RESUMO
Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.
Assuntos
Genes ras , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Adenoma/genética , Adenoma/patologia , Substituição de Aminoácidos , Animais , Diferenciação Celular , Humanos , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias da Glândula Tireoide/patologiaRESUMO
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.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor , Núcleo Celular/metabolismo , Ciclina D3 , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Citoplasma/metabolismo , Genes Supressores de Tumor , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Especificidade por Substrato , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proteína HMGA1a , CamundongosRESUMO
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.
Assuntos
Carcinoma Papilar/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias da Glândula Tireoide/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ativação TranscricionalRESUMO
High mobility group I(Y) [HMGI(Y)] proteins are architectural factors abundantly expressed during embryogenesis, and their overexpression is known to be closely associated with neoplastic transformation of cells. This study was performed to investigate whether determination of HMGI(Y) expression level could assist in (a) differential diagnosis between colorectal carcinoma, adenoma, and normal tissue and (b) determination of the prognosis of patients with colorectal cancer. To this end, HMGI(Y) expression was determined at both the protein and mRNA levels in 30 colorectal carcinomas, 26 adenomas, and 23 normal mucosa samples, and further correlations between the protein expression levels and various clinicopathological parameters, such as depth of tumor invasion, lymphatic and/or venous involvement, regional lymph node metastasis, and Dukes' stage, were determined in 30 carcinoma cases. The expression of HMGI(Y) proteins was significantly increased in carcinoma and adenoma with severe atypia compared with that in adenoma with less atypia and normal colorectal mucosa. This increase in HMGI(Y) protein expression was found to be because of an increase in its mRNA expression by RNA in situ hybridization analysis. Clinicopathological analysis revealed that the level of HMGI(Y) protein expression was significantly correlated with parameters known to be indicative of a poor prognosis in colorectal cancer patients. These findings indicate that the determination of the HMGI(Y) protein expression level could be a potential marker for the diagnosis of colorectal neoplasias and can be of great value in predicting the prognosis of patients with colorectal cancer.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/metabolismo , Proteínas de Grupo de Alta Mobilidade/biossíntese , Fatores de Transcrição/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Proteína HMGA1a , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.
Assuntos
Adenoma/genética , Cloranfenicol O-Acetiltransferase/genética , Genes ras , Camundongos Transgênicos/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/enzimologia , Adenoma/patologia , Amitrol (Herbicida)/farmacologia , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/análise , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Percloratos/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Compostos de Sódio/farmacologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/enzimologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
The high mobility group I (HMGI) family of proteins in mammals belongs to a group of nonhistone nuclear proteins known as architectural transcriptional factors. They function in vivo as both structural components of chromatin and auxiliary gene transcription factors. In an earlier study (N. Abe et al, Cancer Res., 59: 1169-1174, 1999), we demonstrated that the expression level of the HMGI(Y) gene/proteins was significantly increased in colorectal adenocarcinoma and colorectal adenoma with severe cellular atypia. In the current study, we analyzed HMGI(Y) expression in several human pancreatic lesions to investigate (a) whether HMGI(Y) overexpression is also observed in pancreatic carcinoma, and (b) the role of HMGI(Y) in the diagnosis of pancreatic neoplasms. To this end, HMGI(Y) expression was determined at the protein level by immunohistochemistry using a HMGI(Y)-specific antibody in 6 surgically resected specimens of nonneoplastic tissue (4 specimens of normal pancreatic tissue and 2 specimens of chronic pancreatitis tissue), 8 pancreatic cystic neoplasms (5 intraductal papillary mucinous adenomas, 1 serous cystadenoma, and 2 solid pseudopapillary tumors), and 15 duct cell carcinomas of the pancreas. Immunohistochemical analysis revealed intense nuclear staining in the pancreatic carcinoma cells, whereas only very faint nuclear staining was seen in the nonneoplastic cells. There was a strong correlation between HMGI(Y) protein overexpression and a diagnosis of carcinoma (P = 0.000018). Thus, an increased expression level of the HMGI(Y) proteins was clearly associated with the malignant phenotype in pancreatic tissue. In addition, a low level of protein expression was also apparent in two of the cystic neoplasms that exhibited cellular atypia, but not in those that did not exhibit cellular atypia. Based on these findings, we propose that the HMGI(Y) proteins could be closely associated with tumorigenesis in the pancreas and that HMGI(Y) could serve as a potential diagnostic molecular marker for distinguishing pancreatic malignancies unambiguously from normal tissue or benign lesions.
