RESUMO
Persistent infections with some types of human papillomavirus (HPV) constitute the major etiological factor for cervical cancer development. Nanog, a stem cell transcription factor has been shown to increase during cancer progression. We wanted to determine whether Nanog could modulate transcription of E6 and E7 oncogenes. We used luciferase reporters under the regulation of the long control region (LCR) of HPV types 16 and 18 (HPV16/18) and performed RT-qPCR. We found that Nanog increases activity of both viral regulatory regions and elevates endogenous E6/E7 mRNA levels in cervical cancer-derived cells. We demonstrated by in vitro mutagenesis that changes at Nanog-binding sites found in the HPV18 LCR significantly inhibit transcriptional activation. Chromatin immunoprecipitation (ChIP) assays showed that Nanog binds in vivo to the HPV18 LCR, and its overexpression increases its binding as well as that of c-Jun. Surprisingly, we observed that mutation of AP1-binding sites also affect Nanog's ability to activate transcription, suggesting cooperation between the two factors. We searched for putative Nanog-binding sites in the LCR of several HPVs and surprisingly found them only in those types associated with cancer development. Our study shows, for the first time, a role for Nanog in the regulation of E6/E7 transcription of HPV16/18.
Assuntos
Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteína Homeobox Nanog/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Humanos , Proteína Homeobox Nanog/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND: Mammals have limited epithelial regeneration capacity. The K6b-E6/E7 mice model has been described as useful for the study of epithelial regeneration. The objective of this study is to compare the expression of E6/E7 oncogenes with those of cell proliferation and apoptosis during epithelization. The hypothesis of this study is that alterations in cell proliferation and apoptosis in K6b-E6/E7 mice will only occur during epithelization. METHODS: Deep 2 mm punches were performed in the middle of transgenic and control mice's ears. A biopsy was collected from the epithelization zone 72 hours and 2 weeks post-injury. Assays for cell proliferation and apoptosis were carried out by immunohistochemistry and TUNEL techniques, respectively. RT-PCR in situ was performed to compare E6/E7 expressions in the areas studied. RESULTS: Transgenic strain K6b-E6/E7 presented more proliferative cells and less apoptotic cells in epithelizated zones. This effect was limited to suprabasal stratum only, and correlates with E6/E7 oncogenes expression. Two weeks post-injury, cell proliferation and apoptosis were similar in both samples as the E6/E7 expression went down. CONCLUSION: K6b-E6/E7 mouse model is useful for epithelial regeneration. Its mechanisms should be considered for the treatment of deep wounds.
Assuntos
Orelha Externa/fisiologia , Células Epiteliais/citologia , Animais , Apoptose/genética , Biópsia , Divisão Celular/genética , Orelha Externa/lesões , Epitélio/fisiologia , Regulação da Expressão Gênica , Papillomavirus Humano 16/genética , Marcação In Situ das Extremidades Cortadas , Queratina-6/genética , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/biossíntese , Proteínas E7 de Papillomavirus/genética , Regeneração , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , TransgenesRESUMO
Activator protein-1 (AP1) is a dimeric protein, consisting either of homodimers between c-Jun, JunB, and JunD of by heterodimers with members of the Fos-family by physically interacting via a "leucine zipper" region. AP1 is an important transcription factor initially identified as a DNA binding protein that bound to enhancer sequences of the human metallothionein IIA gene. The protein components of AP1 are encoded by a set of genes known as "immediate-early" genes that can be activated by a variety of growth factors and mitogens through several different signaling pathways. Until recently, AP1 was considered a transcription factor expressed in most tissues to regulate cellular and viral genes now, it is becoming evident that AP1 can be involved in tissue-specific regulation of target genes due to the differential combination of the components of this important transcription factor. AP1 plays a crucial role during human papillomavirus (HPV) early gene expression, in particular of the expression of E6 and E7 oncoproteins. The HPV are a group of DNA viruses consisting of more than 80 different genotypes. Some of these HPV, know as high risk HPV, are important etiologic agents of uterine-cervical cancer (CaCu). Of the different types of cancer, CaCu is one of the most frequent among women worldwide, constituting the second death cause due to neoplasia. During cellular transformation, HPV infect basal cells in stratified epithelium; their DNA integrate into the host genome usually through the E2 gene; as these cells differentiate and migrate into the upper layer of the epithelium, viral oncogene are expressed blocking their differentiation. Mutagenesis in AP1 sites belonging to the HPV promoter region (LCR) completely abolished the HPV promoter activity in different cell lines; these results and biochemistry assays on this AP1 transcription factor, that includes protein-protein interactions between AP1 and another factors as E7 from HPV, and YY-1; the post-translattional modification and, the retinoic acid interaction; suggest a role for this AP1 factor in tissue-specific transcription of the human papillomavirus.
Assuntos
Genes Precoces , Genes Virais , Proteínas Imediatamente Precoces/biossíntese , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Fator de Transcrição AP-1/fisiologia , Proteínas Estruturais Virais/genética , Células Epiteliais/virologia , Feminino , Regulação Viral da Expressão Gênica , Humanos , Modelos Biológicos , Mutagênese , Proteínas Oncogênicas Virais/biossíntese , Especificidade de Órgãos , Papillomaviridae/classificação , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/virologia , Neoplasias Uterinas/virologia , Replicação ViralRESUMO
Hemos estudiado la organozación genómica del proto-oncogen myc celular (c-myc) en 48 tumores de mama primarios de humano. Dos tipos de alteraciones (amplificación y rearreglo) se observó en 27 de los tumores estudiados (56%). El proto-oncogen c-myc, apareció amplificado de 2 a 15 veces en el DNA de 20 tumores (41%). Fragmentos relacionados a c-myc no germinal (rearreglos) de tamaño variable fueron detectados en 7 tumores de mama primarios (6 malignos, 1 benigno); 4 de estos tumores presentaron tanto arreglo como amplificación y los otros 3 presentaron únicamente rearreglo. La mayoría de los tumores analizados fueron adenocarcinomas ductal invasivo; 58% de éstos mostraron alteraciones genéticas en el locus c-myc. Aunque las alteraciones de c-myc descritas aquí no aparecen correlacionarse con el comportamiento agresivo de los tumores de mama primarios, parecen estar asociados con el desarrollo de carcinoma mamario
