RESUMO
Transforming growth factor beta (TGF-beta) is a physiological regulator of thyroid epithelial cell growth and differentiation. This factor signals through a heteromeric complex composed of type I (TGF-beta receptor type I) and type II [TGF-beta receptor type II (TbetaRII)] receptors. Loss of TbetaRII expression has been related to resistance to TGF-beta inhibition of cell proliferation. In the present work, we analyzed the TbetaRII expression in a series of human thyroid tumors, from benign lesions (adenomas) to neoplastic lesions of increasing aggressiveness (papillary and follicular carcinomas) up to the extremely aggressive anaplastic tumors. Results obtained indicated a clear reduced expression of TbetaRII mRNA only in the group of thyroid carcinomas when compared with their relative normal tissues. Immunohistochemical analyses with specific anti-TbetaRII antibodies confirm these observations. These data indicate that loss of expression of TbetaRII can contribute to thyroid cancer progression, inducing cancer cells to escape the growth-inhibitory effect of TGF-beta.
Assuntos
Adenocarcinoma Folicular/metabolismo , Carcinoma Papilar/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/ultraestrutura , Adulto , Idoso , Northern Blotting , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The development of cancer is due to the accumulation of multiple somatic mutations, in some cases following germline mutations, which occur in hereditary malignancies such as retinoblastomas or multiple endocrine neoplasia (MEN 2A and B). Genetic alterations or changes in the expression of growth regulatory genes can lead to the initiation of malignant transformation and to eventual tumor progression. Cells that have undergone these cumulative alterations in either the structure or expression of these regulatory genes generally possess a selective growth and/or metastatic advantage over other normal non-transformed cells. Thus, activation of dominantly transforming oncogenes by point mutations, gene amplification, chromosomal translocation or insertional mutagenesis can lead to uncontrolled cellular growth or to a disruption in normal differentiation or apoptosis. Equally contributory to the process of malignant progression is the inactivation of recessive tumor suppressor genes due to point mutations and/or loss of heterozygosity in one allele, which can ultimately lead to a reduction of homozygosity in both alleles. Thyroid tumors in humans represent a particularly suitable multistage model of epithelial tumorigenesis. In fact, even though most thyroid neoplasms originate from a single cell type, i.e. the thyroid follicular cell, they include a broad spectrum of tumors with different phenotypic characteristics and variable biological and clinical behaviour. Multiple degrees of malignancies have been defined: from the benign colloid adenomas through the slowly progressive differentiated papillary and follicular carcinomas to the invariably fatal anaplastic carcinomas, although these histological changes are not necessarily sequential. In this review an effort has been made to summarize and integrate new data published on genetic lesions and altered expression of genes involved in the tumorigenesis of the follicular type of thyroid cancer. We have focused our interest only on gene alterations inducing gain or loss of function, that have been studied in vivo in human thyroid tumor specimens by the use of different techniques, such as PCR mediated DNA analyses, sequencing, mRNA level evaluation and protein expression by immunohistochemical staining.
Assuntos
Genes Supressores de Tumor , Oncogenes , Neoplasias da Glândula Tireoide/genética , HumanosAssuntos
Antígenos CD/genética , Antígenos CD55/genética , Técnicas de Transferência de Genes , Espermatozoides , Animais , Animais Geneticamente Modificados , Antígenos CD/biossíntese , Blastocisto/fisiologia , Antígenos CD55/biossíntese , Embrião de Mamíferos , Feminino , Humanos , Inseminação Artificial , Masculino , Mórula/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Suínos , Transplante HeterólogoAssuntos
Antígenos CD55/genética , Técnicas de Transferência de Genes , Miocárdio/imunologia , Espermatozoides , Animais , Animais Geneticamente Modificados , Antígenos CD/análise , Antígenos CD/genética , Antígenos CD55/análise , Humanos , Imuno-Histoquímica , Masculino , Miocárdio/citologia , Suínos , Transplante Heterólogo/métodosAssuntos
Antígenos CD55/genética , Técnicas de Transferência de Genes , Transcrição Gênica , Transplante Heterólogo/imunologia , Animais , Animais Geneticamente Modificados , Antígenos CD55/fisiologia , Feminino , Terapia Genética , Humanos , Inseminação Artificial/veterinária , Masculino , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Gravidez , Regiões Promotoras Genéticas , EspermatozoidesRESUMO
