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Alexis GRANDE

Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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Pubblicazioni

2023 - The Capacity of Magnesium to Induce Osteoclast Differentiation Is Greatly Enhanced by the Presence of Zoledronate [Articolo su rivista]
Ricchiuto, Silvia; Palumbo, Rossella; Lami, Francesca; Gavioli, Francesca; Caselli, Lorenzo; Montanari, Monica; Zappavigna, Vincenzo; Anesi, Alexandre; Zanocco-Marani, Tommaso; Grande, Alexis
abstract

Simple Summary A number of skeletal disorders, all characterized by a metabolic or neoplastic loss of bone tissue, are cured with drugs called Bisphosphonates (BPs), which exert their therapeutic effect by suppressing cells named osteoclasts, normally mediating bone resorption. Unfortunately, these drugs can also provoke a dangerous side effect known as osteonecrosis of the jaw (ONJ), a bone infection localized in the oral cavity and characterized by gingival ulceration, sometimes accompanied by suppuration and pain. This condition, occasionally arising spontaneously, is more often started by a tooth extraction. The reduced number of osteoclasts, determined by BPs, is thought to favor the bacterial invasion of healthy bone and the incapacity to eliminate infected bone, that are in turn responsible for the appearance of ONJ. Here we show that Magnesium, used for decades as dietary supplement, can invert the effect of BPs, transforming them, through a sort of paradox effect, into powerful activators of osteoclast production. These results suggest that Magnesium might be used in a topical approach aimed to cure or prevent ONJ. Notably, the capacity of Magnesium to activate osteoclast production was even observed in absence of BPs, suggesting its application also in ONJ forms caused by agents distinct to BPs.Abstract Bisphosphonates (BPs) are successfully used to cure a number of diseases characterized by a metabolic reduction in bone density, such as Osteoporosis, or a neoplastic destruction of bone tissue, such as multiple myeloma and bone metastases. These drugs exert their therapeutic effect by causing a systemic osteoclast depletion that, in turn, is responsible for reduced bone resorption. Unfortunately, in addition to their beneficial activity, BPs can also determine a frightening side effect known as osteonecrosis of the jaw (ONJ). It is generally believed that the inability of osteoclasts to dispose of inflamed/necrotic bone represents the main physiopathological aspect of ONJ. In principle, a therapeutic strategy able to elicit a local re-activation of osteoclast production could counteract ONJ and promote the healing of its lesions. Using an experimental model of Vitamin D3-dependent osteoclastogenesis, we have previously demonstrated that Magnesium is a powerful inducer of osteoclast differentiation. Here we show that, surprisingly, this effect is greatly enhanced by the presence of Zoledronate, chosen for our study because it is the most effective and dangerous of the BPs. This finding allows us to hypothesize that Magnesium might play an important role in the topical therapy of ONJ.

2022 - Pulsed Electro-Magnetic Field (PEMF) Effect on Bone Healing in Animal Models: A Review of Its Efficacy Related to Different Type of Damage [Articolo su rivista]
Di Bartolomeo, M.; Cavani, F.; Pellacani, A.; Grande, A.; Salvatori, R.; Chiarini, L.; Nocini, R.; Anesi, A.
abstract

Biophysical energies are a versatile tool to stimulate tissues by generating biopotentials. In particular, pulsed electromagnetic field (PEMF) stimulation has intrigued researchers since the 1970s. To date, many investigations have been carried out in vivo, but a gold standard treatment protocol has not yet been defined. The main obstacles are represented by the complex setting of PEMF characteristics, the variety of animal models (including direct and indirect bone damage) and the lack of a complete understanding of the molecular pathways involved. In the present review the main studies about PEMF stimulation in animal models with bone impairment were reviewed. PEMF signal characteristics were investigated, as well as their effect on molecular pathways and osseous morphological features. We believe that this review might be a useful starting point for a prospective study in a clinical setting. Consistent evidence from the literature suggests a potential beneficial role of PEMF in clinical practice. Nevertheless, the wide variability of selected parameters (frequency, duration, and amplitude) and the heterogeneity of applied protocols make it difficult to draw certain conclusions about PEMF effectiveness in clinical implementation to promote bone healing. Deepening the knowledge regarding the most consistent results reported in literature to date, we believe that this review may be a useful starting point to propose standardized experimental guidelines. This might provide a solid base for further controlled trials, to investigate PEMF efficacy in bone damage conditions during routine clinical practice.

