RESUMEN
The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteínas Supresoras de Tumor , Núcleo Celular/metabolismo , Ciclina D3 , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Citoplasma/metabolismo , Genes Supresores de Tumor , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Especificidad por Sustrato , Neoplasias de la Tiroides/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The down regulation of protein p27(kip1) (p27) in most cases of thyroid cancer has relevant diagnostic and prognostic implications. However, the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma expresses more p27 than benign oxyphilic lesions do. AIM: To evaluate the mechanism underlying this difference in expression of p27. METHODS: Because high levels of cyclin D3 lead to p27 accumulation in cell lines and clinical samples of thyroid cancer, the immunocytochemical pattern of cyclin D3 in oxyphilic (n = 47) and non-oxyphilic (n = 70) thyroid neoplasms was investigated. RESULTS: In the whole study sample, there was a significant correlation between p27 and cyclin D3 expression (Spearman's r: 0.64; p<0.001). The expression of cyclin D3 and p27 was significantly higher in the oxyphilic variant of follicular carcinomas than in non-oxyphilic carcinomas (p<0.001). In the former, cyclin D3 overexpression and p27 accumulation were observed in a median of 75% and 55% of cells, respectively. In co-immunoprecipitation experiments, the level of p27-bound cyclin D3 was much higher in oxyphilic neoplasias than in normal thyroids and other thyroid tumours. CONCLUSION: These results show that increased p27 expression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma results from cyclin D3 overexpression.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Neoplasias de la Tiroides/metabolismo , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patología , Adenoma/metabolismo , Adenoma/patología , Adenoma Oxifílico/metabolismo , Adenoma Oxifílico/patología , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Ciclina D3 , Humanos , Inmunoprecipitación , Proteínas de Neoplasias/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.
Asunto(s)
Proteínas de Ciclo Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Glándula Tiroides/citología , Proteínas Supresoras de Tumor , Animales , Northern Blotting , Western Blotting , Línea Celular , Inhibición de Contacto , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Citometría de Flujo , Genes mos/genética , Humanos , Ratones , Microscopía de Contraste de Fase , Proteínas Asociadas a Microtúbulos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oligonucleótidos Antisentido/genética , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Fenotipo , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Tirosina Fosfatasas/genética , Ratas , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Retroviridae/genética , Retroviridae/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/patología , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/genética , TransfecciónRESUMEN
The high-mobility group I (HMGI) nonhistone chromosomal proteins HMGI(Y) and HMGI-C have been implicated in defining chromatin structure and in regulating the transcription of several genes. These proteins have been implicated in adipocyte homeostasis: a severe deficiency of fat tissue is found in mice with targeted disruption of the HMGI-C locus, and lipomagenesis in humans is frequently associated with somatic mutations of HMGI genes. The aim of this study was to examine the role of HMGI(Y) proteins in adipocytic cell growth and differentiation. First, we found that differentiation of the preadipocytic 3T3-L1 cell line caused early induction of HMGI(Y) gene expression. Suppression of HMGI(Y) expression by antisense technology dramatically increased the growth rate and impaired adipocytic differentiation in these cells. The process of adipogenic differentiation involves the interplay of several transcription factors, among which is the CCAAT/enhancer-binding protein (C/EBP) family of proteins. These factors are required for the transcriptional activation of adipocyte-specific genes. We also tested the hypothesis that HMGI(Y) might participate in transcriptional control of adipocyte-specific promoters. We found that HMGI(Y) proteins bind C/EBPbeta in vivo and in vitro. Furthermore, we show that HMGI(Y) strongly potentiates the capacity of C/EBPbeta to transactivate the leptin promoter, an adipose-specific promoter. Taken together, these results indicate that the HMGI(Y) proteins play a critical role in adipocytic cell growth and differentiation.
Asunto(s)
Adipocitos/citología , Adipocitos/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Proteína HMGA1a , RatonesRESUMEN
BACKGROUND: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. METHODS: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. RESULTS: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. CONCLUSIONS: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.
