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Biomedical subjects

A Fusco

Publications and source records attributed to A Fusco.

At least 19 recordsLinked to original sources
PubMed2010

CCDC6 represses CREB1 activity by recruiting histone deacetylase 1 and protein phosphatase 1.

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation.

Amphiregulin↗
PubMed2005

High Mobility Group A1 (HMGA1) proteins interact with p53 and inhibit its apoptotic activity.

HMGA gene overexpression and rearrangements are frequent in several tumours, but their oncogenic function is still unclear. Here we report of a physical and functional interaction between High Mobility Group A1 (HMGA1) protein and p53 oncosuppressor. We found that HMGA1 binds p53 in vitro and in vivo, and both proteins are present in the same complexes bound to the Bax gene promoter. HMGA1 interferes with the p53-mediated transcription of p53 effectors Bax and p21(waf1) while cooperates with p53 in the transcriptional activation of the p53 inhibitor mdm2. This transcriptional modulation is associated with a reduced p53-dependent apoptosis in cells expressing exogenous HMGA1 and p53, or in cells expressing endogenously the proteins and in which p53 was activated by UV-irradiation. Furthermore, antisense inhibition of HMGA1b expression dramatically increases the UV-induced p53-mediated apoptosis. These data define a new physical and functional interaction between HMGA1 and p53 that modulates transcription of p53 target genes and inhibits apoptosis.

Apoptosis↗
PubMed2001

Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models.

TSH via cAMP, and various growth factors, in cooperation with insulin or IGF-I stimulate cell cycle progression and proliferation in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, PC Cl3) and primary cultures of rat, dog, sheep and human thyroid. The available data on cell signaling cascades, cell cycle kinetics, and cell cycle-regulatory proteins are thoroughly and critically reviewed in these experimental systems. In most FRTL-5 cells, TSH (cAMP) merely acts as a priming/competence factor amplifying PI3K and MAPK pathway activation and DNA synthesis elicited by insulin/IGF-I. In WRT cells, TSH and insulin/IGF-I can independently activate Ras and PI3K pathways and DNA synthesis. In dog thyroid primary cultures, TSH (cAMP) does not activate Ras and PI3K, and cAMP must be continuously elevated by TSH to directly control the progression through G(1) phase. This effect is exerted, at least in part, via the cAMP-dependent activation of the required cyclin D3, itself synthesized in response to insulin/IGF-I. This and other discrepancies show that the mechanistic logics of cell cycle stimulation by cAMP profoundly diverge in these different in vitro models of the same cell. Therefore, although these different thyrocyte systems constitute interesting models of the wide diversity of possible mechanisms of cAMP-dependent proliferation in various cell types, extrapolation of in vitro mechanistic data to TSH-dependent goitrogenesis in man can only be accepted in the cases where independent validation is provided.

Animals↗
PubMed1994

Description of a human papillary thyroid carcinoma cell line. Morphologic study and expression of tumoral markers.

BACKGROUND: The establishment of cell lines from thyroid carcinomas can provide an in vitro model of oncogenesis. B-CPAP is a new cell line that has been obtained from a differentiated papillary thyroid carcinoma. The data presented give a broader characterization and expression of tumoral markers of this cell line and identify the differentiated functions that are preserved. METHODS: An ultrastructural study was performed to confirm the thyroid nature of the new cell line. The cellular markers (thyroglobulin, S100, neuron-specific enolase [NSE]) and the oncogenes (mutated p53, H-ras, c-myc, PTC, trk) were studied by immunohistochemistry, Southern blot, or in situ hybridization. RESULTS: The cells were of a differentiated ultrastructural thyroid type. All of the cells proved immunoreactive with antibodies specific to thyroglobulin, S100 proteins, NSE, and mutant p53 protein. Mutations of H-ras, PTC, and trk were not observed. The c-myc gene was not amplified. CONCLUSIONS: The cell line described in these data provides a suitable model for the study of thyroid carcinogenesis, given that the cells present thyroid characteristics, and metabolic disorders not previously found in such cell lines. In addition, the coexpression of S100 proteins and mutant p53 proteins in the cells should permit the study of the interaction between these two proteins.

