RESUMEN
The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.
Asunto(s)
Carcinoma/diagnóstico , Carcinoma/mortalidad , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/mortalidad , Enzimas Ubiquitina-Conjugadoras/genética , Carcinoma/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/patología , Pronóstico , Interferencia de ARN , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Enzimas Ubiquitina-Conjugadoras/análisis , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
Previous studies suggest the expression of UbcH10 gene, that codes for a protein belonging to the ubiquitin-conjugating enzyme family, as a valid indicator of the proliferative and aggressive status of tumors of different origin. Therefore, to look for possible tools to be used as diagnostic markers in astrocytic neoplasias, we investigated UbcH10 expression in normal brain, gliosis and low-grade and high-grade astrocytic tumors by immunohistochemistry. UbcH10 expression was observed in low-grade astrocytoma and in glioblastoma. Our data indicate a clear correlation between UbcH10 expression and the histological grade of the astrocytic tumors. Moreover, the analysis of UbcH10 expression allows the differentiation between gliotic and malignant tissues. Finally, since proteasome inhibitors have recently been considered as possible drugs in the chemotherapy of various tumors, our results would suggest new perspectives for the treatment of brain malignancies based on the suppression of the UbcH10 function.
Asunto(s)
Astrocitoma/diagnóstico , Astrocitoma/metabolismo , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Enzimas Ubiquitina-Conjugadoras/biosíntesis , Expresión Génica , Humanos , InmunohistoquímicaRESUMEN
Two rat thyroid epithelial differentiated cell lines, PC Cl 3 and PC myc, were infected with the polyoma murine leukemia virus (PyMLV) carrying the Middle-T-antigen gene of polyomavirus. After infection, both cell lines acquired the typical markers of neoplastic transformation; however, the PC myc cells showed a greater malignant phenotype. Furthermore, the thyroid differentiated functions were completely suppressed in PC myc cells transformed by PyMLV, whereas they were, at least partially, retained in PC Cl 3 cells transformed by PyMLV, and in particular, thyroglobulin synthesis and secretion were not affected at all. Since no differences in the expression of the middle-T-antigen gene were observed in the two PyMLV-transformed cell lines, the different properties shown by these two infected cell lines must be ascribed to the expression of the c-myc oncogene.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica/genética , Genes Virales , Oncogenes , Poliomavirus/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Línea Celular , Epitelio , Humanos , Poliomavirus/genética , Proteínas Proto-Oncogénicas c-myc , Ratas , Ratas Endogámicas F344 , Tiroglobulina/metabolismo , Glándula TiroidesRESUMEN
A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.
Asunto(s)
Transformación Celular Viral , Oncogenes , Retroviridae/patogenicidad , Glándula Tiroides/citología , Animales , Diferenciación Celular , Línea Celular Transformada , Células Epiteliales , Epitelio/fisiología , Yoduros/metabolismo , Neoplasias Experimentales/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Ratas , Retroviridae/genética , Tiroglobulina/biosíntesis , Glándula Tiroides/fisiologíaRESUMEN
Elevated expression of the three high-mobility group I (HMGI) proteins (HMGI, HMGY, and HMGI-C) has previously been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells and in experimental thyroid, lung, mammary, and skin carcinomas. Northern (RNA) blot and run-on analyses demonstrated that the induction of HMGI genes in transformed thyroid cells occurs at the transcriptional level. An antisense methodology to block HMGI-C protein synthesis was then used to analyze the role of this protein in the process of thyroid cell transformation. Transfection of an antisense construct for the HMGI-C cDNA into normal thyroid cells, followed by infection with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus, generated cell lines that expressed significant levels of the retroviral transforming oncogenes v-mos or v-ras-Ki and removed the dependency on thyroid-stimulating hormones. However, in contrast with untransfected cells or cells transfected with the sense construct, those containing the antisense construct did not demonstrate the appearance of any malignant phenotypic markers (growth in soft agar and tumorigenicity in athymic mice). A great reduction of the HMGI-C protein levels and the absence of the HMGI(Y) proteins was observed in the HMGI-C antisense-transfected, virally infected cells. Therefore, the HMGI-C protein seems to play a key role in the transformation of these thyroid cells.