OBJECTIVE: To analyze the role of the transforming growth factor (TGF)-beta pathway in renal tumors and to verify whether alterations in TGF-beta 1 pathway expression are associated with the grade of tumor differentiation and pathologic stage in renal cell carcinomas. STUDY DESIGN: The expression of TGF-beta 1 and TGF-beta receptors (T beta RI and T beta RII), SMAD-2 and SMAD-4 was investigated by immunohistochemistry in normal peritumoral and tumoral tissue from 53 renal cell carcinomas (clear cell type). The gene expression of SMAD-2 and SMAD-4 was also studied by reverse transcription polymerase chain reaction (RT-PCR) in normal peritumoral and tumoral tissue from 6 of 56 primary tumors. RESULTS: TGF-beta 1, T beta RI and T beta RII immunoreactivity was more frequent in tumoral than in normal peritumoral renal tissue (96.22%, 79.25% and 75.41% vs. 88.37%, 69.76% and 62.69%), whereas SMAD-2 and SMAD-4 immunoreactivity was more frequent in normal peritumoral than in tumoral tissue (23.25% and 30.23% vs. 15.09% and 7.54%). In tumor areas, immunohistochemical scores were lower for T beta RII than for T beta RI and TGF-beta 1 and higher than SMAD-4 and SMAD-2 scores. TGF-beta 1, T beta RI, T beta RII and SMAD-4 histologic scores correlated with neither the histologic grade of malignancy nor TNM clinical stage, whereas SMAD-2 protein levels were significantly lower in grade 3 than in grade 1 tumors. In the samples of normal kidney and carcinoma studied, RT-PCR detected the correct transcripts for SMAD-2 and SMAD-4, indicating that the RNA of the samples analyzed contained RNA sequences coding for these genes. CONCLUSION: Our data support the concept that the reduction of T beta RII and SMAD proteins in renal cell carcinomas is involved in tumor development and suggest an altered TGF-beta/SMAD signaling pathway in kidney neoplasia.
Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Rim/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/genética , Proteína Smad2 , Proteína Smad4 , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Protein phosphorylation plays an essential role in regulating many cellular processes in eukaryotes. Signal transduction mechanisms that are reversibly controlled by protein phosphorylation require also protein phosphatases (PPs). Okadaic acid (OA), which is a potent inhibitor of protein phosphatase 2A (PP2A) and protein phosphatase 1, elicits phosphorylation of many proteins in unstimulated cells and induces different cellular responses, including transcriptional activation, shape changes, and pseudomitotic state. In this study, the effects of OA on rat thyroid cells (FRTL-5 strain) were analyzed to evaluate the role of serine/threonine phosphatases in hormone-induced thyroid cell proliferation. OA at a concentration range between 0.1 and 1 nM stimulated thyroid cell growth. Furthermore, 0.25 nM OA increased about 3.5-fold the thyrotropin (TSH)-induced DNA synthesis in quiescent cells. OA treatment also stimulated cell proliferation induced by drugs that mimic TSH effect, such as 8Br-cAMP and cholera toxin, suggesting that PP2A activity was relevant in the cAMP pathway activated by the hormone. Flow cytometry experiments showed that OA significantly increased the fraction of TSH-stimulated quiescent cells entering the S phase. In order to define the mechanisms underlying the observed stimulatory effect of OA on thyroid cell growth, expression of genes relevant in the G1-S phase transition was evaluated. A 2-fold increase in the level of cyclin D1 mRNA expression was found by Northern blot analysis in OA-treated cells. Although cdk2 gene expression was not modulated by the same OA treatment, an increase in Cdk2 protein was revealed by immunoprecipitation experiments. Moreover, OA modifies the phosphorylation pattern of the tumor suppressor retinoblastoma protein, a key event in the G1-S phase transition. Therefore, these experiments reveal that PP2A phosphatases play an important role in thyroid cell growth and can act at multiple sites in the TSH pathways driving cells to S phase.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Fase G1/efeitos dos fármacos , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fase S/efeitos dos fármacos , Glândula Tireoide/citologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Toxina da Cólera/farmacologia , Ciclina D1 , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Proteínas Oncogênicas/genética , Fosforilação , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/análise , Ratos , Proteína do Retinoblastoma/metabolismo , Tireotropina/farmacologiaRESUMO
The EGF-like family of proteins, such as epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), amphiregulin (AR), betacellulin (BTC), cripto-1 (CR-1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL-5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL-5H2 cell line, overexpressing erbB-2, Kimol cells, transformed by the wild-type K-ras and A6 clone, transformed by a temperature sensitive K-ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF-like growth factors and corresponding receptors (erbB-2, erbB-3, and erbB-4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL-5 cells, both K-ras and erbB-2-transformed cells expressed elevated levels of erbB-2 receptor. Moreover, both K-ras transformed cells, Kimol and A6 cells, but no FRTL-5H2 cells, were found able to express also high levels of erbB-4 receptor and HRG/NDF ligand. Treatment of K-ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB-2 overexpressing FRTL-5 cells, in K-ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB-2/erbB-4 receptors in an autocrine fashion.
