Search PubMed⌕ Search

Biomedical subjects

S Filetti

Publications and source records attributed to S Filetti.

At least 19 recordsLinked to original sources
PubMed1997

p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells.

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32 degrees C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32 degrees C, a significant reduction in the proliferation rate (approximately or equal to 50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32 degrees C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.

Carcinoma↗
PubMed1996

Changes in sleep spindle activity of subject with chronic somatosensitive and sensorial deficits. Preliminary results.

We investigated the effects of the somatosensitive and sensory afferent inputs on the thalamic generators of sleep spindles (SS) in adult subjects affected by posterior funiculi lesions (five subjects), deafness (four subjects) or blindness (four subjects). The density, duration and frequency of SS, as well as the index of spindling, were analyzed during stage 2 NREM. The results show that the subjects with somatosensitive and sensorial lesions spent much more time on SS activity than the control group (eight subjects), and had a significantly increased density (< .0001), duration (< .0005) and index of spindling (< .0001). On the other hand, the frequency of spindling was little modified (< .05). Moreover, among the three groups of patients, those with somatosensitive deficits showed the greatest SS activity. In conclusion, our results suggest that the thalamic generators of SS are markedly modulated by peripheral inputs in man.

Blindness↗
PubMed1996

Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mamalian cells.

The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investigated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by deleting either the 5' untranslated region (UTR), the 3'UTR, or both UTRs. The 5'UTR contains two false AUG initiation codons followed by a stop codon. The cDNAs in the eukaryotic expression vector pSV2-NEO-ECE, as well as the 5'3'UTR-truncated cDNA in pSVL, were stably transfected into Chinese hamster ovary cells. Pools of more than 100 colonies were studied in order to minimize insertion site-dependent variation in the level of expression. Scatchard analysis of TSH binding indicated that the number of receptors on the surface of Chinese hamster ovary cells expressing the wild-type transcript (approximately 16,000/cell) increased approximately 2-fold with 5'UTR deletion, approximately 5-fold with 3'UTR deletion, and approximately 10-fold with both 5'UTR and 3'UTR deletion. TSH binding affinities of all constructs were in the range of 2-5 x 10(-10) M. No significant difference was evident between the 5'3'UTR truncated cDNAs in the two different vectors, pSV2-NEO-ECE and pSVL. The increase in the amplitude of the cAMP response to TSH stimulation was commensurate with the number of receptors expressed on the surface of the different cell lines. Truncation of the 5'UTR did not alter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as well as the 5'3'UTR-deleted transcript, increased approximately 4-fold independent of the expression vector used. In summary, both the 5'UTR and 3'UTR of the human TSH receptor mRNA influence the level of receptor expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the MRNA. These data explain the greater level of TSH receptor expression in cell lines that are transfected with cDNA lacking these regions of the mRNA transcript.

Animals↗
PubMed1996

Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas.

Forty-four thyroid autonomously hyperfunctioning adenomas were analyzed to assess the frequency of mutations occurring in the TSH receptor (TSHR). PCR-amplified fragments encompassing the entire exon 10 of the TSHR gene were obtained from the genomic DNA extracted from the tumors and their adjacent normal tissues and were examined by direct nucleotide sequencing. Point mutations were found in 9 of the 44 adenomas examined (20%). One mutation occurred in codon 619 (Asp to Gly), four in codon 623 (three were Ala to Ser, one Ala to Val substitution), two in codon 632 (both Thr to Ile), and two in codon 633 (Asp to Tyr or His). All the alterations were located in a part of the gene coding for an area including the third intracellular loop and the sixth transmembrane domain of the TSH receptor. All mutations were somatic and heterozygotic, and none was simultaneous with alterations of ras or gsp oncogenes. Thus, our data show that in our series of 44 hyperfunctioning thyroid adenomas, a somatic mutation of the TSHR, responsible for the constitutive activation of the cAMP pathway, occurs in 20% of the tumors.

Adenoma↗
PubMed1996

Constitutive activation of the Gs alpha protein-adenylate cyclase pathway may not be sufficient to generate toxic thyroid adenomas.