2022 - Targeting Chemotherapy to Decondensed H3K27me3-Marked Chromatin of AML Cells Enhances Leukemia Suppression [Articolo su rivista]
Porazzi, P.; Petruk, S.; Pagliaroli, L.; De Dominici, M.; Deming, D.; Puccetti, M. V.; Kushinsky, S.; Kumar, G.; Minieri, V.; Barbieri, E.; Deliard, S.; Grande, A.; Trizzino, M.; Gardini, A.; Canaani, E.; Palmisiano, N.; Porcu, P.; Ertel, A.; Fortina, P.; Eischen, C. M.; Mazo, A.; Calabretta, B.
abstract

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. SIGNIFICANCE: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359.

2021 - Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis [Articolo su rivista]
Fantini, Sebastian; Rontauroli, Sebastiano; Sartini, Stefano; Mirabile, Margherita; Bianchi, Elisa; Badii, Filippo; Maccaferri, Monica; Guglielmelli, Paola; Ottone, Tiziana; Palmieri, Raffaele; Genovese, Elena; Carretta, Chiara; Parenti, Sandra; Mallia, Selene; Tavernari, Lara; Salvadori, Costanza; Gesullo, Francesca; Maccari, Chiara; Zizza, Michela; Grande, Alexis; Salmoiraghi, Silvia; Mora, Barbara; Potenza, Leonardo; Rosti, Vittorio; Passamonti, Francesco; Rambaldi, Alessandro; Voso, Maria Teresa; Mecucci, Cristina; Tagliafico, Enrico; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

: Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.

2021 - Magnesium favors the capacity of vitamin d3 to induce the monocyte differentiation of u937 cells [Articolo su rivista]
Parenti, S.; Sandoni, L.; Montanari, M.; Zanocco-Marani, T.; Anesi, A.; Iotti, S.; Manfredini, R.; Frassineti, C.; Davalli, P.; Grande, A.
abstract

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.

2021 - Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts [Articolo su rivista]
Bertesi, M.; Fantini, S.; Alecci, C.; Lotti, R.; Martello, A.; Parenti, S.; Carretta, C.; Marconi, A.; Grande, A.; Pincelli, C.; Zanocco Marani, T.
abstract

The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.

2020 - Cadherins down-regulation: towards a better understanding of their relevance in colorectal cancer [Articolo su rivista]
Losi, Lorena; ZANOCCO MARANI, Tommaso; Grande, Alexis
abstract

The down-regulation of cadherin expression in colorectal cancer (CRC) has been widely studied. However, existing data on cadherin expression are highly variable and its relevance to CRC development has not been completely established. This review examines published studies on cadherins whose down-regulation has been already demonstrated in CRC, trying to establish a relationship with promoter methylation, the capacity to influence the Wnt / CTNNB1 (catenin beta 1, beta-catenin) signalling pathway and the clinical implications for disease outcome. Moreover, it also analyses factors that may explain data variability and highlights the importance of considering the altered subcellular localization of the examined cadherins. The results of this survey reveal that thirty of one hundred existing cadherins appear to be down-regulated in CRC. Among these, ten are cadherins, sixteen are protocadherins, equally divided between clustered and non clustered, and four are cadherin - related. These findings suggest that, to better define the role played by cadherin down-regulation in CRC pathogenesis, the expression of multiple rather than individual cadherins should be taken into account and further functional studies are necessary to clarify the relative ability of individual cadherins to inhibit CTNNB1 therefore acting as tumor suppressors.

2020 - Wnt/CTNNB1 signal transduction pathway inhibits the expression of ZFP36 in squamous cell carcinoma, by inducing transcriptional repressors SNAI1, SLUG and TWIST [Articolo su rivista]
Zanfi, E. D.; Fantini, S.; Lotti, R.; Bertesi, M.; Marconi, A.; Grande, A.; Manfredini, R.; Pincelli, C.; Zanocco-Marani, T.
abstract

The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.

