterization as an approach to the understanding of the origin of the blast crisis of chronic myeloid leukemia [Articolo su rivista]
Torelli, Umberto; Torelli, Giuseppe; Cadossi, Ruggero; Ferrari, Stefano; Ferrari, Sergio; Montagna, G; Narni, Franco; Franchini, G; Donelli, A.
abstract

The primary product of transcription, the heterogeneous nuclear RNA, is processed at a very low rate in leukemic blast cells. The proportion of double-helical segments, presumably the recognition sites of cleaving enzymes, is much greater. An alteration of the mechanisms dictating the formation of secondary structure in heterogeneous nuclear RNA might cause the inhibition of genome expression in leukemic cells.

1977 - Immunological assay of double-helical segments in RNA fractions of different molecular size extracted from acute myeloid leukemia blast cells. [Articolo su rivista]
Torelli, Umberto; Ferrari, Sergio; Montagnani, Giuliano; Torelli, Giuseppe; Cadossi, Ruggero; Ferrari, Stefano; Narni, Franco
abstract

Whole-cell RNA, extracted from acute myeloid leukemia blast cells, was fractionated by sedimentation through sucrose gradients. The proportion of double-helical segments present in each fraction was then determined by a quantitative microcomplement fixation assay that specifically measures double-helical RNA. Sizable amounts of double-helical segments were detected in all fractions of cellular RNA corresponding to S values higher than approximately 20. In all cell populations examined the highest proportion of double-helical segments was found in RNA fractions sedimenting faster then the 45 S ribosomal precursors RNA, i.e., in fractions including only heterogeneous nuclear RNA.

1977 - In vitrocleavage of 45S pre-ribosomal RNA and of giant heterogeneous RNA extracted from human leukemic cells. [Articolo su rivista]
Torelli, Umberto; Ferrari, Sergio; Torelli, Giuseppe; Cadossi, Ruggero; Ferrari, Stefano; Montagnani, Giuliano; Narni, Franco
abstract

45S ribosomal precursor RNA and large heterogeneous RNA molecules (greater than 45S) extracted from human leukemic cells were incubated in vitro with purified RNase III, which specifically attacks double-helical RNA regions. About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial 'shift' to a polydisperse distribution in the 10S-45S range

1976 - Accumulation of giant heterogeneous RNA molecules in acute myeloid leukemia blast cells. [Articolo su rivista]
Torelli, Umberto; Torelli, Giuseppe; Cadossi, Ruggero; Ferrari, Stefano; Ferrari, Sergio; Narni, Franco; Montagnani, Giuliano
abstract

Time course and "chase" experiments showed that, after incubation of acute myeloid leukemia blast cells with a labeled RNA precursor, a large proportion of radioactivity remained associated with RNA molecules larger than 45 S even after several hr. Double-labeling experiments with [5-3H]uridine and [methyl-14C]methionine indicated that unmethylated giant heterogeneous RNA larger than 45 S is processed much more slowly than the 45 S ribosomal precursor, so that relatively large amounts of fairly stable RNA of the former class accumulate in the cell. The measurement of labeled giant heterogeneous RNA molecules bound to polyuridylate-fiberglass filters showed that molecules carrying polyadenylate segments seemingly turn over faster than those lacking polyadenylate.