In toxic thyroid adenomas, mutations in the TSH receptor (TSH-R) gene or the gene encoding the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs alpha) have been demonstrated to constitutively activate the cAMP cascade, which subsequently stimulates the growth and function of these tumors. However, the widely varying thyroid phenotypes in patients with TSH-R germline mutations, ranging from only slightly enlarged diffuse to multinodular goiters, suggest that additional mechanisms may be effective in the pathogenesis of toxic adenomas. We have investigated the levels of stimulatory and inhibitory G protein alpha-subunits together with basal and TSH-stimulated adenylate cyclase (AC) activity in toxic adenomas with or without activating mutations and in nodular and extranodular tissues of a toxic goiter due to a germline mutation in the TSH-R gene. Augmented expression of Gs alpha protein was detected in all toxic adenomas, independent of the presence of mutations, and in the nodular tissue of the toxic goiter, but not in the nonnodular hyperplastic tissue of the toxic goiter with the mutated TSH-R. Analogously, the expression of the alpha-subunit of the inhibitory G protein (Gi alpha) was also increased in all adenomas and the nodular tissue of the goiter, but, again, not in the hyperplastic goiter tissue. Basal AC activity was high in all tissues with mutations, but was only slightly increased in adenomas without detected mutations. No correlation was detectable between basal or TSH-stimulated AC activity and the levels of Gs alpha and Gi alpha. Our data suggest that mutational activation of the cAMP cascade may not be sufficient to generate toxic nodules and adenomas, but far more complex mechanisms, including alterations of G protein signaling, may be effective in the pathogenesis of these tumors.

Adenoma↗
PubMed1995

Activating mutations of the TSH receptor in differentiated thyroid carcinomas.

A series of 14 thyroid carcinomas, characterized for their basal adenyl cyclase activity (ACA), was examined for the presence of activating point mutations in the TSH receptor (TSHR) gene. Sequencing of the carboxyl-part of this gene revealed the presence of a somatic and heterozygotic point mutation in codon 623 in three out of six tumors showing a constitutively enhanced ACA and a poor response to TSH stimulation. The mutation determines the substitution of a serine for an alanine in the third intracellular loop of the receptor, in a region critical for signal transduction. One tumor bearing a TSHR mutation presented also a N-ras point mutation. Both mutations were detected also in a lung metastasis of this tumor. Our data represent the first report of alterations in the TSHR gene in thyroid malign neoplasia. TSHR mutations may indeed participate, as well as the G alpha s protein (gsp oncogene), in the oncogenesis of some differentiated thyroid carcinomas presenting increased basal levels of cAMP and a poor response to TSH.

Adenocarcinoma, Follicular↗
PubMed1995

Genetic alterations in thyroid hyperfunctioning adenomas.

Thirty-seven thyroid autonomously hyperfunctioning adenomas were screened for mutations in the TSH receptor (TSHR), G alpha s (gsp), and ras genes. Polymerase chain reaction-amplified fragments of the TSHR C-terminal part (exon 10), the G alpha s (exons 8 and 9), and the three ras genes were obtained from the genomic DNA extracted from 37 tumors and their adjacent normal tissues and were studied by direct nucleotide sequencing and hybridization with synthetic probes. A point mutation in the third intracellular loop (codon 623) of the TSHR was found in 3 of 37 adenomas studied. This mutation codes for a change (Ala to Ser) in the TSHR structure and is somatic and heterozygotic. Constitutive activation of the TSHR was demonstrated by an increase in basal cAMP levels after transfection of Chinese hamster ovary cells with a mutated Ser623-TSHR complementary DNA. Nine gsp[00ae]MDRV[00af]- and one ras-activating mutations were also detected. No simultaneous alteration of the studied genes was present. Thus, in hyperfunctioning thyroid adenomas, our data suggest that a mutational activation of the TSHR and gsp genes may play a tumorigenic role through constitutive activation of the cAMP pathway.

Adenoma↗
PubMed1994

Different molecular mechanisms are involved in the multihormonal control of glucose transport in FRTL5 rat thyroid cells.

We investigated the molecular mechanisms by which TSH, insulin and IGF-1 modulate the glucose transport system in FRTL5 cells. We found that TSH, insulin and IGF-1 increased the glucose transporter Glut-1 specific mRNA levels 6, 8 and 5 fold over control, respectively. The effect on Glut-1 mRNA was evident after 2 hours, followed by an increased Glut-1 protein expression in whole cells, as judged by western blot analysis, after 5 hours of stimulation with all the hormones studied. In contrast, plasma membrane Glut-1 increased (300-400% over control) after 2 hours of stimulation with TSH (10 mU/ml), dibutyryl-cAMP (1mM), IGF-1 (10 ng/ml) and insulin (10 nM). These data indicate that the glucose transport system is under multihormonal control in FRTL5 cells. Two different mechanisms are involved in TSH, IGF-1 and insulin stimulation of the glucose transport: a) neosynthesis of Glut-1 by activation of gene expression; b) recruitment of carriers from the intracellular pool to the plasma membrane.

