RESUMO
Ultraviolet (UV)B radiation affects plant growth inhibiting cell proliferation. This inhibition is in part controlled by the activity of transcription factors from the E2F family. In particular, the participation of E2Fc and E2Fe in UV-B responses in Arabidopsis plants was previously reported. However, the E2Fa and E2Fb contribution to these processes has still not been investigated. Thus, in this work, we provide evidence that, in Arabidopsis, both E2Fa and E2Fb control leaf size under UV-B conditions without participating in the repair of cyclobutane pyrimidine dimers in the DNA. Nevertheless, in UV-B-exposed seedlings, E2Fa, but not E2Fb, regulates primary root elongation, cell proliferation, and programmed cell death in the meristematic zone. Using e2fa mutants that overexpress E2Fb, we showed that the role of E2Fa in the roots could not be replaced by E2Fb. Finally, our results show that E2Fa and E2Fb differentially regulate the expression of genes that activate the DNA damage response and cell cycle progression, both under conditions without UV-B and after exposure. Overall, we showed that both E2Fa and E2Fb have different and non-redundant roles in developmental and DNA damage responses in Arabidopsis plants exposed to UV-B.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dano ao DNA , Fatores de Transcrição E2F/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Ciclo Celular/genética , Divisão Celular/genética , Proliferação de Células/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Perfilação da Expressão Gênica/métodos , Immunoblotting , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Interferência de RNA , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Trombopoetina/farmacologia , Ativação TranscricionalRESUMO
BACKGROUND: Glioblastoma is considered to the most common and malignant brain tumor in adults. Patients have a median survival of approximately one year from diagnosis due to poor response to therapy. OBJECTIVE: We applied bioinformatics approaches to predict transcription factors (TF) that are deregulated in glioblastoma in an attempt to point out molecular targets for therapy. METHODS: Up-regulated genes in glioblastoma selected from public microarray data were submitted to two TF association analyses. Thereafter, the expression levels of TF obtained in the overlap of analyses were assessed by RT-qPCR carried out in seven glioblastoma cell lines (T98, U251, U138, U87, U343, M059J, and M059K). RESULTS: E2F1 and E2F4 were highlighted in both TF analyses. However, only E2F1 was confirmed as significantly up-regulated in all glioblastoma cell lines in vitro. CONCLUSION: E2F1 is a potential common regulator of differentially expressed genes in glioblastoma, despite the genetic heterogeneity of tumor cells.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Glioblastoma/genética , Linhagem Celular Tumoral , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/genética , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1/genética , Prognóstico , Adulto JovemRESUMO
Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation.
Assuntos
Apoptose/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Animais , Proteínas Reguladoras de Apoptose/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Ligação ao Cálcio/genética , Caspases/genética , Regulação para Baixo , Fatores de Transcrição E2F/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Proteínas Repressoras/genética , Ribossomos/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated. METHODS: Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively. RESULTS: Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle. CONCLUSION: The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.
Assuntos
Apoptose/genética , Carcinoma Ductal Pancreático/genética , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/genética , RNA Mensageiro , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Transcrição E2F/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/genética , Reprodutibilidade dos Testes , Proteína do Retinoblastoma/genéticaRESUMO
ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of E2F corresponds to Adenovirus E2 Binding Factor. [corrected]. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.