Assuntos
Carcinoma/metabolismo , Proteínas de Grupo de Alta Mobilidade/biossíntese , Ductos Pancreáticos , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/biossíntese , Cistadenoma Seroso/metabolismo , Proteína HMGA1a , Humanos , Imuno-Histoquímica , Neoplasias Pancreáticas/diagnóstico , Pancreatite/metabolismo , Papiloma/metabolismo , FenótipoRESUMO
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.
Assuntos
Neoplasias Colorretais/metabolismo , Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/biossíntese , Sequência de Aminoácidos , Animais , Anticorpos , Linhagem Celular , Colo/citologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Neoplasias Colorretais/patologia , Epitélio/metabolismo , Feminino , Fibroblastos/metabolismo , Proteína HMGA1a , Proteínas de Grupo de Alta Mobilidade/análise , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Ratos , Valores de Referência , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
Hyperplastic or neoplastic proliferative lesions of thyroid follicular epithelium consist of a spectrum, ranging from nodular hyperplasia to undifferentiated (anaplastic) carcinoma, and usually present as palpable thyroid nodules. Thyroid nodules are a common occurrence in the general population, but only a small proportion of them are eventually diagnosed as carcinoma. The difficulty in objectively identifying those thyroid nodules that are malignant to avoid unnecessary surgery, combined with the range and effectiveness of the available therapeutic options in those patients who do, indeed, have thyroid carcinoma, has prompted the search for tumor markers and prognostic indicators. The high mobility group I (HMGI) proteins represent a class of nuclear proteins involved in the regulation of chromatin structure and function. HMGI(Y), one of the members of this class, is expressed at high levels during embryogenesis and in malignant tumors but at generally low levels in normal adult human tissues. Previous work on a limited number of thyroid samples suggested that the detection of the HMGI(Y) proteins may provide a clinically useful diagnostic tool. To verify this assumption, we analyzed HMGI(Y) expression by a combination of immunohistochemistry and reverse transcription-PCR in 358 thyroid tissue samples that were representative of the spectrum of thyroid tumor pathology. HMGI(Y) was detectable in 18 of 19 follicular carcinomas, 92 of 96 papillary carcinomas, and 11 of 11 undifferentiated (anaplastic) carcinomas but in only 1 of 20 hyperplastic nodules, 44 of 200 follicular adenomas, and 0 of 12 normal tissue samples. The correlation between HMGI(Y) expression and a diagnosis of carcinoma was highly significant (P < 0.0001). We also prospectively collected and analyzed for HMGI(Y) expression by immunohistochemistry and reverse transcription-PCR in 12 fine needle aspiration biopsies from 10 patients who subsequently underwent surgical removal of a solitary thyroid nodule. HMGI(Y) was detectable only in the four fine needle aspiration biopsies, corresponding to the thyroid nodules that were definitively diagnosed as carcinomas after surgery (two follicular carcinomas and two papillary carcinomas). The remaining eight samples (six follicular adenomas and two samples consisting of normal follicular cells) were negative. The findings of this study confirm the differential expression of HMGI(Y) in thyroid neoplasia and indicate the HMGI(Y) protein as a potential marker for thyroid carcinoma.
Assuntos
Adenocarcinoma Folicular/química , Adenoma/química , Carcinoma/química , Proteínas de Grupo de Alta Mobilidade/análise , Proteínas de Neoplasias/análise , Neoplasias da Glândula Tireoide/química , Adenocarcinoma Folicular/diagnóstico , Adenoma/diagnóstico , Adulto , Biópsia por Agulha , Carcinoma/diagnóstico , Carcinoma Papilar/química , Carcinoma Papilar/diagnóstico , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Estudos Prospectivos , Neoplasias da Glândula Tireoide/diagnósticoRESUMO
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.
