Biomedical subjects

Chiara Balducci

Publications and source records attributed to Chiara Balducci.

9 recordsLinked to original sources

PubMed2006

Desmoid and fibroma tumors differently respond to TGFbeta(1) stimulus and ECM macromolecule accumulation.

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.

Blotting, Northern↗

PubMed2006

Cloning and expression of mouse cytosolic alpha-mannosidase (Man2c1).

It is known that the neutral/cytosolic alpha-mannosidase (Man2c1) which can cleave alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino-d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum alpha-mannosidase and the cytosolic alpha-mannosidase. In fact antibodies raised against the soluble alpha-mannosidase recognized the membrane form of the ER alpha-mannosidase. A cDNA encoding the mouse cytosolic alpha-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic alpha-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic alpha-mannosidase was mapped to 170 bases upstream of the ATG codon using 5' RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic alpha-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.

3' Untranslated Regions↗

PubMed2006

Chromium VI-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and a lymphoblastic leukemia cell line (MOLT-4).

Hexavalent chromium compounds are well-documented human carcinogens. In vitro experiments show Cr (VI) induces cell death by apoptosis by activating p53 protein. The aim of this study was to evaluate Cr (VI)-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and in a lymphoblastic leukemia cell line (MOLT-4). Cr (VI) caused a dose- and time-dependent increase in the apoptosis rate in both cell lines. Western blotting showed increased p53 protein expression in MOLT-4 cells, but not in BEAS-2B cells, after exposure to 0.5 and 3 muM hexavalent chromium for 12 hours and 4 hours, respectively. Apoptotic cell death induced by Cr (VI) was not decreased by pretreatment with caspase-3, -8, and -9 inhibitors. These preliminary results provide evidence of Cr (VI)-induced apoptosis, which deserves further investigation in occupationally exposed workers.

Apoptosis↗

PubMed2005

Human desmoid fibroblasts: matrix metalloproteinases, their inhibitors and modulation by Toremifene.

BACKGROUND: Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts. METHODS: We investigated collagen accumulation by 3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis. RESULTS: Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts. CONCLUSION: The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation.

Antineoplastic Agents, Hormonal↗

PubMed2005

Identification and characterization of five novel MAN2B1 mutations in Italian patients with alpha-mannosidosis.

Mutation analysis performed on six Italian families with alpha-mannosidosis type II allowed the identification of five new mutations in the MAN2B1 gene: c.157G>T, c.562C>T, c.599A>T, c.293dupA, c.2402G>A (p.E53X, p.R188X, p.H200L, p.Y99VfsX61, p.G801D). Protein residues G801 and H200 are conserved among the four mammalian alpha-mannosidases cloned to date: human, cattle, cat and mouse. In vitro expression studies demonstrated that both missense mutations expressed no residual alpha-mannosidase activity indicating that they are disease-causing mutations. Modelling into the three-dimensional structure revealed that the p.H200L could involve the catalytic mechanism, whereas p.G801D would affect the correct folding of the enzyme.

Animals↗

PubMed2004

Efficacy of enzyme replacement therapy in alpha-mannosidosis mice: a preclinical animal study.

Alpha-mannosidosis is a lysosomal storage disorder which manifests itself in the excessive storage of mannose-containing oligosaccharides in the lysosomes of multiple peripheral tissues and in the brain. Here we report on the correction of storage in a mouse model of alpha-mannosidosis after intravenous administration of lysosomal acid alpha-mannosidase (LAMAN) from bovine kidney, and human and mouse recombinant LAMAN. The bovine and the human enzyme were barely phosphorylated, whereas the bulk of the mouse LAMAN contained mannose 6-phosphate recognition markers. The clearance decreased from bovine to human to mouse LAMAN with plasma half-times of 4, 8 and 12 min, respectively. The apparent half-life of the internalized enzyme was dependent on the enzyme source as well as tissue type and varied between 3 and 16 h. The corrective effect on the storage of neutral oligosaccharides was time-, tissue- and dose-dependent, and the effects were observed to be transient. After a single dose of LAMAN the maximum corrective effect was observed between 2 and 6 days after injection. In general the corrective effect of the human LAMAN was higher than that of the mouse LAMAN and lowest for the bovine LAMAN. Injection of 250 mU human LAMAN/g body weight followed by a subsequent injection 3.5 days later was sufficient to clear liver, kidney and heart from neutral oligosaccharides. Surprisingly a decrease in mannose containing oligosaccharides was also observed in the brain, with storage levels reported at <30% than that found in controls. These data clearly underline the efficacy of enzyme replacement therapy for the correction of storage in alpha-mannosidosis and suggest that this treatment can substantially decrease storage in the brain.