Assuntos
Comunicação Autócrina/fisiologia , Proteínas de Transporte/genética , Fator de Crescimento Epidérmico , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Glicoproteínas de Membrana , Neuregulina-1 , Receptor ErbB-2/genética , Glândula Tireoide/citologia , Proteínas ras/genética , Anfirregulina , Animais , Anticorpos/farmacologia , Antineoplásicos/metabolismo , Betacelulina , Ligação Competitiva/imunologia , Neoplasias da Mama , Proteínas de Transporte/imunologia , Linhagem Celular Transformada/química , Linhagem Celular Transformada/fisiologia , Neoplasias do Colo , Família de Proteínas EGF , Células Epiteliais/química , Células Epiteliais/fisiologia , Receptores ErbB/genética , Proteínas Ligadas por GPI , Expressão Gênica/fisiologia , Glicoproteínas/imunologia , Substâncias de Crescimento/genética , Humanos , Proteínas de Neoplasias/genética , Testes de Neutralização , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Ratos , Receptor ErbB-3 , Receptor ErbB-4 , Células Tumorais CultivadasRESUMO
Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K-ras-transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin-fixed paraffin-embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis.
Assuntos
Ciclina D1/genética , Ciclina E/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Ciclina D1/análise , Ciclina E/análise , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , RNA Mensageiro/análise , Ratos , Tireotropina/farmacologiaRESUMO
Transforming growth factor beta 1 (TGF beta 1) inhibits the growth of normal rat epithelial thyroid cells (FRTL-5 strain) by counteracting thyrotropin (TSH)-stimulated DNA synthesis and by slowing the cells in the G1 phase of the cell cycle. Here, we have studied two clones of FRTL-5 thyroid cell line transformed by the wild type (wt) v-k-ras oncogene (K.M.A1, K.M.A2) and one clone (A6) transformed by a temperature-sensitive (ts) v-k-ras mutant. Anchorage-dependent as well as anchorage-independent growth of these k-ras-transformed cells was not inhibited by TGF beta 1. TGF beta 1 resistance appeared to be dependent by a functional p21 k-ras, because A6 cell growth was partially inhibited at the nonpermissive temperature (39 degrees C). To determine the basis for TGF beta 1 resistance in k-ras-transformed thyroid cells, we looked for possible defects in the expression of type I (T beta R-I/ALK5) and type II TGF beta receptors (T beta R-II). Lower levels of type II receptors were present in all of the k-ras-transformed clones, as revealed by both Northern blot and cross-linking experiments. A partial reversion of the malignant phenotype of the wt k-ras-transformed clone was obtained in two clones isolated after transfection of the malignant thyroid cells (K.M.A1) with a T beta R-II expression vector. These two clones also showed restored levels of exogenous T beta R-II mRNA and protein, and both clones showed a partially reacquired sensitivity to TGF beta 1. Similarly, the reversion of the malignant phenotype of the A6 clone grown at the nonpermissive temperature was accompanied by a restored expression of the T beta R-II receptors. These data indicate that active k-ras oncogene can induce TGF beta 1 resistance in rat thyroid cells and suggest that one of the possible mechanisms of escape from TGF beta 1 growth control in k-ras-induced thyroid carcinogenesis involves a reduced expression of T beta R-II receptors.
Assuntos
Transformação Celular Neoplásica , Genes ras , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Transporte/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Resistência a Medicamentos , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Receptores de Fatores de Crescimento Transformadores beta/genética , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor beta (TGF-beta) type II receptor (TbetaRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with MI at three or more loci (RER+ phenotype). No mutations in the TbetaRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER+), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases.