Guido Rossi | Università degli Studi di Napoli "Federico II" (original) (raw)
Papers by Guido Rossi
Endocrinology, Apr 1, 1997
The expression of integrins of the  1 family and their possible biological effects were investig... more The expression of integrins of the  1 family and their possible biological effects were investigated in normal human thyroid cells in monolayer culture. The expression of  1 and ␣ 1-6 integrin subunits was determined by flow cytofluorometry with specific antibodies. Follicular cells of subconfluent monolayer cultures expressed ␣ 2  1 and ␣ 3  1 at high levels, while ␣ 1  1 was only slightly expressed, and ␣ 4  1 , ␣ 5  1 , and ␣ 6  1 were never detected. Cell attachment assays were performed in fibronectin-, type I collagen-, and laminin-coated microtiter plates. Thyroid cells, while adherent to collagen and fibronectin, showed poor attachment to laminin despite the abundance of their putative receptors ␣ 2  1 and ␣ 3  1. In serum-free medium, collagen and fibronectin induced cytoskeletal organization, change of cell shape from round to flat, and cell spreading. [ 3 H]Thymidine incorporation
La Ricerca in clinica e in laboratorio, 1975
PubMed, Jun 1, 1999
Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironmen... more Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironment involves adhesion of these cells to the BM extracellular matrix molecules fibronectin and laminin. The 67-kDa laminin receptor (67LR) is a nonintegrin protein with high affinity for laminin, which plays a critical role in basement membrane invasion and metastasis of cancer cells. By Western blotting, we documented that 67LR was strongly expressed in myelomonocytic THP1 and histiocytic U937 cells and was weakly expressed in promyelocytic HL-60 cells. In HL-60 cells, 67LR expression almost disappeared after retinoic-induced granulocytic differentiation, whereas it strongly increased after phorbol ester-induced monocytic differentiation. We did not detect 67LR expression in normal BM hematopoietic cells, in precursor-B acute lymphoblastic leukemia, in chronic lymphocytic leukemia, or in chronic myeloid leukemia in chronic phase. By contrast, we detected enhanced 67LR expression in 40% of 53 de novo acute myeloid leukemias (AMLs), which frequently exhibited monocytic or myelomonocytic morphology and expressed CD14 and CD11a (P < 0.05). Using a colorimetric assay, we found that the expression pattern of this receptor corresponded to a higher adhesion to laminin; the adhesion was specific because in vitro addition to laminin-coated wells of recombinant 37-kDa laminin receptor precursor (37LRP), which is the cytoplasmic precursor containing both laminin-binding domains of cell surface 67LR, significantly reduced laminin binding of AML cells. The expression of 67LR on AML cell surface did not correlate with other differentiation and integrin antigens such as CD7, CD13, CD33, CD34, CD11b, CD11c, CD49d, CD49e, CD45RA, and CD45RO. In contrast with 67LR behavior in solid tumors, no statistically significant difference was found between 67LR expression and any hematological characteristic of the disease at diagnosis, nor between 67LR expression and outcome of the disease as measured by complete remission rate, disease-free survival, or overall survival. In conclusion, our results indicate that 67LR expression mediates specific adhesion to laminin and that the detection of this molecule may be a valuable addition to other lineage-associated antigens in identifying monocytic-oriented AML.
International Journal of Cancer, Apr 14, 2003
The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in t... more The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR-transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA-stabilizing factor that binds the coding region of uPAR-mRNA. We now investigate the relevance of uPA-mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up-regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPAR-mRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR-mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR-mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR-stability through a cellular factor that binds the coding region of uPAR-mRNA.
Endocrinology, Feb 1, 1999
The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, ... more The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of lovastatin in a dose-dependent fashion. The geranylgeranyl transferase GGTI-298 inhibitor mimicked the effects of lovastatin on cell morphology and induced cell death, whereas the farnesyl transferase inhibitor FTI-277 was less effective to induce both cell rounding and apoptosis. Resistance to lovastatin-induced apoptosis by expression of the viral serpine CrmA and by the peptide inhibitor of caspases, Z-DEVD-fmk, demonstrated the involvement of CrmA-sensitive, caspase-3-like proteases. Inhibition of endogenous p53 activity did not affect the sensitivity of thyroid cells to lovastatin, demonstrating that this type of apoptosis is p53 independent. We conclude that lovastatin is a potent inducer of apoptosis in proliferating thyroid cells through inhibition of protein prenylation. This type of apoptosis requires protein synthesis, is CrmA sensitive and caspase-3-like protease dependent, and is independent from p53.