Animals↗
PubMed1994

Three new mutations of thyroid hormone receptor-beta associated with resistance to thyroid hormone.

Three novel point mutations at nucleotides 1249, 1282, and 1614 (exons 9 and 10) of the human thyroid hormone receptor-beta gene were observed in six individuals affected by the syndrome of resistance to thyroid hormone. All three mutations occurred in a heterozygous pattern and caused the following changes in the mature form of the receptor protein: Asp322 to Asn, Glu333 to Gln, and Lys443 to Asn, respectively. The first and third point mutations arose in two unrelated families from eastern Sicily, whereas the second concerned an individual from southern Calabria, apparently presenting a sporadic form of the resistance syndrome. The clinical and biochemical features of resistance to thyroid hormone, both before and after the administration of thyroid hormones, highlight the striking intrafamilial heterogeneity in the phenotypical presentation of the syndrome.

Adolescent↗
PubMed1993

Molecular mechanisms of the increased glucose transport in transformed FRTL-5 rat thyroid cells.

We investigated the molecular mechanisms involved in the glucose carrier (Glut 1) regulation in FRTL-5 cells and two derived transformed clones (SRC and Ki-Mol cells). When compared to the wild-type strain, SRC and Ki-Mol cells showed an increase in both glucose consumption and uptake (about 60 fold), associated with 6-8 fold higher Glut 1 mRNA levels. Transcriptional studies revealed a 2- to 3 fold increased activation of the gene in the transformed cells, suggesting that transcription alone cannot fully account for the higher Glut 1 gene expression. Western blot studies showed an increase of the Glut 1 protein in SRC and Ki-Mol cells, associated with a different gel migration pattern and a disparate distribution rate between the plasma membrane and the microsomal fraction. These data indicate that the higher rate of glucose uptake observed in SRC and Ki-Mol cells is associated to an increase in Glut 1 gene expression, and that also changes in the subcellular distribution and probably in the structure of Glut 1 protein are present.

Animals↗
PubMed1991

Lack of relationship between 3',5'cyclic adenosine monophosphate desensitization and thyrotropin receptor down regulation in the rat thyroid cell line FRTL5.

We studied the cAMP response and radioiodinated TSH binding in the FRTL5 rat thyroid cell line after chronic exposure to TSH. In parallel experiments, continuous exposure to TSH induced both a decrease in the cellular cAMP responsiveness to a second acute stimulation by thyrotropin (desensitization) and a reduction in 125I-TSH cellular binding (down-regulation). These two processes appeared to differ in that: a) cAMP desensitization was maximal (45-55% inhibition in comparison to control cells) after 24 h of preexposure to TSH; on the contrary, a loss of the TSH binding to thyroid cells was detected only after 24 h and was maximal (55-65% lower than control cells) after 48 h of preexposure to TSH; b) a different dependence on the TSH dose was observed for cAMP desensitization and TSH down-regulation: the former was maximal at 10 microU/ml of TSH, the latter only at 10 mU/ml of TSH; c) recovery from cAMP desensitization was detected 3 h after TSH withdrawal and was fully achieved at 24 h, whereas the recovery from the TSH receptor down-regulation was not evident for up to 24 h and a full recovery obtained only at 72 h; d) the use of cAMP analogues proved that, unlike desensitization, TSH receptor down-regulation is a cAMP dependent process. These findings support the concept that different patterns of response to prolonged stimulation with TSH, an earlier and cAMP-independent process (desensitization) and a later cAMP-dependent mechanism (down-regulation), occur in the rat thyroid cell.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Recombinant human thyrotropin (TSH) receptor in a radioreceptor assay for the measurement of TSH receptor autoantibodies.

