RESUMO
In this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged.
Assuntos
Regulação para Baixo , Seminoma/genética , Proteínas de Ligação a Telômeros/genética , Neoplasias Testiculares/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Seminoma/patologia , Complexo Shelterina , Telômero/genética , Telômero/patologia , Proteínas de Ligação a Telômeros/análise , Neoplasias Testiculares/patologia , Testículo/metabolismo , Testículo/patologiaRESUMO
Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-ß expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-ß activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-ß to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-ß are involved in TERT gene regulation during carcinogenesis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Mamárias Experimentais/patologia , Proteínas do Leite/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.