The Journal of Clinical Endocrinology and Metabolism, Apr 1, 1995
The expression of intercellular adhesion molecule-1 (ICAM-1) in tumoral tissues may promote their... more The expression of intercellular adhesion molecule-1 (ICAM-1) in tumoral tissues may promote their interaction with the immune system and cytotoxic effect on tumoral cells. This observation led to the investigation of ICAM-1 expression and modulation in different tumoral cell systems in vitro. Recently, retinoic acid-responsive elements have been found in the 5'-regulatory region of the human ICAM-1 gene. In the present study, we investigated, by flow cytometry, the effect of retinoic acid on the surface expression of ICAM-1 in human thyroid carcinoma cell lines. Two papillary (NPA and TPC-1), one follicular (WRO), one anaplastic (ARO) and one immortalized fetal (TAD-2) cell line have been studied. All of them produced constitutively ICAM-1; its surface expression and specific messenger ribonucleic acid (mRNA) levels were increased significantly by retinoic acid in all except the WRO cell line. ICAM-1 hyperexpression by retinoic acid was time dependent, reversible, and dependent on mRNA and protein synthesis. Furthermore, cytokines, such as interferon-gamma and tumor necrosis factor-alpha, both individually and, to a greater extent, in combination with retinoic acid, increased ICAM-1 surface expression and its mRNA levels. In conclusion, retinoic acid is able to induce ICAM-1 up-regulation via mRNA accumulation in human thyroid carcinoma cell lines.
Journal of Cellular and Molecular Medicine, Jun 28, 2012
Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is a... more Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization.
Journal of Biological Chemistry, Nov 1, 2003
Integrin activation generates different signalings in a cell type-dependent manner and stimulates... more Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1. * This work has been supported in part by Ministero dell'Istruzione, dell'Università e della Ricerca (to M. V.) and Fondazione Italiana per la Ricerca sul Cancro (to T. D. M.
International Journal of Immunopathology and Pharmacology, 2010
Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are tw... more Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (ISSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from ISSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form sh...
The Journal of Clinical Endocrinology & Metabolism, 1993
European Journal of Endocrinology, 2001
OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apo... more OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells. METHODS: TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fracti...
European Journal of Heart Failure, Oct 1, 2006
BackgroundIn remodelling ventricles, the progression of heart failure is associated with structur... more BackgroundIn remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra‐cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death.The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors.AimsTo assess Granulocyte Macrophage‐Colony Stimulating Factor (GM‐CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM‐CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy.Methods and resultsGM‐CSFR expression in human cardiac tissue from explanted hearts of ten patients with end‐stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays.Our results demonstrated an increase in GM‐CSFR in cardiomyocytes from end‐stage heart failure tissues as compared to normal control tissues.ConclusionsWe hypothesize that GM‐CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.
FEBS Letters, 1999
In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell de... more In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell death, a process that has been termed`anoikis'. We investigated the role of p53 and other apoptotic proteins in anoikis in thyroid epithelial cells. Western blot analysis demonstrated that neither p53 nor Bcl-2, Bcl-XL and Bax protein expression changed during anoikis. However, loss of endogenous p53 activity in cells transfected with a dominant-negative mutated p53 inhibited anoikis demonstrating the involvement of p53-dependent processes. The phosphatase inhibitor sodium orthovanadate opposed anoikis when added to the cells within 6 h, suggesting a role for phosphorylated proteins.