Asunto(s)
Transformación Celular Neoplásica , Genes mos , Genes ras , Proteínas del Grupo de Alta Movilidad/biosíntesis , Nucleoproteínas/biosíntesis , Glándula Tiroides/metabolismo , Animales , Secuencia de Bases , Northern Blotting , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Cartilla de ADN , Sustancias de Crecimiento/farmacología , Proteína HMGA2 , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas F344 , Supresión Genética , Glándula Tiroides/patología , Tirotropina/farmacología , TransfecciónRESUMEN
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.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Genes ras , Oncogenes , Glándula Tiroides/citología , Animales , Secuencia de Bases , Línea Celular , ADN de Neoplasias , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Fosforilación , Regiones Promotoras Genéticas , Tiroglobulina/biosíntesis , Tiroglobulina/genética , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in a wide range of basic processes such as cell proliferation, development, apoptosis and stress response. It has recently been found that they are also abnormally expressed in many types of human cancer. We analyzed the genome-wide miRNA expression profile in human thyroid papillary carcinomas (PTCs) using a microarray (miRNACHIP microarray) containing hundreds of human precursor and mature miRNA oligonucleotide probes. Using this approach, we found an aberrant miRNA expression profile that clearly differentiates PTCs from normal thyroid tissues. In particular, a significant increase in miRNA (miR)-221, -222 and -181b was detected in PTCs in comparison with normal thyroid tissue. These results were further confirmed by northern blot and quantitative RT-PCR analyses. Moreover, RT-PCR revealed miR-221, -222 and -181b overexpression in fine needle aspiration biopsies corresponding to thyroid nodules, which were eventually diagnosed as papillary carcinomas after surgery. Finally, miR-221, -222 and -181b overexpression was also demonstrated in transformed rat thyroid cell lines and in mouse models of thyroid carcinogenesis. Functional studies, performed by blocking miR-221 function and by overexpressing miR-221 in human PTC-derived cell lines, suggest a critical role of miR-221 overexpression in thyroid carcinogenesis. In conclusion, these data, taken together, indicate an miRNA signature associated with PTCs, and suggest miRNA deregulation as an important event in thyroid cell transformation.
Asunto(s)
Carcinoma Papilar/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias de la Tiroides/genética , Animales , Línea Celular Tumoral , Humanos , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Activación TranscripcionalRESUMEN
Two-dimensional electrophoresis has been used to document changes in nuclear proteins following viral transformation of an epithelial cell line exhibiting differentiation markers. After transformation, these markers are lost, and the cells become tumorigenic and capable of growth in soft agar. A sharp rise in the phosphorylation of histones H1, H2A, and ubiquitinated H2A is seen on transformation, together with the appearance of three phosphorylated proteins that are extractable by perchloric acid and appear related to high mobility group Protein 14, a constituent of active chromatin. Since comparison is made between normal and transformed cells that are each grown to confluence and since there is little difference between their observed growth rates, the changes seen represent intrinsic differences between the cell lines and are thus a direct reflection of the process of transformation.
Asunto(s)
Núcleo Celular/metabolismo , Transformación Celular Viral , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Fosfoproteínas/metabolismo , Animales , Epitelio/metabolismo , Oncogenes , Ratas , Virus del Sarcoma Murino , Glándula Tiroides/citología , Glándula Tiroides/metabolismoRESUMEN
The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.
Asunto(s)
Transformación Celular Neoplásica , Proteínas Proto-Oncogénicas/metabolismo , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Factor Estimulante de Colonias/metabolismo , Enfermedades de la Tiroides/metabolismo , Glándula Tiroides/metabolismo , Línea Celular , Epitelio/metabolismo , Epitelio/patología , Expresión Génica , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-kit , Proteínas Proto-Oncogénicas c-met , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores del Factor Estimulante de Colonias/análisis , Receptores del Factor Estimulante de Colonias/biosíntesis , Valores de Referencia , Enfermedades de la Tiroides/patología , Glándula Tiroides/citología , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Células Tumorales CultivadasRESUMEN
The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.
Asunto(s)
Transformación Celular Viral , Genes MHC Clase I , Antígenos de Histocompatibilidad/fisiología , Animales , Antígenos de Neoplasias/fisiología , Línea Celular , Citometría de Flujo , Regulación de la Expresión Génica , Neoplasias Experimentales/inmunología , Oncogenes , ARN Mensajero/genética , RatasRESUMEN
The high mobility group (HMG) proteins (HMGA1a, HMGA1b, and HMGA2) bind to DNA and interact with various transcriptional factors. Therefore, they play an important role in chromatin organization. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several experimental and human tumors. Blockage of HMGA expression inhibits the transformation of rat thyroid PC Cl 3 cells treated with oncogene-carrying retroviruses, thus implicating HMGA in rat thyroid transformation. To better understand the role of HMGA and to establish whether its up-regulated expression is sufficient to induce the transformed phenotype, we generated PC Cl 3 cells that overexpress the protein. We demonstrate that HMGA1b protein overexpression does not transform normal rat thyroid PC Cl 3 cells, but it deregulates their cell cycle: cells enter S-phase earlier and the G(2)-M transition is delayed. HMGA1-overexpressing cells undergo apoptosis through a pathway involving caspase-3 activation, probably consequent to the conflict between mitogenic pressure and the inability to proceed through the cell cycle. Using various HMGA1b gene mutations, we found that the third AT-hook domain and the acetylation site K60 are the protein regions required for induction of apoptosis in PC Cl 3 cells. In conclusion, although HMGA1 protein overexpression is associated with the malignant phenotype of rat and human thyroid cells, it does not transform normal thyroid cells in culture but leads them to programmed cell death.
Asunto(s)
Apoptosis/fisiología , Proteínas del Grupo de Alta Movilidad/biosíntesis , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , División Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Ciclinas/biosíntesis , ADN Complementario/genética , Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Mapeo Peptídico , Isoformas de Proteínas , Ratas , TransfecciónRESUMEN
The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, we set out to determine the effects of Akt on thyroid cell proliferation, survival, and differentiation. To this end, we expressed constitutively active myristylated Akt (myrAkt) in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to proliferate, even in the absence of hormones, and they were resistant to programmed cell death induced by starvation. These effects were paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt did not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Glándula Tiroides/citología , Animales , Apoptosis/genética , Proteínas Portadoras/metabolismo , División Celular/genética , Línea Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica , Ciclina D3 , Ciclinas/metabolismo , Fragmentación del ADN , ADN Complementario/metabolismo , Proteína Ligando Fas , Etiquetado Corte-Fin in Situ , Glicoproteínas de Membrana/metabolismo , Fenotipo , Plásmidos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Endogámicas F344 , Transducción de Señal , Transfección , Proteína Letal Asociada a bcl , Receptor fas/metabolismoRESUMEN
A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin beta-10 gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin beta-10 gene was overexpressed in five carcinoma cell lines compared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin beta-10 gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin beta-10 gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thymosin beta-10 overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin beta-10 gene was located on chromosome 2q37 by fluorescence in situ hybridization analysis.
Asunto(s)
Carcinoma Papilar/metabolismo , Carcinoma/metabolismo , Timosina/metabolismo , Neoplasias de la Tiroides/metabolismo , Animales , Carcinoma/genética , Carcinoma Papilar/genética , Cromosomas Humanos Par 2 , Expresión Génica , Biblioteca de Genes , Humanos , Ratas , Ratas Endogámicas F344 , Timosina/genética , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Transfección , Células Tumorales CultivadasRESUMEN
The PC Cl 3 cell line is a well characterized epithelial thyroid cell line of Fischer rat origin. This cell line has the peculiarity of retaining in vitro the typical markers of thyroid differentiation (i.e. thyroglobulin synthesis and secretion, iodide uptake and dependence on TSH for growth). The PC Cl 3 cells have been transfected with the E1A gene of Adenovirus 5. The E1A transfected cells, PC E1A, partially lost the dependency on TSH for growth and completely lost the ability to trap iodide and synthesize thyroglobulin; however they did not acquire the typical markers of the neoplastic phenotype. A highly malignant phenotype was achieved after infection of the PC E1A cells with retroviruses carrying the v-raf, v-abl and polyoma virus middle T oncogenes. In contrast, the PC E1A cells transfected with the E1B gene of Adenovirus were not tumorigenic at all, and those infected with retroviruses carrying oncogenes of the ras family displayed a very weak tumorigenic phenotype.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Transformación Celular Neoplásica , Oncogenes , Glándula Tiroides/patología , Animales , Diferenciación Celular , Línea Celular , Epitelio/patología , Ratas , Ratas Endogámicas F344 , TransfecciónRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica , Inhibidores de Crecimiento/genética , Hemaglutininas/genética , Oncogenes , Proto-Oncogenes , Glándula Tiroides/fisiología , Neoplasias de la Tiroides/genética , Proteínas E1A de Adenovirus/genética , Diferenciación Celular , Línea Celular , Línea Celular Transformada , Galactósidos/metabolismo , Galectinas , Genes Virales , Genes myc , Genes ras , Genes src , Humanos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
High Mobility Group I (HMGI) proteins are nuclear proteins involved in the regulation of chromatin structure and function. Elevated expression of the HMGI proteins (HMGI, HMGY and HMGI-C) has been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells, and in several experimental carcinomas. Here, we demonstrate that HMGI and HMGY proteins are expressed in human thyroid carcinomas and thyroid carcinoma cell lines, but not in adenomas, goiters, normal thyroid tissues and cells. These results indicate a correlation between HMGI and HMGY expression and the malignant phenotype of thyroid neoplasias, suggesting that these proteins may be used as markers in thyroid cancer.
Asunto(s)
Carcinoma/genética , Proteínas del Grupo de Alta Movilidad/genética , Neoplasias de la Tiroides/genética , Adenoma/genética , Secuencia de Aminoácidos , Western Blotting , Regulación Neoplásica de la Expresión Génica , Bocio/genética , Proteína HMGA1a , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , ARN Mensajero/genéticaRESUMEN
We have recently reported the identification of a new oncogene, named PTC, frequently activated in human thyroid papillary carcinomas. This gene is a novel rearranged form of the ret proto-oncogene and we have shown that this rearrangement occurred in vivo as a tumor-specific somatic event. In an effort to further examine the role of this oncogene in human malignancies, we have investigated the expression of the ret oncogene in a number of human tumors. We consistently detected expression of normal-sized transcripts of the ret proto-oncogene in human pheochromocytomas and in human medullary thyroid carcinomas (MTC), both of familial and sporadic type. Moreover, we showed that ret mRNA levels were increased following (Bu)2cAMP-induced differentiation of a human MTC cell line (TT). Since the ret gene has been mapped on chromosome 10, close to the gene which predisposes patients to the MEN2A syndrome, we suggest that this region of chromosome 10 might be involved in the proliferative and differentiative patterns of these neuroectodermal tissues.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Proteínas de Drosophila , Feocromocitoma/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras , Neoplasias de la Tiroides/genética , Animales , Northern Blotting , Línea Celular , Transformación Celular Neoplásica , Expresión Génica , Humanos , Ratones , Proteínas Tirosina Quinasas/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , ARN/genética , ARN/aislamiento & purificación , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Valores de Referencia , Transcripción GenéticaRESUMEN
The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG-RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.
Asunto(s)
Proteínas de Drosophila , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células 3T3 , Animales , Línea Celular , Membrana Celular/enzimología , Membrana Celular/metabolismo , Transformación Celular Neoplásica , Activación Enzimática , Células Epiteliales/citología , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Glándula Tiroides/citología , TransfecciónRESUMEN
Overexpression of the high mobility group I (HMGI) proteins is often associated with the malignant phenotype. Moreover, many benign human tumors, mainly of mesenchymal origin, are characterized by rearrangements of the HMGI-C gene. In most cases, HMGI-C alterations involve breaks within the third intron of the gene resulting in aberrant transcripts carrying exons from 1-3, which encode the three DNA binding domains, fused to ectopic sequences. Here, we show that the expression of a truncated form of HMGI-C protein carrying only the three DNA-binding domains, or of a fusion protein carrying the three DNA-binding domains of HMGI-C and the LIM domains of the lipoma preferred partner gene (LPP) protein, causes malignant transformation of NIH3T3 cells. The unrearranged wild-type HMGI-C cDNA did not exert any transforming activity. These findings indicate that rearranged forms of HMGI-C play a role in cell transformation.
Asunto(s)
Transformación Celular Neoplásica , Proteínas del Grupo de Alta Movilidad/genética , Células 3T3 , Animales , División Celular , Ratones , Mutagénesis , Fenotipo , Proteínas Recombinantes de Fusión/genética , TransfecciónRESUMEN
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.