Aged↗
PubMed1994

Cloning and characterization of H4 (D10S170), a gene involved in RET rearrangements in vivo.

H4(D10S170) is a gene which we isolated because of its frequent rearrangement with the RET proto-oncogene in vivo. Its fusion to RET generates the RET/PTC1 oncogene, which has been detected in about 20% of human thyroid papillary carcinomas. We have cloned and sequenced the cDNA corresponding to the H4(D10S170) gene from a human normal thyroid cDNA library. The nucleotide sequence of the H4(D10S170) 3 kb transcript shows no significant homology to known genes and contains an open reading frame (ORF) of 585 amino acids. H4(D10S170) predicted protein has no transmembrane domain and shows extensive regions in the alpha helical conformation, which are 30% homologous to the alpha-helical domains of several proteins including tropomyosin, vimentin, keratin and the tail region of myosin heavy chain. A putative SH3 binding site is present at the carboxy terminus, which suggests that H4(D10S170) might be a cytoskeletal protein.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma.

The RET proto-oncogene encodes a transmembrane receptor of the tyrosine kinase family and has frequently been found activated in human thyroid carcinomas of the papillary subtype. In most cases the activation consisted of the fusion of its tyrosine-kinase domain with the 5'-terminal region of a gene designated H4 or D10S170. We have named the resulting H4/RET chimeric oncogene RET/PTC. Another activated form of the RET oncogene has subsequently been found in a thyroid carcinoma and is now referred to as RET/PTC2. Here we report the identification and cloning of a novel rearranged version of the RET oncogene in a human thyroid papillary carcinoma. In this case the tyrosine-kinase domain of RET was fused to a sequence 790 bp long belonging to a new gene that we have named RFG (RET Fused Gene). This novel chimeric oncogene has been designated RET/PTC3. In order to have more insights into the function of RFG we have completely cloned and sequenced its cDNA. RFG predicted amino-acid sequence does not have any significant homology to any already known genes and is ubiquitously expressed in human and mouse tissues. Finally we provide evidence indicating that the rearrangement leading to the generation of RET/PTC3 occurred in vivo in the original tumor DNA.

Amino Acid Sequence↗
PubMed1992

Characterization of an inversion on the long arm of chromosome 10 juxtaposing D10S170 and RET and creating the oncogenic sequence RET/PTC.

RET/PTC is a transforming sequence created by the fusion of the tyrosine kinase domain of the RET protooncogene with the 5' end of the locus D10S170 designated by probe H4 and is frequently found activated in human papillary thyroid carcinomas. RET and D10S170 have been mapped to contiguous regions of the long arm of chromosome 10: q11.2 and q21, respectively. To identify the mechanism leading to the generation of the oncogenic sequence RET/PTC, a combined cytogenetic and molecular analysis of several cases of papillary thyroid carcinomas was done. In four cases the results indicated that these tumors had RET/PTC activation and a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with breakpoints coincident with the regions where RET and D10S170 are located. Therefore, a chromosome 10q inversion provides the structural basis for the D10S170-RET fusion that forms the hybrid transforming sequence RET/PTC.

Blotting, Southern↗
PubMed1992

Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, named PTC, is a novel rearranged version of the ret proto-oncogene. In fact PTC is the product of the fusion of the tyrosine kinase domain of the ret proto-oncogene with the 5'-terminal region of another gene that we have named H4. The ret proto-oncogene shows a pattern of expression restricted to neuroendocrine tissue. Its fusion with H4 allows the expression of the activated form in thyroid papillary carcinomas. Therefore the detection of ret transcripts is a tool to investigate ret activation in thyroid neoplasms. Here we show the detection by in situ hybridisation, of activated ret transcripts in human thyroid papillary neoplasms that were positive for PTC activation by Southern blot analysis. We did not find any ret transcripts in papillary carcinomas negative for PTC activation, nor in normal thyroid and in non-papillary thyroid neoplasias.