2019 - From Osteoclast Differentiation to Osteonecrosis of the Jaw: Molecular and Clinical Insights [Articolo su rivista]
Anesi, Alexandre; Generali, Luigi; Sandoni, Laura; Pozzi, Samantha; Grande, Alexis
abstract

Bone physiology relies on the delicate balance between resorption and formation of its tissue. Bone resorption depends on a process called osteoclastogenesis in which bone-resorbing cells, i.e., osteoclasts, are produced by the differentiation of more undifferentiated progenitors and precursors. This process is governed by two main factors, monocyte colony-stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). While the former exerts a proliferating effect on progenitors/precursors, the latter triggers a differentiation effect on more mature cells of the same lineage. Bone homeostasis requires a perfect space–time coordination of the involved signals. When osteoclastogenesis is poorly balanced with the differentiation of the bone forming counterparts, i.e., osteoblasts, physiological bone remodelling can turn into a pathological state, causing the systematic disruption of bone tissue which results in osteopenia or osteolysis. Examples of these conditions are represented by osteoporosis, Paget’s disease, bone metastasis, and multiple myeloma. Therefore, drugs targeting osteoclastogenesis, such as bisphosphonates and an anti-RANKL monoclonal antibody, have been developed and are currently used in the treatment of such diseases. Despite their demonstrated therapeutic efficacy, these agents are unfortunately not devoid of side effects. In this regard, a condition called osteonecrosis of the jaw (ONJ) has been recently correlated with anti-resorptive therapy. In this review we will address the involvement of osteoclasts and osteoclast-related factors in the pathogenesis of ONJ. It is to be hoped that a better understanding of the biological mechanisms underlying bone remodelling will help in the design a medical therapeutic approach for ONJ as an alternative to surgical procedures.

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 - Magnesium is a key regulator of the balance between osteoclast and osteoblast differentiation in the presence of vitamin D 3 [Articolo su rivista]
Mammoli, F.; Castiglioni, S.; Parenti, S.; Cappadone, C.; Farruggia, G.; Iotti, S.; Davalli, P.; Maier, J. A. M.; Grande, A.; Frassineti, C.
abstract

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D 3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

2019 - Monocytes-based in vitro assay for a preliminary biocompatibility assessment of blood-contacting devices [Articolo su rivista]
Gemelli, C.; Grande, A.; Ferrari, S.; Tomasi, A.; Cuoghi, A.
abstract

The biological evaluation of biomaterials is currently defined by the ISO-10993 norm in which parts four and five are dedicated to emo-compatibility and cell toxicity, respectively. Our study will provide a novel in vitro experimental approach for the biocompatibility assessment of biomaterials or medical devices using human primary monocytes as cellular model. In these new settings, human monocytes are exposed to a medium containing the extractable compounds derived from materials or devices; subsequently, cell toxicity and pro-inflammatory effects are analysed through MTT assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA) methodologies. These experimental procedures offer the advantage to use a human and primary cell context belonging to the immune system, in order to accurately predict the nature of blood/device interaction occurring during a clinical application. To validate the reliability of this method, we also reported a comparative study between two different membranes showing a different level of biocompatibility. On the bases of these data, it is possible to state that this new experimental model represents a good approach to investigate the effects induced by a biomaterial on cell death and inflammation using human, primary monocytes.

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 - 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.

2016 - A novel 2,3-benzodiazepine-4-one derivative AMPA antagonist inhibits G2/M transition and induces apoptosis in human leukemia Jurkat T cell line [Articolo su rivista]
Parenti, Sandra; Casagrande, Giacomo; Montanari, Monica; Espahbodinia, M.; Ettari, R.; Grande, Alexis; Corsi, Lorenzo
abstract

It has been shown that the antagonism of glutamate receptors activity was able inhibit proliferation and induce apoptosis in several neuronal and non-neuronal cancer cell lines. In addition, it has been shown that glutamate might facilitate the spread and growth of leukemia T cells through interactions with AMPA receptors. The aim of the present study was to investigate the modulation of cell cycle elicited by a novel 2,3-benzodiazepine-4- one non-competitive AMPA antagonist derivative in the human leukemia Jurkat T cells. Our results indicated that the 1-(4-amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4 h-2,3- benzodiazepin-4-one, named 1 g, exerted a significant growth inhibition of leukemia Jurkat T cells in a time and dose dependent manner, arresting the transition of G2/M phase through activation of Myt-1. The molecule also induced apoptosis through the enhanced expression of the pro-apoptotic p53, and the inhibition of Bcl-2, and Bcl-xl, followed by the activation of caspase-3. The results suggested that compound 1 g might act mostly as a cytostatic rather than cytotoxic compound. Al- though further studies are necessary, in order to identify others specific pathways involved in the activity of the present molecule, the presented results identified a novel molecule acting on specific G2/M checkpoint reg- ulation pathway. Finally, our data suggest that compound 1 g might be a good molecule for future development in the cancer research