Asunto(s)
Apoferritinas/metabolismo , Quimiocina CXCL12/metabolismo , Receptores CXCR4/metabolismo , Apoferritinas/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Transición Epitelial-Mesenquimal , Femenino , Silenciador del Gen , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células MCF-7 , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
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
The proteins of the Ets family are transcription factors involved in signal transduction, cell cycle progression, and differentiation. In this study, we report that thyroid cell neoplastic transformation is associated with a dramatic increase in ETS transcriptional activity, which is dependent on the accumulation of Ets-1, Ets-2, and other Ets-related proteins. Inhibition of ETS transactivation activity by the Ets-dominant negative construct (Ets-Z) induced programmed cell death in human thyroid carcinoma cell lines but not in normal thyroid cells. Apoptotic cell death induced by Ets-Z was dependent on the reduction of c-MYC protein levels, because it was prevented by overexpression of c-myc. Taken together, these data indicate that the induction of Ets-1 and Ets-2 transcription factors plays a pivotal role in thyroid cell neoplastic transformation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Represoras , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Transactivadores/biosíntesis , Factores de Transcripción/biosíntesis , Apoptosis/fisiología , Secuencia de Consenso , ADN/metabolismo , Genes myc/genética , Humanos , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ets , Glándula Tiroides/patología , Neoplasias de la Tiroides/patología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Células Tumorales CultivadasRESUMEN
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
A correlation has previously been demonstrated between the presence of the three HMGI proteins (HMGI, HMGY, and HMGI-C) and the expression of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells; this being subsequently extended to experimental thyroid, lung, prostate, mammary, and skin carcinomas. Recently, we have demonstrated that expression of HMGI and HMGY proteins, coded for by the HMGI(Y) gene, is associated with the malignant phenotype of human thyroid neoplasias. Here, we show that HMGI(Y) gene expression is present both at the RNA and protein level in human colorectal carcinoma cell lines and tissues examined in this study. Conversely, no HMGI(Y) proteins were detected in normal intestinal mucosa. Therefore, these results suggest an involvement of HMGI and HMGY proteins overexpression in colorectal tumorigenesis.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Expresión Génica , Proteínas del Grupo de Alta Movilidad/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos , Línea Celular , Colon/citología , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Neoplasias Colorrectales/patología , Epitelio/metabolismo , Femenino , Fibroblastos/metabolismo , Proteína HMGA1a , Proteínas del Grupo de Alta Movilidad/análisis , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Ratas , Valores de Referencia , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Células Tumorales CultivadasRESUMEN
The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced both at the mRNA and at the protein level - in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.
Asunto(s)
Carcinoma/genética , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos , Genes Supresores de Tumor , Proteínas Asociadas a Microtúbulos/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Serina-Treonina Quinasas , Neoplasias de la Tiroides/genética , Proteínas Supresoras de Tumor , Northern Blotting , Western Blotting , Carcinoma/metabolismo , División Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Eliminación de Gen , Expresión Génica , Humanos , Mutagénesis , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Fase S , Neoplasias de la Tiroides/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.
Asunto(s)
Cromosomas Humanos Par 14 , Sustancias de Crecimiento/genética , Neovascularización Patológica , Placenta/fisiología , Proteínas Gestacionales/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Expresión Génica , Genes , Humanos , Intrones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Factor de Crecimiento Placentario , ARN Mensajero/genética , Especificidad de la EspecieRESUMEN
The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Bromodesoxiuridina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Catálisis , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/enzimología , ADN Complementario/metabolismo , Citometría de Flujo , Biblioteca de Genes , Proteína HMGA1a , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Modelos Genéticos , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
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
Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.
Asunto(s)
Carcinoma/patología , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , ARN sin Sentido/genética , Neoplasias de la Tiroides/patología , Animales , Pruebas de Carcinogenicidad , Carcinoma/genética , Carcinoma/metabolismo , División Celular/genética , Factores de Crecimiento Endotelial/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Linfocinas/metabolismo , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Neovascularización Patológica , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
p53 is the gene most frequently found mutated in human neoplasias. In the majority of tumors, p53 mutations contribute to the progression towards stages of increasing malignancy with the appearance of an undifferentiated phenotype. Also in thyroid cancerogenesis, p53 mutations correlate with the loss of the differentiated phenotype. The results presented here, suggest a direct involvement of p53 in the molecular mechanisms regulating cellular differentiation in thyroid since a mutated p53 gene markedly affects the growth potential and differentiated functions of the rat thyroid cell line PC Cl 3. Blockage in the expression of the PAX-8 transcription factor seems to be a key event in the loss of thyroid differentiated functions induced by the mutated p53 gene. Thyroid cells carrying a mutated p53 gene did not form colonies in soft agar or tumors in athymic mice, suggesting that a mutation of the p53 gene is not sufficient for the induction of the malignant phenotype and probably a cooperation with other oncogenes is necessary to accomplish full malignancy. No effect on either growth or differentiation of thyroid cells was exerted either by overexpression of the wild-type p53 gene, or by the vector alone.
