Biomedical subjects

F Curcio

Publications and source records attributed to F Curcio.

At least 19 recordsLinked to original sources

PubMed2006

Differential expression of the components of the plasminogen activating system in human thyroid tumour derived cell lines and papillary carcinomas.

We characterised the expression of the plasminogen activators (uPA and tPA), the uPA receptor (uPAR) and the PAs inhibitors (PAI-1 and PAI-2) in human thyroid cell lines derived from normal thyroid, follicular adenoma, follicular, papillary and anaplastic carcinomas. Urokinase PA activity was detected in the supernatant of normal thyrocytes and augmented in those of all tumour cells. Quantitative RT-PCR analysis showed that uPA, uPAR and PAI-1 mRNAs increased in all carcinoma cells. Similar results were found in 13 papillary thyroid carcinoma (PTC) tissues which were mirrored in Western blot experiments. A correlation was found between tumour size and uPA mRNA increase, and higher levels of uPA and uPAR mRNAs were found in metastatic PTC. In conclusion, thyroid carcinoma cell lines and PTC overexpress uPA, uPAR and PAI-1 and the correlation of uPA and its cognate receptor with tumour size and metastasis may suggest their potential prognostic relevance in thyroid cancer.

Blotting, Western↗

PubMed2004

Expression of matrix metalloproteinases and their specific inhibitors in normal and different human thyroid tumor cell lines.

In the present study we investigated, by means of zymography and reverse transcription-polymerase chain reaction (RT-PCR), the expression of different matrix metalloproteinases (MMPs) and of the specific tissue inhibitor of metalloproteinases [TIMPs] in human cell lines derived from normal thyrocytes (HTU5), follicular adenoma (HTU42), and follicular (FTC-133), papillary (B-CPAP), and anaplastic (CAL-62, 8305C) thyroid carcinomas. We demonstrated that normal thyrocytes constitutively express MMP-1, MMP-2, MMP-10, MMP-14, and TIMP-1, TIMP-2, TIMP-3, and TIMP-4, and this pattern of expression is profoundly modified in all thyroid tumor-derived cell lines. Analysis of the gelatinolytic activity in the different cell supernatants showed that the expressions of MMP-2 and MMP-9 are, respectively, increased or induced in all the neoplastic cell lines, except in CAL-62. Caseinolytic activity was found only in the supernatants of the 8305C and B-CPAP cells. Using RTPCR analysis we detected an increased expression of MMP-1 in cell lines derived from papillary and from one (8305C) of the two anaplastic carcinomas. MMP-13 mRNA was expressed only in the 8305C, FTC-133, and BCPAP cells. Among stromelysins, MMP-3 mRNA could not be detected in any cell line, while MMP-10 mRNA was expressed in all of them, although at variable levels. MMP-11 mRNA was absent in normal and follicular adenoma derived thyrocytes and induced in all carcinoma cell types. The expression of MMP-14 (MT1-MMP) mRNA was found significantly increased in all thyroid tumor cell lines with respect to HTU5 and HTU42 cells. The expression of TIMP-1 and TIMP-2 mRNAs was maintained in all cell lines tested, while that of TIMP-3 was lost in both anaplastic carcinoma cell lines and that of TIMP-4 was absent in the CAL-62. In conclusion, our data demonstrated a differential expression of MMPs and TIMPs in different thyroid tumor cell types with respect to normal thyrocytes. In particular, the induction of MMP-11 in all thyroid-derived carcinoma cell lines studied and of MMP-13 in all but one may represent, if confirmed in other thyroid tumor-derived cell lines and in thyroid tumor tissues, a new marker of thyrocyte transformation.

Cell Line↗

PubMed2002

Atrial natriuretic factor and C-type natriuretic peptide induce retraction of human thyrocytes in monolayer culture via guanylyl cyclase receptors.