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 - 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 - Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database [Articolo su rivista]
Manzotti, Gloria; Parenti, Sandra; Ferrari, Giovanna; Soliera, Angela Rachele; Cattelani, Sara; Montanari, Monica; Grande, Alexis; Calabretta, Bruno; Ertel, Adam; Cavalli, Daniel
abstract

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.

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 - Cytotoxic effect of hemin in colonic epithelial cell line: Involvement of 18kDa translocator protein (TSPO). [Articolo su rivista]
Gemelli, Claudia; Dongmo, Bm; Ferrarini, F; Grande, Alexis; Corsi, Lorenzo
abstract

Aims The aim of this study is to investigate the effect of hemin in colonic epithelial cells (Caco-2) cell proliferation and if this effect was due to a direct modulation of 18-kDa translocator protein (TSPO) and/or heme oxygenase type 1 (HO-1). Main methods The main methods are as follow: cell proliferation and cell cytotoxic assays on Caco-2 cell lines treated with hemin in the presence or not of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) and Sn-protoporphyrin IX (Sn-PPIX), and immunoblotting for TSPO and HO-1 protein analysis, siRNA directed against TSPO. Key findings Hemin was shown to be toxic for the Caco-2 cell line in a concentration and time dependent manner. Although hemin was able to induce HO-1 in a dose dependent manner, a specific HO-1 inhibitor, Sn-PPIX, was unable to interfere with the effect of hemin on Caco-2 cells. Instead, PK 11195, a specific TSPO ligand, was able to counteract the effect of hemin, suggesting an important role of TSPO in the hemin activity. Cell viability assay further confirms the high cytotoxic effects exerted by hemin on Caco-2 cells expressing TSPO compared to the siRNA-TSPO targeted cells. In addition, hemin was able to decrease significantly the TSPO protein density in a dose dependent manner after 24 h of incubation. Significance The interaction and the consecutive down regulation of TSPO by hemin played an important role in the control of Caco-2 cell viability. The presented data suggest that TSPO might contribute to protect cells from potential toxic compounds such as free tetrapyrroles, candidating this receptor as a survival receptor protein.

2014 - Involvement of μ-protocadherin in colorectal carcinogenesis: a promise for clinico-pathological evaluation. [Articolo su rivista]
Losi, Lorena; Grande, Alexis
abstract

Cell-cell adhesion is a fundamental activity to allow the maintenance of epithelial integrity, and defects impairing this process promote the formation of tumors such as colorectal cancer (CRC). In this regard, a crucial role is played by adhesion molecules, named cadherins, which exert their function through the inhibition of the beta-catenin signaling proliferation pathway, constitutively activated in CRC. A number of reports, published over the last decade, have highlighted the existence of a novel cadherin family member, called μ-protocadherin, to underline the hybrid nature of its extra-cellular region, including both cadherin-like and mucin-like domains. Is has been shown that this protein plays an important role in inter-cellular adhesion processes, inhibits beta-catenin activity in normal colorectal mucosa, undergoes a down-regulated expression in CRC and is up-regulated upon treatment with chemoprevention agents against this tumor.

2014 - Le basi cellulari delle malattie onco-ematologiche [Monografia/Trattato scientifico]
Grande, Alexis
abstract

Il libro propone una trattazione dettagliata a livello molecolare di processi cellulari quali smistamento delle proteine, vie di segnalazione, ciclo cellulare, differenziamento, apoptosi, protooncogeni e antioncogeni, evidenziando come le loro alterazioni possano causare numerose malattie ad esempio ereditarie, ma soprattutto neoplastiche e in particolare onco-ematologiche.

