RESUMO
Epstein-Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.
Assuntos
Linfócitos B/virologia , Herpesvirus Humano 4/metabolismo , Receptor Notch2/metabolismo , Telomerase/metabolismo , Latência Viral/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Ativação Enzimática , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/enzimologia , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Receptor Notch2/antagonistas & inibidores , Receptor Notch2/genética , Transdução de Sinais , Transativadores/biossínteseRESUMO
In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6-8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post-transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5-dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal Gr protein deficiency in 5-hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development.
Assuntos
Desenvolvimento Embrionário/genética , RNA Mensageiro Estocado/genética , Receptores de Glucocorticoides/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Apoptose/fisiologia , Sequência de Bases , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro Estocado/metabolismo , Receptores de Glucocorticoides/metabolismo , Peixe-Zebra/metabolismoRESUMO
Flow cytometry allows an easy quantitation of reactive oxygen intermediate production and of C3bi receptor expression by granulocytes, thus providing a clinical tool to evaluate the hemodialysis-related granulocyte activation. In this flowcytometric study, we analyzed the effects of cellulosic, synthetic polyacrylonitrile, and ethylene-vinyl-alcohol dialyzers on granulocyte membrane and function. Our results confirmed the data reported in the literature on granulocyte activation secondary to cellulosic membranes and the better biocompatibility of synthetic dialyzers that did not increase C3bi receptor expression and reactive oxygen intermediate generation by granulocytes. The flow-cytometric analysis of granulocyte activation might be the method of choice to identify the best patient/membrane interaction in every single patient.