The natriuretic peptides signal through three receptor subtypes, of which two (NPR-A and NPR-B) are membrane-bound guanylyl cyclases for which the principal ligands are respectively atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP). In the human thyroid cell, a third receptor, NPR-C, has been implicated in the regulation of thyroglobulin, but functional roles for NPR-A and NPR-B have not yet been defined. In the present study we used RT-PCR to identify transcripts of all three receptor subtypes, both in human thyroid and in HTU-5 cells, a long-term culture of thyroid-derived cells. Both ANF and CNP induced a twofold increase in intracellular cGMP content in HTU-5 cells. Morphologic changes (a significant increase in cells of the retracted phenotype) were observed in ANF- and CNP-treated cells within 3 and 5 h of treatment respectively. Significant increases in retracted cell number were induced by ANF and CNP, but not the NPR-C-specific ring-deleted ANF analog, C-ANF(4-23), during a 15-day treatment. All three natriuretic peptides, however, induced a small (15-20%) but significant (P<0 small middle dot001) increase in DNA content per well. The stable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), also increased the number of retracted HTU-5 cells, and was equipotent with the cAMP analog, 8-BrcAMP, in this effect. The cGMP-dependent protein kinase inhibitor, KT5823, however, had no significant effect on the ANF-induced increase in numbers of retracted cells. These results suggest that the actions of NPR-A and NPR-B, functional receptors in the human thyroid cell, may in part be mediated by cGMP-induced alterations in the cytoskeleton.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C).

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.

Atrial Natriuretic Factor↗

PubMed2000

A new model of human aortic endothelial cells in vitro.

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.

Aorta↗

PubMed2000

Transcriptional regulation of the MHC II gene DRA in untransformed human thyrocytes.

MHC class II molecules are heterodimeric, polymorphic transmembrane glycoproteins physiologically expressed on cells of the immune system and pathologically expressed on the affected target cells of autoimmunity. Their function is to present processed peptides to antigen-specific CD4(+) T cells. To understand the molecular mechanism of the regulation of class II genes in autoimmune target cell thyrocytes, we investigated the transcriptional regulation of DRA on untransformed, differentiated human thyroid cells following IFN-gamma stimulation, which is potentially relevant to the inappropriate class II expression found in Graves' disease. Data from this study show that IFN-gamma enhances a promoter Y box binding protein and induces an X box binding protein in untransformed thyrocytes, but not in SV-40-transfected thyrocytes. Initial characterization of the proteins has indicated that the Y box binding protein is approximately 132 kDa in size while the X box binding protein binds to the X2 region and is approximately 116 kDa. The X box binding protein may correspond to poly(ADP-ribose) polymerase, a recently described component of the X2 box binding protein, X2BP. In addition, the signal transducer and activator of transcription 1alpha protein (STAT1alpha) is also induced by IFN-gamma in these cells. These results further suggest that there are differences in class II gene regulation between differentiated cells and transformed cell lines.

Base Sequence↗

PubMed2000

Thyroid-specific gene expression is differentially influenced by intracellular glutathione level in FRTL-5 cells.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

Animals↗

PubMed2000

Induction of follicle formation in long-term cultured normal human thyroid cells treated with thyrotropin stimulates iodide uptake but not sodium/iodide symporter messenger RNA and protein expression.

Iodide uptake by the sodium/iodide symporter (NIS) in thyrocytes is essential for thyroid hormone production. Reduced NIS activity has been reported in thyroid diseases, including thyroid cancer and congenital hypothyroidism. The study of iodide uptake in thyrocytes has been limited by the availability of appropriate in vitro models. A new culture technique was recently developed that allows normal human thyroid primary culture cells to grow as monolayer cells and express differentiated functions for more than 3 months. We used this technique to study the effect of follicle formation and TSH on iodide uptake in these cells. Iodide uptake by the cells grown in monolayer was very low. Follicle formation was induced from monolayer cells, and electron micrographs demonstrated cell polarity in the follicles. No significant increase in iodide uptake was observed after TSH treatment of cells in monolayer or when follicle formation was induced without TSH. TSH stimulation of follicles, however, significantly increased iodide uptake ( approximately 4. 4-fold; P<0.001). Compared with iodide uptake in monolayers, the combination of follicle formation and TSH treatment stimulated iodide uptake synergistically to 12.0-fold (P<0.001). NIS messenger RNA (mRNA) and protein levels were almost the same in both monolayer cells and follicles. TSH treatment of monolayers and follicles produced significant (P<0.05) stimulation of mRNA ( approximately 4. 8- and approximately 4.3-fold respectively) and protein ( approximately 6.8- and 4.9-fold respectively). TSH stimulated NIS protein levels in both monolayer and follicles, however, stimulation of functional iodide uptake was only seen with TSH stimulation of follicles. The function of NIS may involve post-transcriptional events, such as intracellular sorting, membrane localization of NIS or another NIS regulatory factor. Polarized functions, such as iodide efflux into follicular lumina, may also contribute to the increased iodide concentration after follicle formation.

