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A Fusco

Publications and source records attributed to A Fusco.

At least 127 records · Page 7Linked to original sources
PubMed1998

Glial cell line-derived neurotrophic factor differentially stimulates ret mutants associated with the multiple endocrine neoplasia type 2 syndromes and Hirschsprung's disease.

Ret is a receptor tyrosine kinase involved in several neoplastic and developmental diseases affecting the thyroid gland and tissues of neuroectodermal origin. Different ret mutations are associated with different disease phenotypes. Gain-of-function of ret is caused by gene rearrangements in thyroid papillary carcinomas and by point mutations in multiple endocrine neoplasia (MEN) type 2A syndrome (MEN2A), in familial medullary thyroid carcinoma (FMTC), and in the more severe MEN2B syndrome. Conversely, Hirschsprung's disease (HSCR) is associated with loss of function of ret. Recently, it has been shown that glial cell line-derived neurotrophic factor (GDNF), by binding to the accessory molecule GDNFR-alpha, acts as a functional ligand of Ret and stimulates its tyrosine kinase and biological activity. To ascertain whether the biological effects of ret mutations are modulated by GDNF, we have investigated the responsiveness to GDNF of ret mutants in cell lines coexpressing GDNFR-alpha and MEN2A-, MEN2B-, FMTC-, or HSCR-associated ret mutants. Here, we show that triggering of GDNF affected only ret/MEN2B, i.e. it stimulated ret/MEN2B mitogenic and kinase activities, as well as its ability to phosphorylate Shc, a bona fide Ret substrate. In contrast, ret mutants associated with MEN2A or FMTC (carrying Cys634 or Cys620 mutations) were unresponsive to GDNF. HSCR mutations, by affecting either the extracellular or the intracellular Ret domain, impaired responsiveness to GDNF. These data suggest that the phenotype of human diseases caused by ret mutations can be differentially influenced by GDNF.

3T3 Cells↗
PubMed1998

The ret/ptc1 oncogene is activated in familial adenomatous polyposis-associated thyroid papillary carcinomas.

Familial adenomatous polyposis (FAP) is caused by germ-line mutations of the apc gene, and it is associated with an increased risk of developing papillary thyroid carcinomas. We have previously reported that a significant fraction of sporadic human papillary thyroid carcinomas is characterized by gene rearrangements affecting the ret protooncogene. These rearrangements generate chimeric transforming oncogenes designated ret/ptc. By a combined immunohistochemical and RT-PCR approach, we analyzed, for ret/ptc oncogene activation, papillary thyroid carcinomas occurred in two FAP kindreds, both showing typical apc gene mutations. Kindred 1 had seven members affected by FAP, and among these, three patients showed papillary thyroid carcinomas. Kindred 2 had two patients, mother and daughter, affected by colonic polyposis; the 20-yr-old daughter showed also a papillary carcinoma. Here we report that ret/ptc1 oncogene was activated in two of the three papillary carcinomas of FAP kindred 1 and in the papillary carcinoma of FAP kindred 2. These findings document that loss of function of apc coexists with gain of function of ret in some papillary thyroid carcinomas, suggesting that ret/ptc1 oncogene activation could be a progression step in the development of FAP-associated thyroid tumors.

Adenomatous Polyposis Coli↗
PubMed1998

Novel mutation of the PTEN gene in an Italian Cowden's disease kindred.

Cowden disease (CD) is an autosomal dominant multiple hamartoma syndrome with an elevated risk of thyroid and breast cancers. The CD susceptibility gene has recently been identified as the PTEN/MMAC1/TEP1 gene localized at 10q23 and coding for a dual specificity protein phosphatase. We report the mutational analysis of the PTEN gene in one Italian CD kindred. By using the single strand conformation polymorphism technique and subsequent direct DNA sequencing of the polymerase chain reaction product, we identified a novel mutation in the exon 5 of the PTEN gene. A heterozygous germline TGT-TAT transition was detected at the nucleotide 407; this causes the amino acid substitution cys136-tyr136 and the generation of a new NSI I restriction site. This mutation was not detected in the unaffected member of the family thereby indicating that it is causally linked to the disease. We ruled out that this mutation is a polymorphic variant because it was not detected in over 100 chromosomes analyzed. Using reverse trancriptase-polymerase chain reaction, we detected the expression of the mutant allele in lymphocytes and pathological tissues from an affected member of the family.

Adolescent↗
PubMed1998

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Animals↗
PubMed1998

Expression of the RET/PTC1 oncogene impairs the activity of TTF-1 and Pax-8 thyroid transcription factors.

