RESUMO
BACKGROUND: The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma. METHODS: Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays. RESULTS: CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α. CONCLUSIONS: MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.
Assuntos
Carcinoma de Células Escamosas , Metaloproteinase 9 da Matriz , Metalotioneína , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Fator de Crescimento Epidérmico/genética , Humanos , Antígeno Ki-67/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Metalotioneína/genética , Fator de Crescimento Transformador alfa/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
ABSTRACT Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway Materials and methods: The cell line was treated with T3 at a physiological dose (10−9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.
Assuntos
Humanos , Feminino , Tri-Iodotironina/genética , Neoplasias da Mama/genética , Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Fator de Crescimento Transformador alfa/genética , Sistema de Sinalização das MAP Quinases/genética , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Proto-Oncogenes/genética , Neoplasias da Mama/metabolismo , RNA Mensageiro/genética , Adenocarcinoma/metabolismo , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Linhagem Celular Tumoral/metabolismo , Células MCF-7/metabolismoRESUMO
OBJECTIVE: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway. MATERIALS AND METHODS: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. RESULTS: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. CONCLUSION: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.
Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Fator de Crescimento Transformador alfa/genética , Tri-Iodotironina/genética , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/metabolismo , Feminino , Humanos , Células MCF-7/metabolismo , Proto-Oncogene Mas , Proto-Oncogenes/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
O objetivo do presente estudo foi verificar a presença de associação fenótipo-genotípico entre TGFA (Fator de Crescimento Transformante α) (C/T rs1523305), IRF6 (Fator Regulatório Interferon 6) (A/C rs2013162) e MSX1 (Segmento Muscular Homeobox 1) (A/G rs12532) e anomalias dentárias em pacientes com malclusão esquelética. Para tanto, dois estudos prévios foram realizados com os objetivos de (I) determinar se indivíduos com discrepâncias esqueléticas de Classe II ou III apresentam uma maior frequência de anomalias dentárias em comparação com indivíduos com maloclusão de Classe I e (II) comparar quatro diferentes métodos de coleta salivar e de células bucais [Expectoração da Saliva (1), Expectoração da Saliva com Estímulo Lingual (2), Raspagem com Escova Citológica (3) e Raspagem com Escova Citológica mais Expectoração da Saliva (4)] para extração de DNA genômico, avaliando sua concentração e pureza. Na revisão sistemática, foi realizada uma busca nas principais bases de dados da literatura científica médica eletrônica. Estudos observacionais foram selecionados se fossem mencionadas anomalias dentárias nos diferentes padrões de maloclusão esquelética. Foi adotado um conjunto de critérios para elegibilidade do estudo, com base na estratégia PECOS (População: maxila e mandíbula; Exposição: discrepâncias esqueléticas; Comparação: padrão de normalidade; e Resultado: anomalias dentárias). Cinco estudos retrospectivos contendo 7679 participantes foram classificados e considerados elegíveis para a revisão sistemática. A amostra do estudo II foi composta por 20 participantes. As coletas foram realizadas com intervalo de no mínimo um dia entre elas e foram armazenadas a -20°C até a extração do DNA ser realizada (Qiagen®). O estudo III foi composto por uma amostra de 505 registros ortodônticos. Dezenove pontos cefalométricos, serviram para a obtenção dos ângulos e medidas utilizados para a caracterização esquelética, utilizando-se o Software Dolphin. Amostras de saliva foram coletadas de todos os participantes e o DNA foi extraído, diluído e quantificado. Os genes TGFA, IRF6 e MSX1 foram usados para reações de PCR em tempo real. Os testes Razão de chance, Qui-quadrado, Exato de Fisher, T independente e coeficiente de correlação (nível de significância = 95%) foram realizados. A partir da revisão sistemática, concluiu-se que indivíduos com padrões de maloclusão esquelética têm mais anomalias dentárias. Após a análise estatística, não foi observado dimorfismo sexual em relação à concentração de