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 - Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro. [Articolo su rivista]
Italiani, P; Mazza, Emilia Maria Cristina; Lucchesi, D; Cifola, I; Gemelli, C; Grande, Alexis; Battaglia, C; Bicciato, Silvio; Boraschi, D.
abstract

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements

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 - Chemoprevention of colorectal cancer [Brevetto]
Grande, Alexis; Ferrarini, Fabrizio; Parenti, Sandra
abstract

In the present invention, a new combination is disclosed comprising (i) 5 -aminosalicylic acid (5 -ASA) or a derivative thereof, or a pharmacologically acceptable salt thereof, and (ii) a group D vitamin, a derivative thereof, a metabolite or analogue, for use in the prevention and/or treatment of colorectal cancer (CRC). A further aspect of the invention is directed to pharmaceutical compositions comprising said combination together with at least one physiologically acceptable excipient and the use thereof in the prevention and/or in the treatment of the colorectal cancer.

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.

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 - HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin. [Articolo su rivista]
Salsi, Valentina; Ferrari, Silvia; Ferraresi, Roberta; Cossarizza, Andrea; Grande, Alexis; Zappavigna, Vincenzo
abstract

HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G(1) phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.

2009 - PPARδ is a ligand-dependent negative regulator of Vitamin D3-induced monocyte differentiation [Articolo su rivista]
A., Lymboussaki; Gemelli, Claudia; Testa, Anna; Facchini, Giulia; Ferrari, Francesco; Mavilio, Fulvio; Grande, Alexis
abstract

A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.

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 - 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 - Indwelling ureteral stents and sexual health: a prospective, multivariate analysis [Articolo su rivista]
Sighinolfi, M. C.; Micali, Salvatore; De Stefani, Stefano; Mofferdin, A.; Grande, A.; Giacometti, M.; Ferrari, N.; Rivalta, M.; Bianchi, Giampaolo
abstract

PURPOSE: Ureteral stents are common devices in urological practice. However, a stent may provoke lower urinary tract symptoms that severely affect quality of life. We evaluated the relationship between ureteral stents and male erection/female sexuality. MATERIALS AND METHODS: A total of 30 men and 20 women undergoing ureteral stent positioning were considered. Patients affected by risk factors for erectile dysfunction or hormonal and metabolic alterations were excluded. Hystero-ovariectomy and menopause were considered exclusion criteria. Three questionnaires were administered before stenting and 45 to 60 days after stent positioning, including the International Prostate Symptom Score, the International Index of Erectile Function-5 for men and the Female Sexual Function Index for women. RESULTS: Mean age was 45 years in men and 39 years in women. The mean+/-SD International Index of Erectile Function-5 score was 23.2+/-1.27 and the mean Female Sexual Function Index score was 32.15+/-2.71 before stent positioning. No lower urinary tract symptoms were reported before the procedure. After the ureteral stent was indwelling the mean International Index of Erectile Function-5 score was 13.5+/-4.01 and the mean Female Sexual Function Index score was 23.6+/-14.66 (p=0.000 and 0.007, respectively). Of 30 men 25 reported a pathological International Index of Erectile Function-5 score and 6 of 20 women denied any sexual activity due to stent related anxiety, resulting in the minimum Female Sexual Function Index score. In the remaining 14 women sexual life was not significantly impaired by the ureteral stent (p=0.08). CONCLUSIONS: Ureteral stents impaired the quality of sexual life in male and female subjects. In men the most important distress was in regard to erectile function, probably related to lower urinary tract symptoms. Conversely female sexuality appeared to be severely impaired due to stent related psychological concerns.

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 - 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 - Competitive engraftment of hematopoietic stem cells genetically modified with a truncated erythropoietin receptor [Articolo su rivista]
URBINATI, Fabrizia; F., Lotti; G., Facchini; M., Montanari; G., Ferrari; MAVILIO, Fulvio; GRANDE, Alexis
abstract

Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (similar to 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34(+) stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection.

