Biomedical subjects

A Fusco

Publications and source records attributed to A Fusco.

At least 181 records · Page 10Linked to original sources

PubMed1995

Expression of the RET oncogene induces differentiation of SK-N-BE neuroblastoma cells.

Expression of the RET proto-oncogene, a cell surface receptor for an as yet unknown ligand, is associated with tumors, tissues, and cell lines of neural crest origin. Accumulating evidence suggests that RET activity is involved in the process of neuronal differentiation. Moreover, induction of phenotypic differentiation of neuroblastoma cell lines is associated with the rapid accumulation of RET transcripts. To verify the role of RET in neuronal differentiation, we introduced into the human neuroblastoma cell line SK-N-BE four versions of the RET oncogene, activated by different mechanisms: RET/PTC1 and RET/PTC3, which are activated by rearrangement with heterologous genes; and two activated RET mutants, which carry the single amino acid substitution found associated to the inheritance of the multiple endocrine neoplasia type 2A (retMEN2A allele) and type2B (retMEN2B allele), respectively. We demonstrate that, after transfection with the RET oncogenes, SK-N-BE cells display a reduced growth rate and acquire a neurite-bearing phenotype accompanied by enhanced expression of the axonal growth-associated protein, GAP-43, and the high molecular weight neurofilament, NF200. These results indicate that, when activated, RET is able to cause growth inhibition and to promote neuronal differentiation of neuroblastoma cells.

Blotting, Northern↗

PubMed1994

Frequent activation of ret protooncogene by fusion with a new activating gene in papillary thyroid carcinomas.

Tumor specific rearrangements of ret gene are frequently detected in papillary thyroid carcinomas. These rearrangements result in the formation of chimeric genes showing the tyrosine kinase domain of ret fused with the 5' end sequences of different genes. We examined a series of 52 patients and identified 10 cases of ret fusion with D10S170 locus resulting in the generation of ret/PTC1 oncogene, 2 cases with the gene encoding the regulatory subunit RI alpha of PKA (ret/PTC2), and finally 6 cases, here described, with a newly discovered gene called ele1 localized on chromosome 10 and leading to the formation of ret/PTC3 oncogene. Our results show the expression of the ret/PTC3 hybrid gene in all the 6 cases and demonstrated its association with the synthesis of 2 constitutively phosphorylated isoforms of the oncoprotein (p75 and p80). The chromosome 10 localization of both ret and ele1 and the detection, in all cases, of a sequence reciprocal to that generating the oncogenic rearrangements, strongly suggest that ret/PTC3 formation is a consequence of an intrachromosomal inversion of chromosome 10.

3T3 Cells↗

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

Expression of the soluble lectin L-14 gene is induced by TSH in thyroid cells and suppressed by retinoic acid in transformed neural cells.

Expression of the soluble lectin L-14 is low in normal and very high in transformed thyroid cells. We show that L-14 gene expression is transiently induced upon thyrotropin stimulation of normal quiescent FRTL-5 rat thyroid cells. Permanent activation of L-14 gene expression is obtained in the same cells infected with a wild-type and a temperature sensitive mutant of Kirsten murine sarcoma virus, both at the permissive and non permissive temperature for transformation. We also find that L-14 mRNA is undetectable in rat brain but is abundant in rat oligodendrocytes precursors transformed by polyoma middle T oncogene. Retinoic acid treatment of these transformed cells leads to acquisition of a differentiated phenotype accompanied by a 30-fold decrease of L-14 mRNA levels. Removal of retinoic acid restores both the transformed undifferentiated phenotype and high L-14 expression. Taken together these results indicate that growth stimulation and induction of cell differentiation are accompanied by strong modulation of L-14 gene expression.

Animals↗

PubMed1994

Cloning of the rat tissue inhibitor of metalloproteinases type 2 (TIMP-2) gene: analysis of its expression in normal and transformed thyroid cells.

We have recently reported that the introduction of the E1A gene of the adenovirus 5 gene into the rat thyroid PC Cl 3 epithelial cell line causes loss of the differentiated thyroid phenotype without the appearance of the typical markers of neoplastic transformation. It is well known that the E1A gene is able either to induce or to repress the transcription of various endogenous cellular genes. In order to characterize the genes involved in the transformation of PC Cl 3 cells by the E1A gene, we have isolated several cDNA clones, whose expression was induced by E1A. One of these clones was found to be the rat homologue of the human tissue inhibitor of metalloproteinase type 2 (TIMP-2) gene. Here we show the complete sequence of the rat TIMP-2. Its homology to the human TIMP-2 is 98% at the amino acid level and, like its human counterpart, the rat TIMP-2 is transcribed in two different mRNAs of 1.0 and 3.5 kb. The expression of TIMP-2, in PC Cl 3 cells, was significantly induced by the E1A gene and also by other oncogenes. Finally TIMP-2 was shown to be significantly overexpressed in human thyroid neoplastic cell lines and some tumoral thyroid samples.

Adenovirus E1A Proteins↗

PubMed1994

Tissue-specific expression of two alternatively spliced isoforms of the human insulin receptor protein.