DNA (p = 0,76), idade (p = 0,91) e etnias (p = 0,72). Não houve diferença significativa entre os métodos de coleta em relação à quantidade e pureza do DNA extraído (p≥0,05). O gene IRF6 foi associado com a ME Classe III (p = 0,04, OR = 0,69, IC 0,49-0,98) e o gene MSX1 foi associado ao padrão de crescimento hipodivergente (p=0,003, OR=0,5, IC 0,36-0,74) e ME Classe II (p=0,0001, OR=0,6, IC 0,46-0,78). Em relação a AD, o gene MSX1 também foi associado à impactação (p=0,03, OR=0,67, IC 0,47-0,96) e dilaceração (p=0,04, OR=0,27, IC 0,07-0,977). MSX1 associouse ao padrão de crescimento hipodivergente e Classe II, e impactação e dilaceração, mostrando que existe uma associação genética entre anomalias dentárias e maloclusões esqueléticas. (AU)
The objective of the present study was to verify the phenotype-genotype association between TGFA (Transforming Growth Factor α) (C/T rs1523305), IRF6 (Interferon Regulatory Factor 6) (A/C rs2013162) and MSX1 (Muscle Segment Homeobox 1) (A/G rs12532) and dental anomalies in patients with skeletal malocclusion. Two previous studies were carried out with the objective of (I) to determine if individuals with Class II or III skeletal discrepancies present a higher frequency of dental anomalies compared to individuals with Class I malocclusion and (II) to compare four different types of saliva and oral buccal cell collecting methods for genomic DNA extraction: (1)Expectoration of saliva, (2)Expectoration of saliva with lingual stimulation, (3)Scraping with cytological brush, and (4)Scraping with cytological brush and expectoration of saliva, evaluating its concentration and purity. In the systematic review, a search of the main electronic medical scientific literature databases was conducted. Observational studies were selected if mentioning dental anomalies in the different skeletal malocclusion patterns. A set of criteria for the eligibility of the study was adopted, based on the PECOS strategy (Population: maxilla and mandible, Exposure: skeletal discrepancies, Comparison: normality pattern, and Outcome: dental anomalies). Five retrospective studies containing 7679 participants were classified and considered eligible for the systematic review. The sample of study II was composed by 20 participants. The biological samples were collected at intervals of at least one day between them and were stored at -20 ° C until DNA extraction was performed (Qiagen®). Study III was composed of a sample of 505 orthodontic records. Nineteen cephalometric points were used to obtain the angles and measurements used for the skeletal characterization, using Dolphin Software. Saliva samples were collected from all participants and the DNA was extracted, diluted and quantified. The TGFA, IRF6 and MSX1 genes were used for real-time PCR reactions. The odds ratio, chi-square, Fisher's exact, independent T and correlation coefficient (significance level = 95%) tests were performed. From the systematic review, it was concluded that individuals with skeletal malocclusion patterns have more dental anomalies. After the statistical analysis, no sexual dimorphism (p = 0.76), age (p = 0.91) and ethnicity (p = 0.72) was observed in relation to DNA concentration. There was no significant difference between the collection methods in relation to the amount and purity of the extracted DNA (p≥0.05). IRF6 was associated with Class III skeletal malocclusion (p=0.04, OR=0.69, C.I. 0.49-0.98), and MSX1 was associated with hypodivergent growth pattern (p=0.003, OR=0.5, 95% C.I. 0.36-0.74), and Class II skeletal malocclusion (p=0.0001, OR=0.6, C.I. 0.46-0.78). In regards to dental anomalies, MSX1 was also associated with tooth impaction (p=0.03, OR=0.67, 95% C.I. 0.47-0.96) and root dilaceration (p=0.04, OR=0.27, 95% C.I. 0.07-0.97). MSX1 was associated with both hypodivergent growth pattern and Class II skeletal malocclusion (p=0.0001), and tooth impaction (p=0.03) and root dilaceration (p=0.04), whereas IRF6 was associated with Class III skeletal malocclusion, (AU)