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 - 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 - Deletion of a negatively acting sequence in a chimeric GATA-1 enhancer-long terminal repeat greatly increases retrovirally mediated erythroid expression [Articolo su rivista]
A., Testa; F., Lotti; L., Cairns; Grande, Alexis; S., Ottolenghi; G., Ferrari; A., Ronchi
abstract

The locus control region of the beta-globin gene cluster has been used previously to direct erythroid expression of globin genes from retroviral vectors for the purpose of gene therapy. Short erythroid regulatory elements represent a potentially valuable alternative to the locus control region. Among them, the GATA-1 enhancer HS2 was used to replace the retroviral enhancer within the 3'-long terminal repeat (LTR) of the retroviral vector SFCM, converting it into an erythroid-specific regulatory element. In this work, we have functionally studied an additional GATA-1 enhancer, HS1. HS1 participates in the transcriptional autoregulation of GATA-1 through an essential GATA-binding site that is footprinted in vivo. In this work we identified within HS1 a new in vivo footprinted region, and we showed that this sequence indeed binds a nuclear protein in vitro. Addition of HS1 to HS2 within the LTR of SFCM significantly improves the expression of a reporter gene. The deletion of the newly identified footprinted sequence in the retroviral construct further increases expression up to a level almost equal to that of the wild type retroviral LTR, without loss of erythroid specificity, suggesting that this sequence may act as a negative regulatory element. An improved vector backbone, MDeltaN, allows even better expression from the new GATA cassette. These results suggest that substantial improvement of overall expression can be achieved by the combination of multiple changes in both regulatory elements and vectors.

2004 - Selective engraftment of genetically modified hematopoietic stem cells by a truncated erythropoietin receptor [Relazione in Atti di Convegno]
Urbinati, F; Grande, Alexis; Lotti, F; Facchini, Gl; Ferrari, G; Mavilio, Fulvio
abstract

Genetic modification of hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Transplantation of HSCs, however, requires toxic myeloablation regimens which render this approach questionable for non life-threatening disorders. A potential alternative is the use of transgenes allowing positive selection of HSCs in vivo. We used MLV-derived retroviral vectors and HIV-derived lentiviral vectors to express a truncated form of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. The tEpoR molecule carries a deletion of the 91 carboxy-terminal amino acids, which enhances its proliferative response due to the elimination of a negative regulatory domain. Murine HSCs expressing retrovirally-transferred tEpoR at different levels (1,500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into co-isogenic mouse recipients. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR significantly increase their marrow repopulation capacity upon xenotransplantation into sub-lethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival and differentiation properties. Long-term analysis of serially transplanted mice showed that expression of tEpoR at physiological levels (i.e., comparable with, or slightly higher than, those of the wild-type EpoR in erythroblastic cells) has no effect on steady-state hematopoiesis, and induces no further expansion of transduced cells after the engraftment period. However, significant overexpression of tEpoR (>8-fold the physiological levels) causes mild anemia and erythrocyte morphological abnormalities. These data indicate that expression of tEpoR is a potential alternative for in vivo selection of murine and human repopulating HSCs.

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 - 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.

2003 - Safety of retroviral gene marking with a truncated NGF receptor [Articolo su rivista]
Bonini, C; Grez, M; Traversari, C; Ciceri, F; Martkel, S; Ferrari, G; Dinauer, M; Sadat, M; Aiuti, A; Deola, S; Raddrizzani, M; Hagenbeek, A; Apperley, J; Ebeling, S; Martens, A; Kolb, Hj; Weber, M; Lotti, F; Grande, Alexis; Weissinger, E; Bueren, Ja; Lamana, M; Falkenburg, Jhf; Heemskeerk, Mhm; Austin, T; Kornblau, S; Marini, F; Benati, C; Magnani, Z; Cazzaniga, S; Toma, S; GALLO STAMPINO, C; Introna, M; Slavin, S; Greenberg, Pd; Bregni, M; Mavilio, Fulvio; Bordignon, C.
abstract

Random integration into the host cell genome and inappropriate transgene expression are major safety concerns for the clinical use of retroviral vectors.

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 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 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.

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.