3T3 Cells↗
PubMed1992

Multiple mechanisms of interference between transformation and differentiation in thyroid cells.

Transformation of the thyroid cell line FRTL-5 results in loss or reduction of differentiation as measured by the expression of thyroglobulin and thyroperoxidase, two proteins whose genes are exclusively expressed in thyroid follicular cells. The biochemical mechanisms leading to this phenomenon were investigated in three cell lines obtained by transformation of FRTL-5 cells with Ki-ras, Ha-ras, and polyomavirus middle-T oncogenes. With the ras oncogenes, transformation leads to undetectable expression of the thyroglobulin and thyroperoxidase genes. However, the mechanisms responsible for the extinction of the differentiated phenotype seem to be different for the two ras oncogenes. In Ki-ras-transformed cells, the mRNA encoding TTF-1, a transcription factor controlling thyroglobulin and thyroperoxidase gene expression, is severely reduced. On the contrary, nearly wild-type levels of TTF-1 mRNA are detected in Ha-ras-transformed cells. Furthermore, overexpression of TTF-1 can activate transcription of the thyroglobulin promoter in Ki-ras-transformed cells, whereas it has no effect on thyroglobulin transcription in the Ha-ras-transformed line. Expression of polyoma middle-T antigen in thyroid cells leads to only a reduction of differentiation and does not severely affect either the activity or the amount of TTF-1. Another thyroid cell-specific transcription factor, TTF-2, is more sensitive to transformation, since it disappears in all three transformed lines, and probably contributes to the reduced expression of the differentiated phenotype.

Animals↗
PubMed1992

[Description of cell line established from human thyroid papillary cancer and secreting human chorionic gonadotropin hormone].

One of the difficulties in characterization of the oncogenes involved in thyroid carcinogenesis is the production of cell lines. Arising from a poorly differentiated thyroid papillary carcinoma we have established a cell line synthesizing the thyroglobulin and human chorionic gonadotropin (alpha and beta subunits) (HCG) hormones. These cells will allow research of the oncogenes involved or potentially involved in thyroid papillary carcinomas and evaluation of the role of the autocrine secretion of HCG.

Carcinoma, Papillary↗
PubMed1992

Identification of the product of two oncogenic rearranged forms of the RET proto-oncogene in papillary thyroid carcinomas.

In papillary thyroid carcinomas, we have identified two tumor-specific rearrangements of the RET proto-oncogene leading to the formation of different transforming fusion products sharing the tyrosine kinase (tk) domain of the proto-oncogene and designated ptc-1 and ptc-2. We have analysed ptc-1 and ptc-2 products by immunoprecipitation with specific anti-RET antibodies followed by immunoblotting with the same reagent or with antibodies specific for phosphotyrosine (P-tyr) residues. The anti-RET antibodies were reactive with 64-kDa (p64ptc-1) and 81-kDa (p81ptc-2) proteins from lysates of ptc-1 and ptc-2 transformed cells, respectively, and identified two proteins of 140 kDa and 160 kDa from extracts of SK-N-SH, a neuroblastoma cell line previously shown to express two differently glycosylated forms of the normal RET product. The anti P-tyr antibodies, while detecting the same p64ptc-1 and p81ptc-2 proteins from ptc-1 and ptc-2 extracts, did not show any specific band in the neuroblastoma lysates. An additional set of experiments led us to conclude that, whereas the normal product of the RET proto-oncogene is a membrane-associated receptor-like molecule not intrinsically phosphorylated on tyrosine, both oncogenic forms of RET, ptc-1 and ptc-2, are constitutively phosphorylated on tyrosine, display an 'in vitro' autophosphorylation activity, are translocated from the membrane to the cytoplasm and are apparently unaffected by protein kinase C modulation.

3T3 Cells↗
PubMed1992

Increased expression of the negative growth factor, galactoside-binding protein, gene in transformed thyroid cells and in human thyroid carcinomas.