Assuntos
Carcinoma/genética , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos , Genes Supressores de Tumor , Proteínas Associadas aos Microtúbulos/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Proteínas Serina-Treonina Quinases , Neoplasias da Glândula Tireoide/genética , Proteínas Supressoras de Tumor , Northern Blotting , Western Blotting , Carcinoma/metabolismo , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27 , Deleção de Genes , Expressão Gênica , Humanos , Mutagênese , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fase S , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
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.
Assuntos
Carcinoma Papilar/genética , Proteínas de Drosophila , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-retRESUMO
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.
Assuntos
Diferenciação Celular/genética , Genes p53/genética , Mutação/genética , Proteínas Nucleares , Glândula Tireoide/citologia , Sequência de Aminoácidos , Animais , Divisão Celular/genética , Linhagem Celular , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Expressão Gênica , Humanos , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/genética , Camundongos , Dados de Sequência Molecular , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Receptores da Tireotropina/biossíntese , Receptores da Tireotropina/genética , Tireoglobulina/biossíntese , Tireoglobulina/genética , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiologia , Transativadores/biossíntese , Transativadores/genética , TransfecçãoRESUMO
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.
Assuntos
Carcinoma/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Western Blotting , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Bócio/genética , Bócio/metabolismo , Humanos , Técnicas Imunoenzimáticas , Fator de Crescimento Placentário , Testes de Precipitina , Proteínas da Gravidez/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Neoplasias da Glândula Tireoide/genética , Células Tumorais Cultivadas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
RET/PTC1 is a chimeric oncogene created by the fusion of the tyrosine kinase domain of RET to the 5'-terminal region of another gene named H4. So far, this oncogene has been found activated only in human papillary thyroid carcinomas. In order to investigate its transforming properties in vivo, we have produced transgenic mice carrying RET/PTC1 under the control of the H4 promoter. The transgene was expressed in several tissues, consistently with the ubiquitous expression of the wild type H4 gene. Mammary adenocarcinomas and, less frequently, hyperplasia of sebaceous glands and rare benign skin tumors, named pilomatrixomas, developed in these mice. The tumors were shown to express the transgene both at the RNA and protein level. These results demonstrate that the transforming ability of the RET/PTC1 oncogene is not restricted to the thyroid epithelium in vivo. Despite its ubiquitous expression, however, RET/PTC1 was able to induce only a limited number of tumor types; specifically mammary epithelium was affected by transgene expression, thus suggesting that RET/PTC1 is able to couple with transforming pathways specific for these glandular cells.
Assuntos
Proteínas de Drosophila , Neoplasias Mamárias Animais/genética , Camundongos Transgênicos/genética , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores Etários , Animais , Carcinoma/genética , Carcinoma/patologia , Transformação Celular Neoplásica/genética , Proteínas do Citoesqueleto , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-ret , Proteínas Recombinantes de Fusão/genética , Glândulas Sebáceas/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Distribuição TecidualRESUMO
The HMGI protein family includes three proteins, named HMG-I, HMG-Y and HMGI-C. The first two proteins are coded for by the same gene, HMGI (Y), through an alternative splicing mechanism. Their expression is elevated in neoplastic tissues and cells and this overexpression has a causal role in the process of cellular neoplastic transformation. We demonstrate that the HMGI (Y) gene is expressed at very low levels in normal adult tissues, whereas in embryonic tissues it is expressed at high levels comparable to those detected in neoplastic tissues. Specifically, a very high expression of the HMGI (Y) gene was detected in all embryonic tissues at 8.5 dpc. Then in the following days, even though the gene is expressed essentially in all tissues, an abundant gene expression was restricted to some tissues. These results indicate an important role of the HMGI (Y) gene in development.