We studied the suitability of using the recombinant human TSH receptor expressed in Chinese hamster ovary cells in a TSH binding inhibition (TBI) assay for autoantibodies against the TSH receptor. Purified immunoglobulin G (IgG) containing potent thyroid-stimulating immunoglobulin bioactivity competed for radiolabeled TSH binding to recombinant TSH receptor in parallel to inhibition by unlabeled TSH. Using polyethylene glycol-prepared IgG, this assay discriminated very well between sera from normal individuals [TBI, 0.98 +/- 0.04 (+/- SD); range, 0.92-1.08; n = 35] and patients with untreated Graves' disease (TBI, 0.49 +/- 0.23; range, 0.06-0.98; n = 93). Only four of the sera from the untreated Graves' patients were TBI negative (greater than or equal to 0.92), providing a sensitivity of 96%. In sera from patients with Graves' disease receiving antithyroid drug therapy, TBI was 0.63 +/- 0.18 (range, 0.19-0.97; n = 75). In this group of treated patients, 2 of 75 were TBI negative. Four of 12 patients with Hashimoto's thyroiditis (33%) were positive for TBI activity. All 18 patients with nonautoimmune thyroid diseases (toxic nodular goiter, single toxic adenoma, subacute thyroiditis, or thyroid cancer) were TBI negative. Correlation of the TBI values with thyroid-stimulating immunoglobulin bioactivity revealed a generally positive correlation (r = 0.31; P less than 0.05); however, there were many discrepancies among individual sera. TSI bioactivity was undetectable in all 4 patients with Hashimoto's thyroiditis who were TBI positive. These data indicate that the recombinant TSH receptor in Chinese hamster ovary cells is a suitable system for detecting TBI activity in the sera of patients with autoimmune thyroid disease.

Animals↗
PubMed1991

The biological activity of bovine and human thyrotropin is differently affected by trypsin treatment of human thyroid cells: thyroid-stimulating antibody is related to human thyrotropin.

Pretreatment of cultured human thyroid cells with trypsin decreased the cAMP response to bovine TSH (bTSH) (by 50-60%). In striking contrast, in trypsin treated cells the cAMP stimulation by both human TSH (hTSH) and thyroid-stimulating antibodies (TSab) was unimpaired, indicating a similar behavior for these two stimulators. The effect of trypsin on inhibiting cAMP stimulation by bTSH was: 1) dose dependent; 2) present at a trypsin concentration as low as 3.3 mg/L; 3) fully reversible within 24 h after removal of the enzyme. In accordance with the altered biological activity in human thyroid cells exposed to trypsin the binding of labeled bTSH was reduced (about 40%). On the contrary, in the same cells, the binding of labeled human TSH was enhanced (about 3-fold). The cAMP response to cholera toxin and forskolin was unaffected in trypsin treated cells, indicating that the tryptic treatment did not alter any other component of the adenylate cyclase complex. The medium obtained from trypsin-treated human thyroid cells was able to neutralize the biological activity of bTSH but not that of hTSH or TSab. Our study demonstrates that in human thyroid cells: 1) trypsin impaires bovine, but not human TSH or TSab biological activity; 2) bovine and human TSH may bind to different components of the TSH receptor.

Animals↗
PubMed1990

Effect of thyrotropin and cAMP on FRTL5 cell growth in a serum free medium.

TSH-induced proliferation of FRTL5 cells was studied in a chemically serum-free, defined medium. FRTL5 cells incubated for several days in a medium lacking of serum and hormones were fully able to respond to TSH in terms of cAMP production and iodine uptake. In the same medium, TSH stimulated FRTL5 cell growth as assessed by thymidine incorporation, DNA content and cell count. In these experimental conditions the cellular doubling time was of 7.5 days as compared to 40 h when calf serum was present together with TSH. Cholera toxin, forskolin and (bu)2cAMP, substances able to selectively increase intracellular cAMP levels, were not as efficient as TSH in inducing FRTL5 cell growth. However, both TSH and (bu)2cAMP, in a similar way, induced c-myc gene expression and cellular progression through the prereplicative phase of cell division. These data demonstrate that cAMP-dependent mechanisms are only partially responsible for TSH-induced FRTL5 cell growth, thus suggesting a role of cAMP-independent mechanisms.

Animals↗
PubMed1990

Recombinant human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease.