2001 - Interleukin-11 induces Th2 polarization of human CD4(+) T cells [Articolo su rivista]
Curti, A; Ratta, M; Corinti, S; Girolomoni, G; Ricci, F; Tazzari, P; Siena, M; Grande, A; Fogli, M; Tura, S; Lemoli, R M
abstract

Exploration of the immunomodulatory activities of the multifunctional cytokine interleukin-11 (IL-11) has prompted several therapeutic applications. The immunomodulatory effects of IL-11 on human antigen-presenting cells and on T cells were investigated. IL-11 inhibited IL-12 production by activated CD14(+) monocytes, but not by mature dendritic cells (DCs) stimulated via CD40 ligation. Moreover, IL-11 did not affect either DC maturation, as demonstrated by phenotypic analysis and evaluation of cytokine production, or DC generation from progenitor cells in the presence of specific growth factors. Molecular analysis demonstrated the expression of IL-11 receptor messenger RNA in highly purified CD14(+) monocytes, CD19(+) B cells, CD8(+), and CD4(+) T cells, and CD4(+)CD45RA(+) naive T lymphocytes. In keeping with this finding, IL-11 directly prevented Th1 polarization of highly purified CD4(+)CD45RA(+) naive T cells stimulated with anti-CD3/CD28 antibodies, as demonstrated by significant increases of IL-4 and IL-5, by significantly decreased interferon-gamma production and by flow cytometry intracellular staining of cytokines. Coincubation of naive T cells with DCs, the most potent stimulators of Th1 differentiation, did not revert IL-11-mediated Th2 polarization. Furthermore, parallel experiments demonstrated that the activity of IL-11 was comparable with that induced by IL-4, the most effective Th2-polarizing cytokine. Taken together, these findings show that IL-11 inhibits Th1 polarization by exerting a direct effect on human T lymphocytes and by reducing IL-12 production by macrophages. Conversely, IL-11 does not exert any activity on DCs. This suggests that IL-11 could have therapeutic potential for diseases where Th1 responses play a dominant pathogenic role.

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.

1999 - Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells [Articolo su rivista]
Grande, Alexis; B., Piovani; A., Aiuti; S., Ottolenghi; Mavilio, Fulvio; G., Ferrari
abstract

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.PMID: 10233879 [PubMed - indexed for MEDLINE]

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.

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 - 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 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 - 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 - Normal Human Keratinocytes Express Nerve Growth-Factor Receptor And Proliferate In Response To Nerve Growth-Factor [Abstract in Rivista]
Pincelli, Carlo; C., Sevignani; Grande, Alexis; F., Fantini; L., Aloe; Giannetti, Alberto
abstract

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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.

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 - Abundance of the Primary Transcript and Its Processed Product of Growth-related Genes in Normal and Leukemic Cells during Proliferation and Differentiation [Articolo su rivista]
Ferrari, S.; Tagliafico, E.; Manfredini, R.; Grande, A.; Rossi, E.; Zucchini, P.; Torelli, G.; Torelli, U.
abstract

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was deter-mined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the SI 4 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells. © 1992, American Association for Cancer Research. All rights reserved.

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 - THE USE OF THE ALKALINE PHOSPHATASE-ANTIALKALINE PHOSPHATASE TECHNIQUE FOR IMMUNOPHENOTYPING ACUTE MYELOID-LEUKEMIA [Articolo su rivista]
Sacchi, Stefano; Marietta, M; Vecchi, A; Morselli, S; Longo, R; Grande, Alexis; Torelli, U.
abstract

The leukemic cells from 31 cases of acute myeloid leukemia were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) technique, using 7 monoclonal antibodies reactive with cells of myeloid origin. We found a good correlation between the results obtained using the APAAP method and indirect immunofluorescence. In most cases, we observed a slight degree of variation in the percentages of reacting cells when comparing the two methods. Nevertheless, taking 20% of cells being immunolabelled as a threshold for defining a case as positive, we found no discrepancies in the final classification of each case. The main advantages of the APAAP method are: (1) its use with routinely prepared peripheral or blood marrow samples, and (2) the possibility of correlating immunological characterization with morphology. Since the results with the APAAP method were comparable with those obtained using indirect immunofluorescence, we suggest that this former technique can complement, and sometimes substitute, other methods of immunological evaluation.

1989 - Determinazione dell'immunofenotipo delle leucemie acute non linfatiche mediante la tecnica della immunofosfatasi alcalina: vantaggi ed applicazioni cliniche. [Abstract in Rivista]
Vecchi, A; Morselli, M; Longo, R; Grande, A; Zucchini, Patrizia; Marietta, M; Montagnani, G; Sacchi, Stefano
abstract

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