RESUMO
Arrest of proliferation is one of the prerequisites for differentiation of cytotrophoblasts into syncytiotrophoblasts, and thus during differentiation telomerase activity, as well as human telomerase reverse transcriptase (hTERT) expression, is down-regulated. Considering this, it is of interest to investigate whether syncytium formation can be delayed by prolonging the expression of telomerase in cytotrophoblasts. BeWo cells were transfected with pLPC-hTERT retroviral vector and the reverse transcription-polymerase chain reaction analysis for hTERT mRNA concentrations in the transfected cells revealed a several-fold increase in hTERT mRNA compared with the cells transfected with empty vector, and this confirmed that the transfection was successful. An increase in the proliferation, as assessed by bromodeoxyuridine incorporation assay, as well as an increase in mRNA and protein concentration of various cyclins and proliferating cell nuclear antigen, was noticed. The effect of hTERT transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cell-cell fusion was delayed and differentiation did not occur in hTERT-transfected cells. However, the effects seen were only transient as stable transfection was not possible and the cells were undergoing apoptosis after 72 h, which suggested that apart from hTERT other factors might be important for immortalization of BeWo cells.
Assuntos
Diferenciação Celular , Coriocarcinoma/patologia , Telomerase/genética , Transfecção , Apoptose , Sequência de Bases , Western Blotting , Proliferação de Células , Coriocarcinoma/enzimologia , Primers do DNA , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
During the first trimester of pregnancy, the human placenta is an actively dividing and highly invasive tumour-like tissue, while near term, it represents a fully developed, non-invasive unit. In order to understand the molecular basis of this marked difference in the placental phenotypes, an approach based on a differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) was adopted to analyse changes in gene expression, using total RNA isolated from first-trimester and term placental villi. Using this approach, T-plastin was initially identified as being differentially expressed in the human first-trimester placenta. T-plastin is an actin-bundling protein and is known to be highly expressed in actively dividing cells and up-regulated in several carcinomas. Using a homogenous population of cytotrophoblasts and syncytiotrophoblasts isolated from human placentae, the present authors demonstrate the differential expression of T-plastin in cytotrophoblasts compared with the terminally differentiated syncytiotrophoblasts. The down-regulation of T-plastin expression is further demonstrated in human trophoblastic BeWo cells induced to differentiate using transforming growth factor (TGF)beta1, a growth factor known for its anti-proliferative and anti-invasive response in placental cells. These studies suggest that expression of T-plastin in the placental context may indeed be associated with the enhanced replicative potential of placental trophoblasts.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fosfoproteínas/genética , Trofoblastos/citologia , Trofoblastos/fisiologia , Western Blotting , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , DNA Complementar/genética , Endométrio/citologia , Endométrio/fisiologia , Feminino , Glicodelina , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana , Proteínas dos Microfilamentos , Gravidez , Proteínas da Gravidez/genética , Primeiro Trimestre da Gravidez/fisiologia , RNA Mensageiro/análise , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
The rat R2C Leydig tumor cell line is constitutively steroidogenic in nature, while the mouse MA-10 Leydig tumor cell line synthesizes large amounts of steroids only in response to hormonal stimulation. Earlier studies showed abundant cAMP-independent steroid production and constitutive expression of steroidogenic acute regulatory (StAR) protein in R2C cells. The objective of the current study was to identify possible genetic alterations in the R2C cell line responsible for rendering it a constitutively steroidogenic cell line, especially those that might have altered its cholesterol homeostatic mechanisms. Measurement of the levels of cholesterol esters and free cholesterol, precursors for steroidogenesis, indicated that R2C mitochondria were fourfold enriched in free cholesterol content compared with MA-10 mitochondria. In addition to the previously demonstrated increased expression of StAR protein, we show that R2C cells possess marginally enhanced protein kinase A activity, exhibit higher capacity to take up extracellular cholesterol esters, and express much higher levels of scavenger receptor-type B class 1 (SR-B1) and hormone sensitive lipase (HSL). These observations suggest that the high level of steroid biosynthesis in R2C cells is a result of the constitutive expression of the components involved in the uptake of cholesterol esters (SR-B1), their conversion to free cholesterol (HSL), and its mobilization to the inner mitochondrial membrane (StAR).
Assuntos
Antígenos CD36/metabolismo , Ésteres do Colesterol/metabolismo , Tumor de Células de Leydig/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas/metabolismo , Esteroides/biossíntese , Neoplasias Testiculares/metabolismo , Animais , Sequência de Bases , Transporte Biológico Ativo , Bucladesina/farmacologia , Antígenos CD36/genética , Colesterol/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Tumor de Células de Leydig/genética , Masculino , Camundongos , Mitocôndrias/metabolismo , Fosfoproteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Depuradores , Receptores Depuradores Classe B , Esterol Esterase/metabolismo , Neoplasias Testiculares/genética , Células Tumorais CultivadasRESUMO
The MA-10 mouse Leydig tumor cell line produces large amounts of steroids only in response to hormonal stimulation while the R2C rat Leydig tumor cell line is constitutively steroidogenic in nature. In an effort to uncover the potential reasons for constitutive steroidogenesis in R2C cells, we have recently shown that compared to MA-10 cells, R2C cells express much higher levels of the Scavenger Receptor Class B type 1 which results in a higher capacity for cholesteryl ester uptake through the selective uptake pathway. We also found an enhanced expression of Hormone Sensitive Lipase and the Steroidogenic Acute Regulatory protein in these cells and reasoned that they may further facilitate the conversion of cholesteryl esters to free cholesterol and its mobilization to the inner mitochondrial membrane, thus rendering them constitutively steroidogenic. Given the proposed role of the peripheral-type benzodiazepine receptor (PBR) in conferring a constitutively steroidogenic phenotype to the R2C cells, the current study was conducted to investigate the relationship between its expression in MA-10 and R2C cells and correlate it with the constitutive nature of R2C cell steroidogenesis. Our studies show that PBR expression as measured by PK 11195 ligand binding and Western analysis is much higher in MA-10 cells than R2C cells. We also determined that the affinity of ligand binding to the PBR is comparable in the two cell lines, suggesting that PBR is unlikely to be solely responsible for the constitutive nature of R2C cell steroidogenesis.