El objetivo del presente estudio fue verificar la presencia de asociación fenotipo-genotípica entre TGFA (Factor de crecimiento transformante α) (C / T rs1523305), IRF6 (Factor regulador de interferón 6) (A / C rs2013162) y MSX1 (Segmento muscular 1 de Homeobox 1). ) (A / G rs12532) y anomalías dentales en pacientes con malclusión esquelética. Con este fin, se realizaron dos estudios previos para (I) determinar si las personas con discrepancias esqueléticas de Clase II o III tienen una mayor frecuencia de anomalías dentales en comparación con las personas con maloclusión de Clase I y (II) para comparar cuatro métodos diferentes. y colección de células salivales [Esputo de saliva (1), Estímulo lingual Saliva Esputo (2), Raspado con cepillo citológico (3) y Raspado con cepillo citológico más Esputo de saliva (4)] para extracción de ADN genómico , evaluando su concentración y pureza. En la revisión sistemática, buscamos en las principales bases de datos de la literatura científica médica electrónica. Se seleccionaron estudios de observación si se mencionaban anomalías dentales en los diferentes patrones de maloclusión esquelética. Se adoptó un conjunto de criterios para la elegibilidad del estudio, basado en la estrategia PECOS (Población: maxilar y mandíbula; Exposición: discrepancias esqueléticas; Comparación: patrón de normalidad; y Resultado: anomalías dentales). Cinco estudios retrospectivos con 7679 participantes fueron clasificados y considerados elegibles para una revisión sistemática. La muestra del estudio II consistió en 20 participantes. Se tomaron muestras con al menos un día de diferencia y se almacenaron a -20 ° C hasta que se realizó la extracción de ADN (Qiagen®). El estudio III consistió en una muestra de 505 registros de ortodoncia. Diecinueve puntos cefalométricos se utilizaron para obtener los ángulos y las medidas utilizadas para la caracterización esquelética utilizando Dolphin Software. Se recogieron muestras de saliva de todos los participantes y se extrajo, diluyó y cuantificó el ADN. Los genes TGFA, IRF6 y MSX1 se usaron para reacciones de PCR en tiempo real. Se realizaron las pruebas de odds ratio, Chi-cuadrado, exacta de Fisher, T independiente y coeficiente de correlación (nivel de significancia = 95%). De la revisión sistemática, se concluyó que las personas con patrones de maloclusión esquelética tienen más anomalías dentales. Después del análisis estadístico, no se observó dimorfismo sexual en relación con la concentración de ADN (p = 0,76), la edad (p = 0,91) y el origen étnico (p = 0,72). No hubo diferencias significativas entre los métodos de recolección con respecto a la cantidad y pureza del ADN extraído (p≥0.05). El gen IRF6 se asoció con ME Clase III (p = 0.04, OR = 0.69, CI 0.49-0.98) y el gen MSX1 se asoció con el patrón de crecimiento hipodivergente (p = 0.003, OR = 0.5, CI 0.36-0.74) y EM Clase II (p = 0.0001, OR = 0.6, CI 0.46-0.78). Con respecto a AD, el gen MSX1 también se asoció con impactación (p = 0.03, OR = 0.67, CI 0.47-0.96) y laceración (p = 0.04, OR = 0.27, IC 0,07-0,977). MSX1 se asoció con un patrón de crecimiento hipodivergente y de Clase II, impactación y laceración, lo que demuestra que existe una asociación genética entre las anomalías dentales y las maloclusiones esqueléticas. (AU)
Assuntos
Humanos , Masculino , Feminino , Saliva , Anormalidades Dentárias/genética , DNA/genética , Fator de Crescimento Transformador alfa/genética , Má Oclusão/complicações , Fenótipo , Revisões Sistemáticas como Assunto , GenótipoRESUMO
Breast cancer remains the second most common disease worldwide. Radiotherapy, alone or in combination with chemotherapy, is widely used after surgery as a treatment for cancer with proven therapeutic efficacy manifested by reduced incidence of loco-regional and distant recurrences. However, clinical evidence indicates that relapses occurring after radiotherapy are associated with increased metastatic potential and poor prognosis in the breast. Among the anticarcinogenic and antiproliferative agents, curcumin is a well-known major dietary natural yellow pigment derived from the rhizome of the herb Curcuma longa (Zingiberaceae). The aim of the present study was to analyze the differential expression of metastatic genes in radiation- and estrogen-induced breast cancer cell model and the effect of curcumin on such metastatic genes in breast carcinogenesis. Expression levels of TGF-α and TGFß1 genes were upregulated in MCF-10F and downregulated in Tumor2 cell lines treated with curcumin. Expression levels of other genes such as caspase 9 and collagen 4 A2 were upregulated in both MCF-10F and Tumor2-treated cell lines. Integrin α5 and cathepsin B and D decreased its expression in Tumor2, whereas E-Cadherin, c-myc and CD44 expressions were only increased in MCF-10F. It can be concluded that metastatic genes can be affected by curcumin in cancer progression and such substance can be used in breast cancer patients with advanced disease without side-effects commonly observed with therapeutic drugs.
Assuntos
Antioxidantes/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carcinogênese/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Antioxidantes/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Curcuma/química , Estrogênios/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Prognóstico , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10(-6)). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10(-6)). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10(-6) for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.
Assuntos
Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Fatores Reguladores de Interferon/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Animais , Brasil , Fenda Labial/genética , Fissura Palatina/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Fatores Reguladores de Interferon/genética , Desequilíbrio de Ligação/genética , Camundongos , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Fator de Crescimento Transformador alfa/genética , População BrancaRESUMO
We report a study of TGFA/ Taq I polymorphisms and environmental factors in non-syndromic oral cleft in Southern Brazil. Nonsyndromic cleft case-parent triads were recruited to participate. Clinical data was collected with an emphasis on tobacco and alcohol use during pregnancy. DNA was extracted from peripheral blood and TGFA/ Taq I polymorphisms were analyzed by PCR/RFLP with Taq I restriction enzyme. Association of clefts and TGFA/ Taq I polymorphisms was determined using a transmission disequilibrium test (TDT). Association of environmental factors, clefts, and genotypes was evaluated with Fisher's exact test. The minor allele frequency was 0.064. We found no evidence of association between TGFA/ Taq I polymorphisms and clefting (TDT p = 0.335). We also found no association between TGFA/ TaqI polymorphisms and environmental factors (alcohol and/or tobacco). Therefore, no evidence was found that TGFA/ Taq I polymorphisms play a role in clefting in this population. No evidence was found that tobacco or alcohol exposure during pregnancy was related to clefting, however a larger sample size is needed to confirm these results.
Assuntos
Feminino , Humanos , Masculino , Gravidez , Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Taq Polimerase/genética , Fator de Crescimento Transformador alfa/genética , Brasil , Frequência do Gene , Exposição Materna , Reação em Cadeia da Polimerase , Fatores de Risco , FumarRESUMO
We report a study of TGFA/ Taq I polymorphisms and environmental factors in non-syndromic oral cleft in Southern Brazil. Nonsyndromic cleft case-parent triads were recruited to participate. Clinical data was collected with an emphasis on tobacco and alcohol use during pregnancy. DNA was extracted from peripheral blood and TGFA/ Taq I polymorphisms were analyzed by PCR/RFLP with Taq I restriction enzyme. Association of clefts and TGFA/ Taq I polymorphisms was determined using a transmission disequilibrium test (TDT). Association of environmental factors, clefts, and genotypes was evaluated with Fisher's exact test. The minor allele frequency was 0.064. We found no evidence of association between TGFA/ Taq I polymorphisms and clefting (TDT p = 0.335). We also found no association between TGFA/ TaqI polymorphisms and environmental factors (alcohol and/or tobacco). Therefore, no evidence was found that TGFA/ Taq I polymorphisms play a role in clefting in this population. No evidence was found that tobacco or alcohol exposure during pregnancy was related to clefting, however a larger sample size is needed to confirm these results.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Taq Polimerase/genética , Fator de Crescimento Transformador alfa/genética , Brasil , Feminino , Frequência do Gene , Humanos , Masculino , Exposição Materna , Reação em Cadeia da Polimerase , Gravidez , Fatores de Risco , FumarRESUMO
OBJECTIVE: To test the TGFA/Taq I polymorphism in the development of nonsyndromic cleft lip and palate. DESIGN AND SETTING: The research was based on a case-control study, including nonsyndromic cleft lip and palate patients (140 individuals) and a control sample of unaffected individuals (142) to ascertain the absence or presence of genic mutation at the TGFA locus. INTERVENTIONS: The DNA of carriers of nonsyndromic cleft lip with or without cleft palate was obtained by buccal swab, and the DNA of the control group was extracted from peripheral blood leucocytes. TGFA/Taq I polymorphism was determined genetically by polymerase chain reaction using specific primers and fragment digestion with Taq I restriction enzyme. RESULTS: No significant association was detected when patients and controls were compared with the genotype for TGFA/Taq I polymorphism. CONCLUSION: Mutations in TGFA gene have no association with nonsyndromic cleft lip and palate in the sample from Rio Grande do Sul. Therefore, based on this study, it is not possible to determine the role played by TGFA in the expression of cleft lip and palate.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Fator de Crescimento Transformador alfa/genética , Adenina , Adolescente , Adulto , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 2/genética , DNA/genética , Feminino , Genótipo , Humanos , Lactente , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Deleção de Sequência/genética , Taq Polimerase/genética , Timina , Adulto JovemRESUMO