Carrier Proteins↗

PubMed1999

In vitro cultured cells as probes for space radiation effects on biological systems.

Near future scenarios of long-term and far-reaching manned space missions, require more extensive knowledge of all possible biological consequences of space radiation, particularly in humans, on both a long-term and a short-term basis. In vitro cultured cells have significantly contributed to the tremendous advancement of biomedical research. It is therefore to be expected that simple biological systems such as cultured cells, will contribute to space biomedical sciences. Space represents a novel environment, to which life has not been previously exposed. Both microgravity and space radiation are the two relevant components of such an environment, but biological adaptive mechanisms and efficient countermeasures can significantly minimize microgravity effects. On the other hand, it is felt that space radiation risks may be more relevant and that defensive strategies can only stem from our deeper knowledge of biological effects and of cellular repair mechanisms. Cultured cells may play a key role in such studies. Particularly, thyroid cells may be relevant because of the exquisite sensitivity of the thyroid gland to radiation. In addition, a clone of differentiated, normal thyroid follicular cells (FRTL5 cells) is available in culture, which is well characterized and particularly fit for space research.

Animals↗

PubMed1999

Response to thyrotropin of normal thyroid follicular cell strain FRTL5 in hypergravity.

Thyroid hormones control every cell in the organisms and, as indicated by many hormonal changes in astronauts during and shortly after space missions, its complex regulation may be influenced by gravity. To test in vitro the effects of gravity environment on thyroid, we selected a unique cultured cell system: the FRTL5, a normal follicular thyroid cell strain in continuous culture, originally derived from adult rat thyroids. To establish if modifications of the gravitational environment may interfere with post-receptorial signal transduction mechanisms in normal mammalian cultured cells, following our previous microgravity experiments, we exposed thyrotropin-stimulated and unstimulated FRTL5 cells to hypergravity (5 g and 9 g) in a special low-speed centrifuge. At all thyrotropin doses tested, we found significant increases in terms of cyclic AMP production in FRTL5 thyroid cells. The data here reported correlate well with our previous microgravity data, showing that the FRTL5 cells functionally respond to the variable gravity force in a dose-dependent manner in terms of cAMP production following TSH-stimulation.

Animals↗

PubMed1999

HLA-DMB expression by thyrocytes: indication of the antigen-processing and possible presenting capability of thyroid cells.

Expression of HLA class II molecules on thyrocytes is a characteristic feature of autoimmune thyroid disease and may lead the thyroid cells to present autoantigens to CD4+ T lymphocytes. Since HLA-DM is a critical molecule in class II-restricted antigen processing and presentation, we assessed the expression of HLA-DMB, -invariant chain (Ii), class II transactivator (CIITA) and DRA in an untransformed, pure thyrocyte strain HTV-59A. Here we report that both HLA-DMB mRNA and the protein are expressed in thyrocytes and that CIITA expression is enhanced by interferon-gamma (IFN-gamma) treatment and occurs before DMB, Ii and DRA up-regulation, suggesting CIITA expression is a requirement for antigen processing in thyrocytes. These results indicate that thyrocytes are capable of antigen processing and possibly antigen presentation to T cells.

Antigen Presentation↗

PubMed1997

Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression.

We have investigated the role of the NFkappaB complex in the process of thyroid carcinogenesis by analysing thyroid carcinoma cell lines. A significant increase in p65 NFkappaB mRNA and protein expression, compared to normal thyroid cultures or tissue, was found in all of the cancer cell lines. Conversely, only a modest increase in the p50 NFkappaB mRNA and protein was found in most, but not all carcinoma cell lines. The block of p65 protein synthesis with specific antisense oligonucleotides greatly reduced the ability of two undifferentiated carcinoma cell lines to form colonies in agar and reduced their growth rate. On the other hand, no effect was observed in the same cell lines when treated with p50 specific antisense oligonucleotides. These inhibitory effects seem to be mediated by the suppression of c-myc gene expression, since treatment with antisense oligonucleotides for p65 gene interfered negatively with c-myc gene expression. Our results indicate that activation of the NFkappaB complex by overexpression of p65 plays a critical role in the process of thyroid cell transformation.

Down-Regulation↗

PubMed1997

Expression of differentiation markers in cultured cells from various thyroid diseases.