Assuntos
Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/metabolismo , RNA Mensageiro/metabolismo , Adulto , Animais , Idade Gestacional , Proteína HMGA1a , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Carcinoma/genética , Proteínas de Grupo de Alta Mobilidade/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/genética , Sequência de Aminoácidos , Western Blotting , Regulação Neoplásica da Expressão Gênica , Bócio/genética , Proteína HMGA1a , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Bócio/fisiopatologia , Linfocinas/metabolismo , Proteínas da Gravidez/metabolismo , Tiouracila/farmacologia , Tireotropina/metabolismo , Animais , Antitireóideos/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Doença de Graves/metabolismo , Humanos , Linfocinas/efeitos dos fármacos , Linfocinas/genética , Linfocinas/farmacologia , Neovascularização Patológica , Fator de Crescimento Placentário , Proteínas da Gravidez/efeitos dos fármacos , Proteínas da Gravidez/genética , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro , Ratos , Ratos Endogâmicos , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/efeitos dos fármacos , Receptores de Fatores de Crescimento/imunologia , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Veias Umbilicais/citologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Gene rearrangements activating the RET proto-oncogene are frequently associated with human thyroid carcinomas belonging to the papillary subtype. These arrangements cause the fusion of the tyrosine-kinase domain of RET to the 5'-terminal region of different genes creating the RET/PTC chimeric oncogenes. Here we report the generation of transgenic mice lines expressing the RET/PTC1 oncogene under the control of the thyroid-specific rat thyroglobulin promoter. RET/PTC1-transgenic mice developed thyroid tumors displaying the histological aspect of papillary carcinomas. These tumors were slowly progressive and did not cause premature death of the animals. Two additional mice developed areas of thyroid hyperplasia. Immunohistochemical and reverse-transcriptase polymerase chain reaction analyses confirmed the thyroid-specific expression of the transgene. Given the frequency of activating rearrangements of RET in human papillary thyroid carcinomas we conclude that this animal system could be a good model for studying the neoplastic progression of thyroid carcinomas.
Assuntos
Carcinoma Papilar/genética , Proteínas de Drosophila , Fosfoproteínas Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas de Saccharomyces cerevisiae , Neoplasias da Glândula Tireoide/genética , Animais , Sequência de Bases , Carcinoma Papilar/patologia , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Ratos , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Germinoma/irrigação sanguínea , Linfocinas/biossíntese , Neovascularização Patológica/metabolismo , Proteínas da Gravidez/biossíntese , Neoplasias Testiculares/irrigação sanguínea , Sequência de Bases , Western Blotting , Carcinoma Embrionário/irrigação sanguínea , Carcinoma Embrionário/metabolismo , Hipóxia Celular , Coriocarcinoma/irrigação sanguínea , Coriocarcinoma/metabolismo , Fatores de Crescimento Endotelial/genética , Germinoma/metabolismo , Humanos , Isomerismo , Linfocinas/genética , Masculino , Dados de Sequência Molecular , Fator de Crescimento Placentário , Reação em Cadeia da Polimerase , Proteínas da Gravidez/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Teratocarcinoma/irrigação sanguínea , Teratocarcinoma/metabolismo , Neoplasias Testiculares/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have recently reported that neoplastic transformation of two rat thyroid epithelial cell lines by retroviruses carrying the v-mos and v-ras Ki oncogenes is associated with a drastic increase of AP-1 activity. The most important effects were represented by the dramatic junB and fra-1 gene induction, which was abolished by the block of the transformation-induced HMGI-C protein synthesis. Here, we have further characterized the transformation-dependent AP-1 activity, by analysing the expression of different jun- and fos-related components, in rat thyroid cell lines transformed by several oncogenes, in human thyroid carcinoma cell lines, and in naturally occurring human thyroid tumours. A significant increase of Fra-1 and JunB protein levels was detected in all oncogene transformed rat thyroid cell lines. Fra-1 gene induction was demonstrated to occur also in human thyroid carcinoma cell lines and tissues. Conversely, c-Jun and JunD proteins, rather than JunB, accumulated in human thyroid carcinoma cell lines. An induction of AP-1 target genes was also detected both in rat and human thyroid transformed cell lines. Therefore, in vivo and in vitro thyroid cell transformation is associated with important compositional changes in the AP-1 complex and an increased transcriptional activity.