Recombinant, enzymatically active human thyroid peroxidase (hTPO) generated in nonthyroidal eukaryotic cells was compared with Graves' thyroid microsomes as a source of antigen for the immunological detection of antimicrosomal/anti-hTPO antibodies. Enzyme-linked immunosorbent assay of 51 sera, selected to produce a balanced distribution of antimicrosomal antibody (anti-MSA) levels, revealed (at 1:100 serum dilution) a moderately good correlation between anti-MSA and anti-hTPO antibody levels (r = 0.668; P less than 0.001). However, a number of sera with high anti-MSA levels yielded markedly discordant values between the two assays. A much lower correlation was observed between antithyroglobulin and anti-hTPO antibody levels (r = 0.315; P less than 0.05). At higher serum dilutions (1:1,000 and 1:10,000), at which low affinity, high capacity binding reactions will be reduced, the correlation between anti-MSA and anti-hTPO antibody values was greatly improved (r = 0.906 and 0.902, respectively; P less than 0.001), and there were no longer widely discrepant values between the two assays. In summary, the present study indicates that recombinant hTPO expressed in nonthyroidal cells provides an unlimited source of human TPO of unvarying quality for anti-hTPO antibody assays. This material offers increased specificity over standard anti-MSA assays that use thyroid cell microsomes as antigen.

Antigen-Antibody Reactions↗
PubMed1990

Increased aggressiveness of thyroid cancer in patients with Graves' disease.

To evaluate whether coexistence of Graves' disease affects the prognosis of thyroid cancer we examined the clinical and pathological characteristics of 22 differentiated thyroid carcinomas concomitant with hyperthyroidism; 13 were associated with Graves' disease, and 9 with autonomous thyroid nodules. Carcinomas were identified in a consecutive series of 359 hyperthyroid patients (132 with Graves' disease and 227 with autonomous thyroid nodules) who underwent surgery during a 6-yr period. One hundred and thirty-seven thyroid carcinomas were found in the 582 euthyroid patients operated on in the same period. In Graves' patients, carcinomas were more often multifocal (46.1% vs. 0%), locally invasive (61.5% vs. 11.1%), and metastatic to lymph nodes (61.5% vs. 11.1%) or to distant sites (23.0% vs. 0%) than in patients with autonomous thyroid nodules. In addition, carcinomas concomitant with Graves' disease were larger (3.3 +/- 1.8 vs. 1.0 +/- 0.7 cm) than the ones associated with autonomous thyroid nodules and showed a high recurrence rate. In euthyroid patients, aggressiveness of thyroid cancer was intermediate. Serum TSH levels were suppressed in all hyperthyroid patients with thyroid cancer. However, circulating thyroid-stimulating antibodies were present in 12 of 13 cancer patients with Graves' disease, but were absent in patients with autonomous thyroid nodules. Our study suggests, therefore, that TSAb may play a role in determining the high aggressiveness of thyroid cancer in Graves' disease patients and indicates that a vigorous treatment should be pursued in this subgroup of patients.

Graves Disease↗
PubMed1989

Thyrotropin regulation of thyroid peroxidase messenger ribonucleic acid levels in cultured rat thyroid cells: evidence for the involvement of a nontranscriptional mechanism.

The influence of TSH on thyroid peroxidase (TPO) gene expression was investigated in FRTL5 rat thyroid cells. Cultured in the presence of TSH, these cells express a TPO mRNA species of 3.1 kilobases. TSH withdrawal from the culture medium led to a decline in TPO mRNA levels over 24 h. In contrast, no decline in beta-actin mRNA levels occurred after 24 h of incubation in TSH-free medium. TSH (1 mU/ml) added to FRTL5 cells cultured in the absence of TSH increased TPO mRNA levels 7- to 9-fold compared to levels in control cells. This effect of TSH on TPO mRNA accumulation in FRTL5 cells was time related (it was already present after 4 h and was maximal after 24 h of cell exposure to TSH), dose related (0.01 and 1 mU/ml were, respectively, the lowest and the maximally effective doses), and independent of new protein synthesis, in that it was not prevented by cycloheximide (100 microM). cAMP analogues [8-bromo-cAMP and (Bu)2cAMP] also increased TPO mRNA levels, although to a lesser degree than TSH. Run-on transcription analysis in nuclei prepared from FRTL5 cells previously cultured in the presence or absence of TSH did not reveal any difference in TPO mRNA transcripts. These results suggest that TSH regulates the level of TPO mRNA in FRTL5 cells, in part via the second messenger cAMP and by a nontranscriptional mechanism. This TSH effect may represent a primary site of TSH action in regulating TPO bioactivity.

8-Bromo Cyclic Adenosine Monophosphate↗