The use of cell cultures as a model system for studying thyroid diseases requires establishment of appropriate culture conditions that allow in vitro propagation of populations that correspond to in vivo ones. We have defined these conditions and verified functional parameters such as thyrotropin-dependent cyclic adenosine monophosphate (cAMP) production and thymidine incorporation, and molecular markers such as thyroglobulin (by radioimmunoassay [RIA] and Northern blot), thyroperoxidase (by Northern blot), thyroid-specific transcription factor 1 (by immunohistochemistry and Northern blot) and PAX-8 (by Northern blot). The "in vitro profile" (functional parameters and molecular markers) was found to correlate with the degree of differentiation of the starting specimens and the pathological diagnosis. The data presented suggest that our culture technique allows in vitro growth of cell populations that may be used to perform functional assays and may facilitate the molecular characterization of pathological samples. This approach could be especially useful to define prognosis and also help to develop innovative therapies.

Biomarkers↗

PubMed1997

In vitro cultures of pathological thyroid cells: a powerful diagnostic technique.

In order to get a correct predictivity from molecular and functional markers in neoplastic disease, cell cultures, correspondent to in vivo existing populations, should be available. The difficulty, as yet, to correlate in vivo conditions with in vitro molecular and functional markers, represents a hurdle for a better prognosis in several neoplastic diseases. We tackled the problem establishing cultures from surgical samples of human thyroid glands bearing various pathologies (pathological diagnosis were obtained for all samples). Cells were frozen after 2 passages, and molecular markers (thyroglobulin, TPO, TTF-1 and PAX-8) and functional parameters (TSH-dependent cAMP production and thymidine incorporation) were investigated after thawing. The "in vitro profile" (functional parameters and molecular markers) was found to correlate with the pathological diagnosis and the degree of differentiation of the starting specimens. The data presented suggest that our culture technique allows in vitro growth of cell populations that may be used to perform functional assays and may make the molecular characterization of pathological samples easier. These findings could be especially useful to better define prognosis and also help to develop innovative therapies.

Journal Article↗

PubMed1996

Glucose may induce cell death through a free radical-mediated mechanism.

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20mM glucose. We also found that, when glutathione production was inhibited, 10mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).

Animals↗

PubMed1995

The defence against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor 1 over-production.

It has been shown that hyperglycaemia may stimulate plasminogen activator inhibitor 1 (PAI-1) over-production in human endothelial cells in culture. At the same time, it has been shown that glucose may enolize, producing free radicals. In this study, the possibility that hyperglycaemia stimulates PAI-1 over-production in human endothelial cells in culture by generating free radicals has been evaluated. For this purpose two experimental models were used: human endothelial cells transfected to express high glutathione peroxidase levels cultured in hyperglycaemic media, and human endothelial cells cultured in hyperglycaemic media with the antioxidant GSH. Cells grown in 20 mM glucose produced higher values of PAI-1 with respect to controls. The production of PAI-1 was not influenced by hyperglycaemia in transfected cells. GSH in the medium reduced hyperglycaemia-induced PAI-1 over-production, but also reduces the basal production of PAI-1 in the cells grown in normal glucose concentration. These data show that antioxidant defences may reduce hyperglycaemia-induced PAI-1 over-production in human endothelial cells in culture. The hypothesis that oxidative stress may play an important role in the pathogenesis of diabetic complications is then supported by this study.

Antioxidants↗

PubMed1995

Sod and GSH inhibit the high glucose-induced oxidative damage and the PDGF increased secretion in cultured human endothelial cells.

Poor control of blood glucose has been established as a key pathogenetic mechanism in the vascular complications of diabetes. It has been reported that glucose may autooxidize generating free radicals which have been suggested to delay proliferation, to modify mobility, to influence platelet-derived growth factor and other secretory protein production in a variety of cell systems. Platelet-derived growth factor, in turn, may induce proliferation and migration of vascular smooth muscle cells and thus play a role in atherogenesis. In the present study the effects of antioxidants on the high glucose-dependent oxidative cell damage and increased platelet-derived growth factor secretion have been investigated using cultured human endothelial cells. Our findings show that rising the glucose concentration in the culture medium from 5 mM to 20 mM, increased the production of free radicals cell damage markers, such as malondialdehyde and conjugated dienes, as well as the production of platelet-derived growth factor. The addition of superoxide dismutase or glutathione prevents both such effects. These results suggest that antioxidants may be a helpful therapeutic adjuvant to reduce the vascular complications of diabetes.

Antioxidants↗

PubMed1994

Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.

Cell Division↗