FEBS Letters, 1999
The urokinase-type plasminogen activator uPA-R can regulate integrin functions by associating wit... more The urokinase-type plasminogen activator uPA-R can regulate integrin functions by associating with several types of L Lsubunit. We have recently shown that normal thyroid TAD-2 cells express both a native and a cleaved form of uPA-R which lacks the binding domain for uPA. We found this cleaved form to be present in reduced amounts in papillary and follicular thyroid carcinoma cells and completely absent in cells derived from an anaplastic thyroid carcinoma (ARO). We now report that in normal thyroid cells the intact form of uPA-R strongly associates with L L-1 integrins, whereas its cleaved form does not. uPA-R expressed by ARO cells shows a stronger resistance to the cleavage mediated by uPA, plasmin and chymotrypsin than does uPA-R expressed by normal thyroid cells. This resistance to cleavage correlates with the higher level of glycosylation of uPA-R of ARO cells as compared to that of cleavable uPA-R of normal thyroid cells. These results suggest that uPA-R cleavage, which occurs in several cell types, represents a mechanism regulating the interactions of uPA-R with integrins and, possibly, the subsequent integrin-mediated cell adhesion. Moreover we hypothesize that glycosylation regulates uPA-R cleavage and, indirectly, its interaction with integrins.
Experimental Cell Research, 1989
We studied the relationship between differentiation, transformation, and uPA production in a syst... more We studied the relationship between differentiation, transformation, and uPA production in a system of rat thyroid cells in vitro. The fully differentiated FRTL5 cells did not produce detectable amounts of uPA, even after stimulation with phorbol esters, potent inducers of uPA expression. All the other cell lines (i.e., FRT, cells which have lost the characteristics of the differentiated thyroid cells; l-5G and FRA, transformed cells derived from rat thyroid tumors) produced uPA, the l-5G line being the highest producer. Also the FRTL line became positive for uPA production after viral transformation (clone RM4). The lack of uPA expression in FRTL5 cells was not due to the presence of inhibitors and these cells did not produce an inactive molecule, as shown by immunoprecipitation with anti-uPA antibody. However, in PRTLr cells Northern analysis showed the presence of a small amount of uPA-specific mRNA that increased appreciably after phorbol ester stimulation. In conclusion, in our system uPA expression was a property of undifferentiated and transformed cells; in fully differentiated cells uPA expression was switched off by a still unclear mechanism. Q 1sss Academic PWS, I~C.
European Journal of Endocrinology, 2006
Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate su... more Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAFV600E), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) f...
Endocrinology, 1982
The production of thyroglobulin (TG) by cultured rat thyroid cells was demonstrated at the level ... more The production of thyroglobulin (TG) by cultured rat thyroid cells was demonstrated at the level of a single cell using a modified reverse plaque-forming assay. Thyroid cells were cultured in the presence of sheep erythrocytes (SE) coated with hybrid antibody specific for both SE ...
Endocrinology, 2000
Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; ... more Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas
Endocrinology, 1999
The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, ... more The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of lovastatin in a dose-dependent fashion. The geranylgeranyl transferase GGTI-298 inhibitor mimicked the effects of lovastatin on cell morphology and induced cell death, whereas the farnesyl transferase inhibitor FTI-277 was less effective to induce both cell rounding and apoptosis. Resistance to lovastatin-induced apoptosis by expression of the viral serpine CrmA and by the peptide inhibitor of caspases, Z-DEVD-fmk, demonstrated the involvement of CrmA-sensitive, caspase-3-like proteases. Inhibition of endogenous p53 activity did not affect the sensitivity of thyroid cells to lovastatin, demonstrating that this type of apoptosis is p53 independent. We conclude that lovastatin is a potent inducer of apoptosis in proliferating thyroid cells through inhibition of protein prenylation. This type of apoptosis requires protein synthesis, is CrmA sensitive and caspase-3-like protease dependent, and is independent from p53.