Murine beta-galactoside-binding protein has been shown to be a cell growth regulatory molecule and a cytostatic factor. We analysed the beta-galactoside-binding protein gene expression in a thyroid cell system including two normal cell lines (FRTL-5 and PC Cl 3) and the same cells transfected by several oncogenes that induce different degrees of malignancy and differentiation. We show that beta-galactoside-binding protein mRNA levels correlate with the expression of the malignant phenotype. Run-on experiments suggest that a transcriptional effect accounts at least in part for such a difference. We also show that the beta-galactoside-binding protein gene expression is increased in most human papillary thyroid carcinomas compared with normal thyroid.

Adenovirus E1A Proteins↗
PubMed1991

Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor gene is expressed in two variant isoforms which differ by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the extracellular alpha-subunit as a consequence of alternative splicing of exon 11. Expression of the two variant isoforms is regulated in a tissue-specific manner. In this study, we have measured the levels of the two receptor variants in isolated adipocytes from 10 non-insulin-dependent diabetes mellitus (NIDDM) and 11 normal subjects using an immunological assay, based on the ability of a human anti-receptor autoantibody to discriminate between HIR-A and HIR-B. Results indicate that levels of HIR-B variant are increased in NIDDM patients.

3T3 Cells↗
PubMed1991

Comparison of multiple forms of the high mobility group I proteins in rodent and human cells. Identification of the human high mobility group I-C protein.

The class I of the high mobility group (HMG) proteins is formed by phosphoproteins which are associated with AT-rich DNA sequences in the nucleus. Three HMGI proteins have previously been described in proliferating rodent cells (HMG Y, HMG I and HMGI-C). All three proteins exhibit microheterogeneity. The microheterogeneity of mouse HMG Y has been investigated in detail and shown to be due to phosphorylation of the protein which is sensitive to alkaline-phosphatase treatment. HMG I is similarly modified. Human cells have up to now only been found to contain HMG Y and HMG I. A search for the third protein, HMGI-C, in human cells was carried out and the protein was found in a hepatoma cell line, but not in normal or transformed T-cells. This HMGI-C protein was found to be modified by phosphorylation, part of which was found to be phosphatase insensitive. An unexpected additional finding in this study was that human cells contain two HMG17 proteins which differ in their N-terminal primary sequences.

Alkaline Phosphatase↗
PubMed1991

Refined localization to contiguous regions on chromosome 10q of the two genes (H4 and RET) that form the oncogenic sequence PTC.

In this report we confirm the localization of the human RET proto-oncogene to chromosome 10q11.2, both by Southern blot analysis of a panel of human-rodent somatic cell hybrids and by in situ hybridization on human metaphase chromosomes. Previously, we had assigned to the same chromosome region the gene termed H4. In about 25% of papillary thyroid carcinomas, this gene was shown to rearrange with RET to give rise to the transforming sequence PTC. The analysis of different cell hybrids containing subfragments of chromosome 10, in conjunction with pulse field gel electrophoresis, established that H4 is mapped distally to RET at a distance not less than 280 kb. These findings suggest that intrachromosomal rearrangements are responsible for PTC activation in papillary thyroid carcinomas.

Carcinoma, Papillary↗
PubMed1990

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

Animals↗
PubMed1990

PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas.

We recently detected a novel activated oncogene by transfection analysis on NIH 3T3 cells in five out of 20 primary human thyroid papillary carcinomas and in the available lymph node metastases. We designated this transforming gene PTC (for papillary thyroid carcinoma). Here we describe the molecular cloning and sequencing of the gene. The new oncogene resulted from the rearrangement of an unknown amino-terminal sequence to the tyrosine kinase domain of the ret proto-oncogene. This gene rearrangement was detected in all of the transfectants and in all of the original tumor DNAs, but not in normal DNA of the same patients, thus indicating that this genetic lesion occurred in vivo and is specific to somatic tumors. Moreover, the transcript coded for by the fused gene was detected in an additional PTC-positive human papillary carcinoma for which mRNA was available.

Amino Acid Sequence↗