OBJECTIVES: To examine the effects of triiodothyronine (T3), 17beta-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. METHODS AND RESULTS: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3- mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. CONCLUSION: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Tamoxifeno/farmacologia , Fator de Crescimento Transformador alfa/genética , Tri-Iodotironina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma/patologia , Técnicas de Cultura de Células , Regulação para Baixo/efeitos dos fármacos , Interações Medicamentosas , Estradiol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
BACKGROUND: Nonsyndromic cleft lip/palate (NSCLP) is a congenital malformation with the characteristics of a complex genetic trait. Based on experimental evidences as well as on association and linkage studies candidate genes TGFA, RARA and BCL3 have been postulated as being involved in the genetic etiology of this pathology. AIM: To test the possible association due to linkage disequilibrium between microsatellite markers located at less than 1cM from the three candidate genes and nonsyndromic cleft lip/palate using the case-parents trio design. PATIENTS AND METHODS: The sample consisted of 58 case-parents trios. Two microsatellite markers, flanking each one of the candidate genes were analyzed by means of the polymerase chain reaction (PCR) with fluorescent labeled microsatellite markers. Electrophoresis of the PCR products was performed on a laser-fluorescent automatic DNA sequencer. Nonparametric ETDT was used to analyze the genotype data. RESULTS: Significant linkage disequilibrium was detected between D2S443 (TGFA) and NSCLP. Significance was almost reached between D17S800 (RARA) and NSCLP. Alleles 239bp (D2S443) and 172bp (D17S800) showed significant preferential transmission from heterozygous parents to affected offspring. In the case of BCL3 both markers showed no significant results. CONCLUSIONS: The results of the present study do not show clear evidence that TGFA or RARA could be involved in the genetic etiology of NSCLP. Even though the importance of retinoic acid in the development of the embryo is well documented the results obtained for RARA are difficult to analyze. In relation to the possible role of BCL3 in NSCLP, recent information postulates that other genes located in the same chromosome region could be involved in NSCLP.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Desequilíbrio de Ligação/genética , Repetições de Microssatélites/genética , Alelos , Proteína 3 do Linfoma de Células B , Chile , Marcadores Genéticos , Genótipo , Humanos , Proteínas Proto-Oncogênicas/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Fatores de Transcrição , Fator de Crescimento Transformador alfa/genéticaRESUMO
Background: Nonsyndromic cleft lip/palate (NSCLP) is a congenital malformation with the characteristics of a complex genetic trait. Based on experimental evidences as well as on association and linkage studies candidate genes TGFA, RARA and BCL3 have been postulated as being involved in the genetic etiology of this pathology. Aim: To test the possible association due to linkage disequilibrium between microsatellite markers located at less than 1cM from the three candidate genes and nonsyndromic cleft lip/palate using the case-parents trio design. Patients and Methods: The sample consisted of 58 case-parents trios. Two microsatellite markers, flanking each one of the candidate genes were analyzed by means of the polymerase chain reaction (PCR) with fluorescent labeled microsatellite markers. Electrophoresis of the PCR products was performed on a laser-fluorescent automatic DNA sequencer. Nonparametric ETDT was used to analyze the genotype data. Results: Significant linkage disequilibrium was detected between D2S443 (TGFA) and NSCLP. Significance was almost reached between D17S800 (RARA) and NSCLP. Alleles 239bp (D2S443) and 172bp (D17S800) showed significant preferential transmission from heterozygous parents to affected offspring. In the case of BCL3 both markers showed no significant results. Conclusions: The results of the present study do not show clear evidence that TGFA or RARA could be involved in the genetic etiology of NSCLP. Even though the importance of retinoic acid in the development of the embryo is well documented the results obtained for RARA are difficult to analyze. In relation to the possible role of BCL3 in NSCLP, recent information postulates that other genes located in the same chromosome region could be involved in NSCLP.