Endocrinology, Apr 1, 1997
The expression of integrins of the  1 family and their possible biological effects were investig... more The expression of integrins of the  1 family and their possible biological effects were investigated in normal human thyroid cells in monolayer culture. The expression of  1 and ␣ 1-6 integrin subunits was determined by flow cytofluorometry with specific antibodies. Follicular cells of subconfluent monolayer cultures expressed ␣ 2  1 and ␣ 3  1 at high levels, while ␣ 1  1 was only slightly expressed, and ␣ 4  1 , ␣ 5  1 , and ␣ 6  1 were never detected. Cell attachment assays were performed in fibronectin-, type I collagen-, and laminin-coated microtiter plates. Thyroid cells, while adherent to collagen and fibronectin, showed poor attachment to laminin despite the abundance of their putative receptors ␣ 2  1 and ␣ 3  1. In serum-free medium, collagen and fibronectin induced cytoskeletal organization, change of cell shape from round to flat, and cell spreading. [ 3 H]Thymidine incorporation
La Ricerca in clinica e in laboratorio, 1975
PubMed, Jun 1, 1999
Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironmen... more Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironment involves adhesion of these cells to the BM extracellular matrix molecules fibronectin and laminin. The 67-kDa laminin receptor (67LR) is a nonintegrin protein with high affinity for laminin, which plays a critical role in basement membrane invasion and metastasis of cancer cells. By Western blotting, we documented that 67LR was strongly expressed in myelomonocytic THP1 and histiocytic U937 cells and was weakly expressed in promyelocytic HL-60 cells. In HL-60 cells, 67LR expression almost disappeared after retinoic-induced granulocytic differentiation, whereas it strongly increased after phorbol ester-induced monocytic differentiation. We did not detect 67LR expression in normal BM hematopoietic cells, in precursor-B acute lymphoblastic leukemia, in chronic lymphocytic leukemia, or in chronic myeloid leukemia in chronic phase. By contrast, we detected enhanced 67LR expression in 40% of 53 de novo acute myeloid leukemias (AMLs), which frequently exhibited monocytic or myelomonocytic morphology and expressed CD14 and CD11a (P < 0.05). Using a colorimetric assay, we found that the expression pattern of this receptor corresponded to a higher adhesion to laminin; the adhesion was specific because in vitro addition to laminin-coated wells of recombinant 37-kDa laminin receptor precursor (37LRP), which is the cytoplasmic precursor containing both laminin-binding domains of cell surface 67LR, significantly reduced laminin binding of AML cells. The expression of 67LR on AML cell surface did not correlate with other differentiation and integrin antigens such as CD7, CD13, CD33, CD34, CD11b, CD11c, CD49d, CD49e, CD45RA, and CD45RO. In contrast with 67LR behavior in solid tumors, no statistically significant difference was found between 67LR expression and any hematological characteristic of the disease at diagnosis, nor between 67LR expression and outcome of the disease as measured by complete remission rate, disease-free survival, or overall survival. In conclusion, our results indicate that 67LR expression mediates specific adhesion to laminin and that the detection of this molecule may be a valuable addition to other lineage-associated antigens in identifying monocytic-oriented AML.
International Journal of Cancer, Apr 14, 2003
The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in t... more The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR-transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA-stabilizing factor that binds the coding region of uPAR-mRNA. We now investigate the relevance of uPA-mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up-regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPAR-mRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR-mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR-mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR-stability through a cellular factor that binds the coding region of uPAR-mRNA.
Endocrinology, Feb 1, 1999
The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, ... more The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of lovastatin in a dose-dependent fashion. The geranylgeranyl transferase GGTI-298 inhibitor mimicked the effects of lovastatin on cell morphology and induced cell death, whereas the farnesyl transferase inhibitor FTI-277 was less effective to induce both cell rounding and apoptosis. Resistance to lovastatin-induced apoptosis by expression of the viral serpine CrmA and by the peptide inhibitor of caspases, Z-DEVD-fmk, demonstrated the involvement of CrmA-sensitive, caspase-3-like proteases. Inhibition of endogenous p53 activity did not affect the sensitivity of thyroid cells to lovastatin, demonstrating that this type of apoptosis is p53 independent. We conclude that lovastatin is a potent inducer of apoptosis in proliferating thyroid cells through inhibition of protein prenylation. This type of apoptosis requires protein synthesis, is CrmA sensitive and caspase-3-like protease dependent, and is independent from p53.
The Journal of Clinical Endocrinology and Metabolism, Apr 1, 1995
The expression of intercellular adhesion molecule-1 (ICAM-1) in tumoral tissues may promote their... more The expression of intercellular adhesion molecule-1 (ICAM-1) in tumoral tissues may promote their interaction with the immune system and cytotoxic effect on tumoral cells. This observation led to the investigation of ICAM-1 expression and modulation in different tumoral cell systems in vitro. Recently, retinoic acid-responsive elements have been found in the 5'-regulatory region of the human ICAM-1 gene. In the present study, we investigated, by flow cytometry, the effect of retinoic acid on the surface expression of ICAM-1 in human thyroid carcinoma cell lines. Two papillary (NPA and TPC-1), one follicular (WRO), one anaplastic (ARO) and one immortalized fetal (TAD-2) cell line have been studied. All of them produced constitutively ICAM-1; its surface expression and specific messenger ribonucleic acid (mRNA) levels were increased significantly by retinoic acid in all except the WRO cell line. ICAM-1 hyperexpression by retinoic acid was time dependent, reversible, and dependent on mRNA and protein synthesis. Furthermore, cytokines, such as interferon-gamma and tumor necrosis factor-alpha, both individually and, to a greater extent, in combination with retinoic acid, increased ICAM-1 surface expression and its mRNA levels. In conclusion, retinoic acid is able to induce ICAM-1 up-regulation via mRNA accumulation in human thyroid carcinoma cell lines.
Journal of Cellular and Molecular Medicine, Jun 28, 2012
Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is a... more Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kD (PED/PEA-15) is an anti-apoptotic protein whose expression is increased in several human cancers. In addition to apoptosis, PED/PEA-15 is involved in the regulation of other major cellular functions, including cell adhesion, migration, proliferation and glucose metabolism. To further understand the functions of this protein, we performed a yeast two-hybrid screening using PED/PEA-15 as a bait and identified the 67 kD high-affinity laminin receptor (67LR) as an interacting partner. 67 kD laminin receptor is a non-integrin cell-surface receptor for the extracellular matrix (ECM), derived from the dimerization of a 37 kD cytosolic precursor (37LRP). The 67LR is highly expressed in human cancers and widely recognized as a molecular marker of metastatic aggressiveness. The molecular interaction of PED/PEA-15 with 67LR was confirmed by pull-down experiments with recombinant His-tagged 37LRP on lysates of PED/PEA-15 transfected HEK-293 cells. Further, overexpressed or endogenous PED/PEA-15 was co-immunoprecipitated with 67LR in PED/PEA-15-transfected HEK-293 cells and in U-373 glioblastoma cells, respectively. PED/PEA-15 overexpression significantly increased 67LR-mediated HEK-293 cell adhesion and migration to laminin that, in turn, determined PED/PEA-15 phosphorylation both in Ser-104 and Ser-116, thus enabling cell proliferation and resistance to apoptosis. PED/PEA-15 ability to induce cell responses to ECM-derived signals through interaction with 67LR may be of crucial importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic spread and colonization.
Journal of Biological Chemistry, Nov 1, 2003
Integrin activation generates different signalings in a cell type-dependent manner and stimulates... more Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1. * This work has been supported in part by Ministero dell'Istruzione, dell'Università e della Ricerca (to M. V.) and Fondazione Italiana per la Ricerca sul Cancro (to T. D. M.
International Journal of Immunopathology and Pharmacology, 2010
Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are tw... more Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (ISSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from ISSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form sh...
The Journal of Clinical Endocrinology & Metabolism, 1993
European Journal of Endocrinology, 2001
OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apo... more OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells. METHODS: TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fracti...
European Journal of Heart Failure, Oct 1, 2006
BackgroundIn remodelling ventricles, the progression of heart failure is associated with structur... more BackgroundIn remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra‐cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death.The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors.AimsTo assess Granulocyte Macrophage‐Colony Stimulating Factor (GM‐CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM‐CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy.Methods and resultsGM‐CSFR expression in human cardiac tissue from explanted hearts of ten patients with end‐stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays.Our results demonstrated an increase in GM‐CSFR in cardiomyocytes from end‐stage heart failure tissues as compared to normal control tissues.ConclusionsWe hypothesize that GM‐CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.
FEBS Letters, 1999
In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell de... more In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell death, a process that has been termed`anoikis'. We investigated the role of p53 and other apoptotic proteins in anoikis in thyroid epithelial cells. Western blot analysis demonstrated that neither p53 nor Bcl-2, Bcl-XL and Bax protein expression changed during anoikis. However, loss of endogenous p53 activity in cells transfected with a dominant-negative mutated p53 inhibited anoikis demonstrating the involvement of p53-dependent processes. The phosphatase inhibitor sodium orthovanadate opposed anoikis when added to the cells within 6 h, suggesting a role for phosphorylated proteins.