Two insulin receptor mRNA species are expressed in human tissues as a result of alternative splicing of exon 11. This event is regulated in a tissue-specific manner. To date, there is little information about the relative abundance of the two receptor protein isoforms on the cell surface. The aim of the present investigation was to assess whether the tissue-specific expression of the two insulin receptor mRNA species is paralleled by a similar pattern of expression of the two receptor protein isoforms. To this end, we assessed the relative distribution of the two receptor variants in various human tissues at the mRNA and protein levels. A PCR-based technique was used to measure the relative abundance of the two mRNA species, and two immunological assays were used to measure the relative steady-state expression of the two receptor protein isoforms. The expression of the two insulin receptor protein isoforms followed the tissue-specific pattern of expression of the two mRNA species.

3T3 Cells↗

PubMed1994

Aberrant expression, function and localization of connexins in human esophageal carcinoma cell lines with different degrees of tumorigenicity.

We have analyzed the level of mRNA expression and protein localization of the gap-junction protein connexins (Cx) 26, 32, and 43, as well as gap-junctional intercellular communication (GJIC) in seven human esophageal carcinoma cell lines (TE series). These cell lines exhibited various degrees of tumorigenicity in nude mice; two (TE-1 and TE-8) formed progressively growing tumors, four (TE-2, TE-3, TE-9, and TE-13) developed non-progressing tumors and one (TE-10) showed no tumorigenicity. We found that normal human esophageal tissue expressed both Cx26 and Cx43 and that most of the cell lines expressed lower amounts of Cx26 and Cx43 mRNAs than normal human esophageal tissues or none at all. The co-expression of Cx26 and Cx43 mRNAs and proteins was observed only in two cell lines (TE-3 and TE-9) that showed a high level of GJIC and non-progressive tumor development. However, the non-tumorigenic cell line TE-10 did not express either connexin. A possible regulator of GJIC, E-cadherin, was expressed in all cell lines. These results suggest that aberrant expression and function of connexins are common among human esophageal carcinoma cell lines, but there is no quantitative relationship between connexin expression and tumorigenic properties of these cell lines.

Animals↗

PubMed1994

Molecular and cellular characterization of the GABAA receptor in the rat pancreas.

In the present study, we characterize the molecular structure of the GABAA receptor in pancreas, islets, alpha and beta cells, and in RIN 1046-38 cells. Using the polymerase chain reaction and specific primers for 11 out of the 15 subunits known so far, that may contribute to the composition of the GABAA receptors, we demonstrate that pancreas and its cellular components, as well RIN 1046-38 cells, might contain a GABAA receptor resulting from all the possible combinations in a pentameric configuration of the subtypes alpha 1, alpha 2, alpha 3 of the alpha subunit family, beta 1, beta 2, beta 3 subtypes of the beta subunit family, delta subunit and gamma 2 subtype of the gamma subunit family. The presence of the gamma 2 subunit renders the GABAA receptors potentially sensitive to allosteric modulators.

Animals↗

PubMed1994

Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide antibodies.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two HIR alpha-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) directed against a sequence in the carboxy-terminal region of the alpha-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA-12 linearises the Scatchard plot of binding data, and retards the dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.

3T3 Cells↗

PubMed1994

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway.

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

3T3 Cells↗

PubMed1994

Alpha 1A- and alpha 1B-adrenergic receptors mediate the effect of norepinephrine on cytosolic calcium levels in rat PC C13 thyroid cells: thyrotropin modulation of alpha 1B-linked response via a adenosine 3',5'-monophosphate-protein kinase-A-dependent pathway.

The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC C13 rat thyroid cells and identify the molecular mechanisms by which TSH regulates the noradrenergic response. We studied TSH regulation of norepinephrine-induced cytosolic calcium increase by means of the fluorescent probe fura-2. In PC C13 cells grown and maintained in a medium containing TSH (PC C13 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC C13 starved from TSH 5 days before the experiments (PC C13 5H). In both group of cells the calcium response to norepinephrine was concentration dependent and reduced by the removal of extracellular calcium ions. Reintroduction of TSH in the culture medium of the PC C13 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC C13 6H. This effect was complete after a 48-h incubation period and was abolished by the simultaneous treatment of the cells with the protein synthesis inhibitor cycloheximide, suggesting that TSH may stimulate the synthesis of alpha 1-adrenergic receptors in PC C13 cells. Because in these cells we found that TSH increased cAMP levels as well as inositol phosphate production, we tested whether the activation of a protein kinase-A and/or protein kinase-C was involved in TSH regulation of the adrenergic response. We found that the treatment of PC C13 5H cells with forskolin restored the effect of norepinephrine on the calcium level, and that KT5720, an inhibitor of the protein kinase-A, was able to prevent the recovery of the noradrenergic response induced by the readdition of TSH to the culture medium of PC C13 5H. Conversely, treatment of PC C13 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC C13 6H and, to a lesser extent, in PC C13 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize alpha 1-adrenergic receptor subtypes mediating the effects of norepinephrine in PC C13 cells, we used antagonists of alpha 1A and alpha 1B receptors (WB4101 and chlorethylclonidine respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1994

Variant sequences of insulin receptor substrate-1 in patients with noninsulin-dependent diabetes mellitus.

The pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM) is characterized by insulin resistance and insulin deficiency. To search for genetic defects causing NIDDM, we have screened for mutations in the gene encoding insulin receptor substrate-1 (IRS-1), an intracellular protein that is phosphorylated by the insulin receptor and is thought to play an important role in mediating insulin action. The coding sequence of the IRS-1 gene (divided into 12 overlapping fragments) was amplified by polymerase chain reaction and screened for the presence of single stranded conformational polymorphisms. This led to the identification of 6 variants in the nucleotide sequence. There were 3 nonconservative amino acids substitutions: Gly819-->Arg, Gly972-->Arg, and Arg1221-->Cys. In addition, there were three silent polymorphisms: GAC vs. GAT encoding Asp90, GGG vs. GGA encoding Gly235, and GCA vs. GCG encoding Ala805. The previously reported Arg972 substitution was identified in 7 of 31 patients with NIDDM, 4 of 32 normal subjects, and 4 of 16 nondiabetic obese individuals. The 2 novel amino acid substitutions (Arg819 and Cys1221) were both detected in 1 patient with NIDDM, but not in either of the other 2 groups of nondiabetic individuals. All 3 amino acid residues are identically conserved in the amino acid sequences of human, mouse, and rat IRS-1, suggesting that Gly819, Gly972, and Arg1221 are important for the normal function of IRS-1. Furthermore, the prevalence of amino acid substitutions in IRS-1 is increased in patients with NIDDM. These observations suggest that mutations in the IRS-1 gene may play a causal role in the pathogenesis of NIDDM.

Adult↗

PubMed1994

Developmental expression of the RET protooncogene.

The RET protooncogene encodes a transmembrane protein of the receptor-type tyrosine kinase family whose ligand has not yet been identified. Its activation in vivo is restricted to human carcinomas of the thyroid. In order to learn more about the possible role played by RET during normal development, we have examined its expression by performing in situ hybridization experiments on mouse embryos. Here, we show that the RET protooncogene is expressed during mouse embryogenesis in an unusual temporal and spatial manner. In fact, its expression was first detected around day 10 of gestation in the basal plate of the neural tube and in the developing encephalic ganglia, and later its pattern of expression was definitely established in neural structures, mostly in neural crest derivatives (spinal and encephalic ganglia). As far as the central nervous system is concerned, RET expression was confined to the ventral part of the midbrain from 12.5 days postcoitum (dpc) until birth. RET was also found to be expressed within structures of sensory organs such as the ganglial layer of the retina and the olfactory epithelium. A peculiar pattern of RET expression was clearly observed in the wall of the gut and in the nephrogenic zone of the developing kidney cortex, specifically in the metanephrogenic vesicles. Finally, RET was found to be expressed in the liver mostly between 12.5 dpc and 14.5 dpc. In conclusion, its expression in the early stages of embryogenesis suggests that RET may play a role in the differentiation of specific neural structures and the excretory system.

Animals↗

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↗

PubMed1993

Possible role of activated ras genes in human esophageal carcinogenesis.

Activation by point mutation of the H-, K- and N-ras genes is found in many tumors. However, no such mutation has yet been found in human esophageal carcinomas from various parts of the world. We have confirmed the absence of mutation at codons 12, 13 and 61 of K- and N-ras and at codons 12 and 61 of H-ras in 25 primary tumors obtained in France. In contrast, among 7 human esophageal carcinoma cell lines (TE1, TE2, TE3, TE8, TE9, TE10, TE13) with different degrees of tumorigenicity in nude mice, 3 highly tumorigenic lines (TE1, TE2 and TE8) exhibited activation of ras oncogenes; 2 showed a G35 to A35 transition of K-ras gene and one a H-ras G35 to T35 transversion. Since these cell lines had been established from tumors of Japanese patients from Sendai, we examined 3 primary esophageal tumors from Tokyo and 19 from Sendai, including the primary tumors from which the TE cell lines had been derived. No ras mutation was detected, which suggests that the ras gene mutations in the TE cell lines are either due to their long-term culture or that only a small portion of the original tumors contained such mutations. In order to directly examine the effect of ras gene mutation, one of the non-tumorigenic cell lines, TE13, was transfected with a plasmid coding for a mutated H-ras gene (G35 to T35). Transfected clones expressing high levels of mutated ras gene were able to induce tumors in nude mice. Thus, although no primary human esophageal tumor contained mutated ras genes, our studies do not exclude a significant role of mutated ras genes in cell proliferation and malignant transformation of human esophageal cells.

Adult↗

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↗

PubMed1993

Norepinephrine and thyrotropin stimulation of [Ca++]i in PC C13 a rat thyroid epithelial cell line: effect of transformation by E1A gene adenovirus and polyomavirus middle-T antigen gene.

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin.

Adenoviridae↗