Assuntos
Humanos , Fenda Labial/genética , Fissura Palatina/genética , Desequilíbrio de Ligação/genética , Repetições de Microssatélites/genética , Alelos , Chile , Marcadores Genéticos , Genótipo , Proteínas Proto-Oncogênicas/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição , Fator de Crescimento Transformador alfa/genéticaRESUMO
OBJECTIVES AND METHODS: Risks of oral cancer related to a CA microsatellite repeat polymorphism in intron 1 of the epidermal growth factor receptor (EGFR) gene and a TaqI polymorphism in the transforming growth factor-alpha (TGFA) gene were evaluated in a population-based case-control study consisting of 157 cases and 149 controls recruited in Puerto Rico. RESULTS: Carriers of > or = 16 CA repeats in EGFR showed a 1.9-fold increased risk for oral cancer (OR=1.9, 95% CI=1.0-3.5). Risks also tended to increase with decreasing number of alleles with > or = 16 CA repeats (P for trend=0.06). Our data suggested a non-significant reduction in risk for subjects heterozygous for the TGFA polymorphism (OR=0.6, 95% CI=0.2-1.3). CONCLUSIONS: The EGFR-associated risk appeared to be independent of tobacco and alcohol use and may be restricted primarily to subjects who consumed low amounts of fresh fruits and vegetables (OR=5.9, 95%CI: 2.3-15.2). These data implicate dietary and molecular targets for oral cancer prevention.
Assuntos
Genes erbB-1/genética , Repetições de Microssatélites/genética , Neoplasias Bucais/genética , Polimorfismo Genético , Fator de Crescimento Transformador alfa/genética , Adulto , Idoso , Bebidas Alcoólicas , Estudos de Casos e Controles , Feminino , Frutas , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/epidemiologia , Porto Rico/epidemiologia , Fatores de Risco , Fumar , VerdurasRESUMO
OBJECTIVE: Transforming growth factor-alpha (TGFA) was the first gene suggested to be associated with nonsyndromic cleft lip, cleft palate, or both (CL/ P). There are, however, still controversies of the effect of TGFA on the predisposition of this malformation. To contribute to a better understanding of the role of this gene in the occurrence of CL/P we undertook a case-control study including patients and controls ascertained in different regions of the country. DESIGN: We examined the C2/TaqI variant of the TGFA gene in 536 patients with nonsyndromic CL/P and 412 controls. The TGFA genotype frequencies in patients were compared with controls using chi-square or Fisher exact test. DNA, obtained from peripheral blood or buccal swabs, was genotyped for the TaqI polymorphism of TGFA. SETTING: The probands and corresponding controls were ascertained in different centers of Brazil, partly representing the ethnic admixture of our population. RESULTS: The TGFA genotype distribution was very similar in patients with CL/P ascertained in the three different regions of Brazil. However, a discrete difference was observed between controls of Säo Paulo and Ceará (chi-square = 3.605; p = 0.058), with a lower value of the C2/Taq allele frequency in controls of CE (0.04). These data reinforce that this polymorphic system is heterogenous among different ethnic groups. In addition, no evidence was found for an association of TGFA with CL/P in this case-control study. CONCLUSION: These data further suggest that TGFA is not a relevant modifier locus for the occurrence of CL/P.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fator de Crescimento Transformador alfa/genética , Adolescente , Brasil , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Cromossomos Humanos Par 2 , Feminino , Frequência do Gene , Humanos , Masculino , Polimorfismo de Fragmento de RestriçãoRESUMO