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroid-differentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-PTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTC1 oncogene alters the expression of the thyroid-differentiated phenotype by at least two different mechanisms, ie., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level.

Animals↗
PubMed1998

RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.

Malignant tumors of the thyroid gland vary considerably in aggressiveness, ranging from a well-differentiated, clinically indolent, to an undifferentiated, often lethal phenotype. Undifferentiated (anaplastic) thyroid tumors are supposed to be derived, through a process of progression, from previously differentiated neoplasms. A common genetic alteration in thyroid tumors is the rearrangement of the tyrosine kinase-encoding RET proto-oncogene, leading to the generation of chimeric RET/PTC oncogenes. To define the characteristics of the thyroid tumor subset with RET rearrangements, we have investigated its activation by a combined immunohistochemistry and reverse transcription-PCR approach in a series of 316 well-characterized thyroid tumors representative of the main diagnostic groups. RET activation was detected in 81 of 201 (40.3%) papillary carcinomas. It correlated with tumors exhibiting the "classic" morphological features of papillary cancer or with the microcarcinoma subtype (P = 0.017). RET activation in papillary carcinoma was not associated with clinical markers (such as large tumor size, extrathyroidal extension, or metastases) of increased morbidity. Follicular-type neoplasms (61 adenomas and 22 carcinomas), as well as the aggressive poorly differentiated (15 cases) or undifferentiated (anaplastic) carcinomas (17 cases), were negative. This study demonstrates that all thyroid carcinomas harboring activating RET rearrangements exhibit a well-differentiated phenotype, that of papillary carcinoma, and indicates that the subset of RET/PTC-positive papillary carcinomas do not progress to more aggressive, less differentiated tumor phenotypes.

Adolescent↗
PubMed1998

HMGIY is the target of 6p21.3 rearrangements in various benign mesenchymal tumors.

Specific chromosomal abnormalities of chromosomal region 6p21.3 have been described in subsets of many benign mesenchymal tumors. In the presented study, we investigated a series of 36 such cases by FISH, and Southern blot analyses for HMGIY rearrangements. FISH results revealed that the chromosomal breakpoints of 11 pulmonary chondroid hamartomas (PCHs), 12 endometrial polyps (EPs), one lipoma, and two uterine leiomyomas (ULs) were located within a 80 kb region surrounding the HMGIY gene. In 11 PCHs and one UL the breakpoints were located 3' of HMGIY, and one PCH showed a breakpoint 5' of HMGIY. Southern blot analyses with intra- and extragenic probes were performed of primary tumor material or cell lines from one UL, three PCHs, and five EPs. In none of these cases was an intragenic rearrangement found. Finally, we were able to detect expression of truncated HMGIY transcripts by 3'-RACE PCR. Our data clearly show the role of a further member of the HMGI family in the development of benign mesenchymal tumors. Although most of the breakpoints of the chromosomal translocations involving HMGIY are located outside the gene, aberrant transcripts resembling the structure of those observed in the case of HMGIC have been found. Our molecular investigations thus led to the identification of the molecular mechanism by which rearrangements of either of two closely related genes lead to the development of frequent benign mesenchymal tumors in humans.

Chromosome Aberrations↗
PubMed1997

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Animals↗
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

Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation.

Animals↗
PubMed1997

Thyroid cell transformation inhibits the expression of a novel rat protein tyrosine phosphatase.

We have isolated a rat thyroid cDNA encoding a novel rat receptor-type tyrosine phosphatase protein. This gene, on the basis of its homology to another tyrosine phosphatase, the recently isolated human DEP-1/HPTPeta, has been named r-PTPeta. In rat thyroid cells the r-PTPeta gene acts as a differentiation marker. Indeed, the block of thyroid cell differentiation induced by viral and cellular oncogenes is associated with the inhibition or marked reduction of the expression of this gene, and its expression is positively regulated by thyrotropin, the physiological stimulator of thyroid cell growth.

Amino Acid Sequence↗
PubMed1997

Distribution of insulin/insulin-like growth factor-I hybrid receptors in human tissues.

Insulin receptors (IR) and type 1 IGF receptors (IGF-IR) have been shown to form insulin/IGF-I hybrid receptors in tissues expressing both molecules. The biological function of hybrid receptors is still undefined. To date there is no information about the distribution of hybrid receptors in human tissues. We have applied two microwell-based immunoassays which are capable of quantitating hybrid receptors in small samples of human tissues and cells. Results demonstrated that the proportion of total IGF-IR assembled as hybrids varied between 40 and 60%, thus indicating that hybrid receptors account for a large fraction of total IGF-I binding in human tissues. A significant fraction of total IR was assembled as hybrids in the tissues examined, varying from 37% in placenta to 45% in hepatoma, with the exception of adipose tissue where the fraction of insulin receptors forming hybrids was 17%. Because hybrid receptors bind IGF-I, but not insulin, with high affinity, it is likely that in human tissues hybrid receptors may be primarily activated by IGF-I rather than insulin under physiological conditions. Therefore, differences in hybrid receptors distribution may contribute to regulate tissue sensitivity to insulin and IGF-I by sequestering insulin receptor alphabeta-heterodimer in an IGF-I responsive form.

Female↗
PubMed1997

The different RET-activating capability of mutations of cysteine 620 or cysteine 634 correlates with the multiple endocrine neoplasia type 2 disease phenotype.

Distinct point mutations of RET, a tyrosine-kinase receptor encoding gene, are responsible for the inheritance of multiple endocrine neoplasia type 2 syndromes (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In particular, MEN2A is a more complex and aggressive disease than FMTC, being characterized by pheochromocytomas and parathyroid alterations, in addition to medullary thyroid carcinomas. The mutations associated with MEN2A and FMTC affect one of five cysteine residues mapping in the extracellular domain of the Ret protein. However, recent studies have indicated that MEN2A and FMTC disease phenotypes correlate with the position of mutations in RET. Mutations of Cys-634 are more frequent in families with MEN2A, whereas Cys-620 mutations are very rarely found in MEN2A patients and, in contrast, are frequently found in FMTC patients. We have reported previously that mutations of Cys-634 constitutively activate the RET transforming potential by causing a disulfide bridge-mediated homodimerization. Here, we report that the mutation Cys-620 --> Tyr is able to cause a constitutive dimerization of Ret, with consequent activation of its kinase and transforming activities, to a lower extent than mutation of Cys-634. We suggest that the difference in ability to activate RET shown by mutations associated with FMTC and MEN2A represents the molecular basis of the phenotypic diversity between the two syndromes.

3T3 Cells↗
PubMed1997

Thyroid carcinoma associated with familial adenomatous polyposis.

AIMS: Thyroid carcinoma is an extracolonic manifestation that is present in about 1% to 2% of patients with familial adenomatous polyposis (FAP). Less than 100 cases have been reported in detail. We have investigated the suggestion that FAP associated thyroid carcinoma is significantly different morphologically from both papillary and follicular types and can be considered as a separate entity. METHODS AND RESULTS: Specimens from three patients with FAP associated thyroid tumours, all but one having single nodules, have been analysed. All three patients belonged to an extended kindred (23 siblings in four generations) who had genetic analysis and intensive screening for thyroid nodules. Seven patients had the same APC mutation at codon 1061. Pathological examination revealed a typical papillary carcinoma, encapsulated variant, in all patients, with follicular areas in one case. All thyroid specimens, in addition to histological and immunohistological examinations, were also specifically studied for activation of the RET-PTC oncogene, that seems to be restricted to papillary thyroid carcinoma. Two of the three patients had RET-PTC activation (PTC1 isoform). CONCLUSIONS: The findings suggest that the tumours were certainly papillary, at least in the present kindred. Further studies in different families are required for a better understanding of this peculiar tumour and of its biological behaviour.

Adenomatous Polyposis Coli↗
PubMed1997

Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto's thyroiditis.

Hashimoto's thyroiditis is an inflammatory disease of the thyroid gland with autoimmune etiology. Patients afflicted with Hashimoto's have a higher risk of thyroid malignancies such as papillary thyroid carcinoma. In the present study, we investigated the frequency of papillary thyroid carcinoma specific genes in patients diagnosed with Hashimoto's disease. The newly identified oncogenes RET/PTC1 and RET/PTC3 provide useful and specific markers of the early stages of papillary carcinoma as they are highly specific for malignant cells. Using a sensitive and specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we found messenger RNA (mRNA) expression for the RET/PTC1 and RET/PTC3 oncogenes in 95% of the Hashimoto's patients studied. All Hashimoto's patients presenting without histopathologic evidence of papillary thyroid cancer showed molecular genetic evidence of cancer. These data suggest that multiple, independent occult tumors exist in these patients at high frequency.

Adult↗
PubMed1997

Only the substitution of methionine 918 with a threonine and not with other residues activates RET transforming potential.

Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.

3T3 Cells↗
PubMed1997

Oncogene transformation of PC Cl3 clonal thyroid cell line induces an autonomous pattern of proliferation that correlates with a loss of basal and stimulated phosphotyrosine phosphatase activity.

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTP eta, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTP mu, was unchanged. Thus, PTP eta may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.

Animals↗