Asunto(s)
Diferenciación Celular/genética , Genes p53/genética , Mutación/genética , Proteínas Nucleares , Glándula Tiroides/citología , Secuencia de Aminoácidos , Animales , División Celular/genética , Línea Celular , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Expresión Génica , Humanos , Yoduro Peroxidasa/biosíntesis , Yoduro Peroxidasa/genética , Ratones , Datos de Secuencia Molecular , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Receptores de Tirotropina/biosíntesis , Receptores de Tirotropina/genética , Tiroglobulina/biosíntesis , Tiroglobulina/genética , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiología , Transactivadores/biosíntesis , Transactivadores/genética , TransfecciónRESUMEN
Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells in vitro, promotes neoangiogenesis in vivo and increases the permeability of the vascular endothelium. VEGF overexpression occurs in several cultured tumor cell lines and in certain human malignancies. Placenta growth factor (PlGF) is a recently identified growth factor for endothelial cells (EC); PlGF strongly potentiates both the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. To uncover the molecular mechanisms underlying neoangiogenesis in human thyroid tumors, we have analysed VEGF and PlGF expression in a panel of thyroid carcinoma cell lines with different tumorigenic potential as well as in human primary thyroid tumors. We show that a high tumorigenic potential is associated with an elevated VEGF expression in human thyroid tumor cell lines. Furthermore, VEGF overexpression occurs in 5/5 highly malignant anaplastic carcinomas. Papillary and follicular carcinomas express intermediate levels of VEGF mRNA. In contrast, PlGF expression is severely down regulated in the majority of thyroid tumor cell lines and in tumors. Furthermore, we show that both the VEGF receptors, FLT-1 and flk/KDR, are expressed in endothelial cells that line tumor-embedded microvascular vessels, suggesting that VEGF but not PlGF, contributes to thyroid tumor development.
Asunto(s)
Carcinoma/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Linfocinas/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Neoplasias de la Tiroides/metabolismo , Western Blotting , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Bocio/genética , Bocio/metabolismo , Humanos , Técnicas para Inmunoenzimas , Factor de Crecimiento Placentario , Pruebas de Precipitina , Proteínas Gestacionales/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Neoplasias de la Tiroides/genética , Células Tumorales Cultivadas , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
RET/PTC oncogene activation occurs in about 20% of human thyroid papillary carcinomas. However, it is not known yet whether it is an early or late event in the process of thyroid carcinogenesis. Here we demonstrate, by using a combined immunohistochemical and reverse transcriptase-polymerase chain reaction based approach, that RET/PTC activation is present in 11 out of 26 occult thyroid papillary carcinomas analysed. Therefore, we conclude that it represents an early event in the process of thyroid cell transformation.
Asunto(s)
Carcinoma Papilar/genética , Proteínas de Drosophila , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-retRESUMEN
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.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Bocio/fisiopatología , Linfocinas/metabolismo , Proteínas Gestacionales/metabolismo , Tiouracilo/farmacología , Tirotropina/metabolismo , Animales , Antitiroideos/farmacología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Enfermedad de Graves/metabolismo , Humanos , Linfocinas/efectos de los fármacos , Linfocinas/genética , Linfocinas/farmacología , Neovascularización Patológica , Factor de Crecimiento Placentario , Proteínas Gestacionales/efectos de los fármacos , Proteínas Gestacionales/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero , Ratas , Ratas Endogámicas , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/efectos de los fármacos , Receptores de Factores de Crecimiento/inmunología , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Venas Umbilicales/citología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.