The 677 C --> T polymorphism in the 5-10 methylenetetrahydrofolate reductase (MTHFR) gene has been associated with nonsyndromic cleft lip with or without cleft palate (CL/P) in some populations, but not others. Previous studies (ie, case-control and transmission disequilibrium tests (TDT)) in Brazilian families with CL/P have been unable to replicate this putative association. However, our group observed a lower proportion of CT heterozygotes among the mothers of CL/P probands, suggesting that the maternal genotype for this polymorphism might influence predisposition to CL/P. In order to further examine this issue, we performed a case-control study of the 677 C --> T/MTHFR polymorphism in families with CL/P ascertained in two regions of Brazil: 172 from São Paulo (SP) and 252 from Ceará (CE). The control samples included 243 individuals from SP and 401 from CE. TDT was carried out in 102 patients with CL/P and their parents. No evidence of an association was observed between the 677 C --> T/MTHFR polymorphism and CL/P using the case-control design, while borderline significance was obtained with the TDT (P=0.055). We have also looked for an interaction between maternal MTHFR genotypes and the propositi offspring's genotypes at two candidate susceptibility loci for CL/P, TGFA and BCL3. Interestingly, we observed an interaction between the maternal MTHFR and offspring's BCL3 genotypes (OR: 2.3; 95% CI: 1.1-4.8; P=0.03) but not with the offspring's TGFA genotypes. Therefore, our results reinforce the idea that the maternal MTHFR genotype plays a significant role in susceptibility to CL/P, but its teratogenic effect depends on the genotype of the offspring.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Predisposição Genética para Doença , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Proteínas Proto-Oncogênicas/genética , Adulto , Proteína 3 do Linfoma de Células B , Brasil , Estudos de Casos e Controles , Fenda Labial/etnologia , Fissura Palatina/etnologia , Feminino , Frequência do Gene , Genótipo , Heterozigoto , Humanos , Desequilíbrio de Ligação , Masculino , Fatores de Transcrição , Fator de Crescimento Transformador alfa/genéticaRESUMO
BACKGROUND: Cytokines are important modulators of post-transplant, allogeneic immune responses. In heart transplantation, endomyocardial biopsies allow monitoring of histologic and immunologic events that occur inside the graft; their correlation with risk factors condition graft outcome. Recent reports indicate that various cytokine gene allelic polymorphisms control the number of cytokines produced and may be associated with graft outcome. METHODS: We studied 71 heart transplant recipients between December 1985 and December 2000. We used sequence-specific primers (SSP) polymerase chain reaction to study interleukin-10 (IL-10) polymorphisms at -1082 (G/A), -819 (C/T), and -592 (C/A); tumor necrosis factor alpha (TNF-alpha) at -308 (G/A) and -238 (G/A); transforming growth factor beta (TGF-beta) variants at codon 10 (C/T) and codon 25 (G/C); and interferon-gamma (IFN-gamma) polymorphisms at +874 (T/A). We determined the association of allele, genotype, and haplotype frequencies with the presence of histologically proven rejection episodes (according to International Society for Heart and Lung Transplantation criteria) and the presence of Quilty lesions in endomyocardial biopsy specimens. RESULTS: We found no association between the polymorphisms studied and the frequency and severity of acute and chronic rejection episodes. However, the gene frequency of allele A at IL-10 -1082, associated with decreased IL-10 production, was increased in patients with Quilty lesions (p = 0.0027, odds ratio = 2.98). Similarly, we found more AA homozygous individuals, compared with AG heterozygous and GG homozygous individuals (p = 0.0017), among patients with Quilty effect. The ATA and ACC IL-10 haplotypes also were associated with Quilty effect (p = 0.0051). CONCLUSIONS: These results suggest that genetically controlled decreased IL-10 production predisposes to the development of Quilty lesions. The decreased negative regulatory effect of IL-10 on T cells and macrophages may result in enhanced graft infiltration.