FEBS Letters, 1999
The urokinase-type plasminogen activator uPA-R can regulate integrin functions by associating wit... more The urokinase-type plasminogen activator uPA-R can regulate integrin functions by associating with several types of L Lsubunit. We have recently shown that normal thyroid TAD-2 cells express both a native and a cleaved form of uPA-R which lacks the binding domain for uPA. We found this cleaved form to be present in reduced amounts in papillary and follicular thyroid carcinoma cells and completely absent in cells derived from an anaplastic thyroid carcinoma (ARO). We now report that in normal thyroid cells the intact form of uPA-R strongly associates with L L-1 integrins, whereas its cleaved form does not. uPA-R expressed by ARO cells shows a stronger resistance to the cleavage mediated by uPA, plasmin and chymotrypsin than does uPA-R expressed by normal thyroid cells. This resistance to cleavage correlates with the higher level of glycosylation of uPA-R of ARO cells as compared to that of cleavable uPA-R of normal thyroid cells. These results suggest that uPA-R cleavage, which occurs in several cell types, represents a mechanism regulating the interactions of uPA-R with integrins and, possibly, the subsequent integrin-mediated cell adhesion. Moreover we hypothesize that glycosylation regulates uPA-R cleavage and, indirectly, its interaction with integrins.
Experimental Cell Research, 1989
We studied the relationship between differentiation, transformation, and uPA production in a syst... more We studied the relationship between differentiation, transformation, and uPA production in a system of rat thyroid cells in vitro. The fully differentiated FRTL5 cells did not produce detectable amounts of uPA, even after stimulation with phorbol esters, potent inducers of uPA expression. All the other cell lines (i.e., FRT, cells which have lost the characteristics of the differentiated thyroid cells; l-5G and FRA, transformed cells derived from rat thyroid tumors) produced uPA, the l-5G line being the highest producer. Also the FRTL line became positive for uPA production after viral transformation (clone RM4). The lack of uPA expression in FRTL5 cells was not due to the presence of inhibitors and these cells did not produce an inactive molecule, as shown by immunoprecipitation with anti-uPA antibody. However, in PRTLr cells Northern analysis showed the presence of a small amount of uPA-specific mRNA that increased appreciably after phorbol ester stimulation. In conclusion, in our system uPA expression was a property of undifferentiated and transformed cells; in fully differentiated cells uPA expression was switched off by a still unclear mechanism. Q 1sss Academic PWS, I~C.
European Journal of Endocrinology, 2006
Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate su... more Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAFV600E), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) f...
Endocrinology, 1982
The production of thyroglobulin (TG) by cultured rat thyroid cells was demonstrated at the level ... more The production of thyroglobulin (TG) by cultured rat thyroid cells was demonstrated at the level of a single cell using a modified reverse plaque-forming assay. Thyroid cells were cultured in the presence of sheep erythrocytes (SE) coated with hybrid antibody specific for both SE ...
Endocrinology, 2000
Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; ... more Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas
Endocrinology, 1999
The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, ... more The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of lovastatin in a dose-dependent fashion. The geranylgeranyl transferase GGTI-298 inhibitor mimicked the effects of lovastatin on cell morphology and induced cell death, whereas the farnesyl transferase inhibitor FTI-277 was less effective to induce both cell rounding and apoptosis. Resistance to lovastatin-induced apoptosis by expression of the viral serpine CrmA and by the peptide inhibitor of caspases, Z-DEVD-fmk, demonstrated the involvement of CrmA-sensitive, caspase-3-like proteases. Inhibition of endogenous p53 activity did not affect the sensitivity of thyroid cells to lovastatin, demonstrating that this type of apoptosis is p53 independent. We conclude that lovastatin is a potent inducer of apoptosis in proliferating thyroid cells through inhibition of protein prenylation. This type of apoptosis requires protein synthesis, is CrmA sensitive and caspase-3-like protease dependent, and is independent from p53.