Animals↗

PubMed2002

Crouzon's syndrome: differential in vitro secretion of bFGF, TGFbeta I isoforms and extracellular matrix macromolecules in patients with FGFR2 gene mutation.

In the Crouzon's syndrome the cranial morphogenic processes are altered due to the early fusion of cranial sutures. We analysed the phenotype of cultured fibroblasts from normal subjects and from Crouzon patients with a specific fibroblast growth factor receptor 2 mutation resulting in a Cys 342 Tyr substitution within the third immunoglobulin domain. Crouzon fibroblasts differed from normal fibroblasts in their extracellular matrix macromolecule accumulation. In Crouzon fibroblasts glycosaminoglycans and fibronectin were decreased and type I collagen increased. As transforming growth factors beta (TGF beta) and basic fibroblasts growth factor (bFGF) together regulate extracellular matrix deposition, we evaluated TGF beta(1), TGF beta(3) and bFGF production by Crouzon and normal fibroblasts. TGF beta(1), TGFb(3) and bFGF levels were lower while TGF beta(1) mRNA transcripts were higher in Crouzon cells. As the increased TGF beta(1) gene expression did not translate into a parallel increase of secreted TGF beta(1), control of TGF beta(1) secretion may be mainly post-transcriptional. Furthermore, adding bFGF increased TGF beta(1) and TGF beta(3) secretion, suggesting the drop may be due to the altered signal transduction of bFGF. These innovative data suggest the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place.

Blotting, Northern↗

PubMed2002

Effects of transforming growth factor-beta1 and tumour necrosis factor-alpha on cultured fibroblasts from skin fibroma as modulated by toremifene.

To determine how toremifene, an anti-oestrogen triphenylethylene derivate, reduces tumour mass, we investigated its modulation of TGF-beta1 and TNF-alpha in fibroma fibroblasts. Normal and fibroma fibroblasts, isolated from patients affected by Gardner's syndrome without or with fibroma manifestation, were cultured in vitro. Secretion of GAG, collagen and TGF-beta1 was increased in fibroma fibroblasts compared to healthy cells. The increase in TGF-beta1 secretion into the medium was associated with a parallel increase in TGF-beta1 gene expression and receptor number. Receptor cross-linking studies using radiolabelled TGF-beta1 revealed more receptors, particularly types I and II, in fibroma fibroblasts than in normal cells. Normal and fibroma fibroblasts did not synthesise TNF-alpha, but they had TNF-alpha membrane receptors, as shown by TNF-alpha assay. TNF-alpha secreted by human monocytes, which may be present in the peritumoral area, increased cell proliferation and GAG accumulation and was, in turn, enhanced by TGF-beta1 treatment. Both growth factors increased angiogenesis, as shown by the CAM assay. Toremifene reduced TGF-beta1 secretion by fibroma fibroblasts and TNF-alpha secretion by monocytes, thus downregulating cell proliferation, ECM macromolecule accumulation and angiogenic progression. We hypothesise that increased TGF-beta1 gene expression and TGF-beta1 secretion in fibroma fibroblasts as well as the subsequent rise in TNF-alpha production by monocytes may facilitate fibroma growth and that toremifene inhibits autocrine and paracrine growth factor production.

Animals↗

PubMed2002

Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation.

BACKGROUND: We focused on the phenotype of non-mineralizing MG 63 and mineralizing TE 85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated. MATERIALS AND METHODS: Osteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay. RESULTS: Osteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG 63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE 85 cell proliferation and reduced TE 85 procollagen and osteocalcin production. CONCLUSIONS: The different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG 63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

Alkaline Phosphatase↗