Assuntos
Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Interferon gama/genética , Interleucina-10/genética , Polimorfismo Genético/genética , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador beta/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/imunologia , Miocárdio/patologiaRESUMO
The confirmation of supposed genes causing susceptibility to nonsyndromic cleft lip with or without a cleft palate (CL/P) and cleft palate alone (CP) would help to understand the molecular development of the lip, the palate and the jaw, and to predict more accurately the risk of recurrence of such defects. The purpose of this article is to present a brief review of the current state of knowledge regarding the search for genes responsible for the occurrence of CL/P and CP. After ten years of research, approximately twenty candidate genes have already been suggested for CL/P and CP. Some genes or chromosomal regions seem to be frequently associated with these defects and a specific nomenclature (orofacial cleft genes--OFC) has been suggested. Everything indicates that it will still take many years of research before the genes responsible for CL/P and CP are confirmed. These efforts are justified, however, because of the possibility of being able to predict more accurately the risk of such defects occurring.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fatores de Transcrição , Alelos , Proteína 3 do Linfoma de Células B , Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 6/genética , Frequência do Gene , Genes Homeobox/genética , Ligação Genética , Marcadores Genéticos/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição MSX1 , Polimorfismo Genético/genética , Proteínas Proto-Oncogênicas/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3RESUMO
Cowpox virus (CPV) is a member of the Orthopoxvirus genus and has the genetic capacity to encode a multitude of genes that interfere with the host inflammatory and immune response or modulate the physiological state of infected and non-infected cells. Among these CPV factors are receptors homologous to interferon and tumor necrosis factor receptors and also a viral cellular serine-proteinase analog. Here we describe the detection of a CPV gene that encodes a protein homologous to epidermal growth factor, transforming growth factor alpha and poxvirus growth factors, such as the vaccinia growth factor (VGF). The VGF and other poxvirus growth factors are produced early in the infection and are secreted into the medium where they bind to the EGF receptors, generating mytotic responses. The cowpox growth factor (CGF) gene was detected in three copies on the virus genome by PCR, and by northern and southern blot hybridization using VGF nucleotide sequences as primers and probes. The CPV gene has a strong nucleotide and predicted amino acid similarity with VGF, and is also produced early in the infection.
Assuntos
Vírus da Varíola Bovina/genética , Fator de Crescimento Epidérmico/genética , Genes Virais , Substâncias de Crescimento/genética , Peptídeos/genética , Fator de Crescimento Transformador alfa/genética , Proteínas Virais , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Chlorocebus aethiops , DNA Viral , Genoma Viral , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Esteroides/metabolismo , Fator de Crescimento Transformador alfa/biossíntese , Biópsia , Fator de Crescimento Epidérmico/genética , Feminino , Humanos , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptores de Estradiol/metabolismo , Receptores de Progesterona/metabolismo , Estatísticas não Paramétricas , Fator de Crescimento Transformador alfa/genéticaRESUMO
MCF-7 (estrogen receptor positive--ER+) and MDA-MB-231 (estrogen receptor negative--ER-) are human breast cancer cell lines which express functional thyroid hormone receptors (c-erb A alpha1 and c-erb beta1) as indicated by stimulation of mitochondrial alpha-glycerophosphate dehydrogenase. In MCF-7, mimicking E2, T3 stimulated growth in a dose-dependent (10(10) M - 10(-8) M) manner, induced the expression of progesterone receptor and growth factor TGFalpha mRNAs and inhibited that of TGFbeta mRNA; T3 also increased progesterone binding and LDH5 isozyme activities. None of these effects were observed in (ER-) MDA-MB-231 cells. 10(-6) M tamoxifen (TAM) reverted growth stimulation, suppressed progesterone receptor and TGFalpha mRNA induction and restored TGFbeta mRNA to control levels in T3-treated MCF-7 cells. That T3 is acting in MCF-7 cells via its binding to ER is suggested by the immunoprecipitation of pre-bound 125I-T3 from MCF-7 nuclear extracts by an ER-specific monoclonal antibody and by the displacement of 3H-estradiol binding to ER by radioinert T3.