RESUMO
BACKGROUND: Nasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated with the Epstein-Barr virus. The viral genome is contained in the nuclei of all malignant cells with abundant transcription of a family of viral microRNAs called BART miRNAs. MicroRNAs are well known intra-cellular regulatory elements of gene expression. In addition, they are often exported in the extra-cellular space and sometimes transferred in recipient cells distinct from the producer cells. Extra-cellular transport of the microRNAs is facilitated by various processes including association with protective proteins and packaging in secreted nanovesicles called exosomes. Presence of microRNAS produced by malignant cells has been reported in the blood and saliva of tumor-bearing patients, especially patients diagnosed with glioblastoma or ovarian carcinoma. In this context, it was decided to investigate extra-cellular release of BART miRNAs by NPC cells and their possible detection in the blood of NPC patients. To address this question, we investigated by quantitative RT-PCR the status of 5 microRNAs from the BART family in exosomes released by NPC cells in vitro as well as in plasma samples from NPC xenografted nude mice and NPC patients. RESULTS: We report that the BART miRNAs are released in the extra-cellular space by NPC cells being associated, at least to a large extent, with secreted exosomes. They are detected with a good selectivity in plasma samples from NPC xenografted nude mice as well as NPC patients. CONCLUSIONS: Viral BART miRNAs are secreted by NPC cells in vitro and in vivo. They have enough stability to diffuse from the tumor site to the peripheral blood. This study provides a basis to explore their potential as a source of novel tumor biomarkers and their possible role in communications between malignant and non-malignant cells.
Assuntos
Exossomos/química , Herpesvirus Humano 4/patogenicidade , MicroRNAs/sangue , MicroRNAs/metabolismo , RNA Viral/sangue , RNA Viral/metabolismo , Adulto , Idoso , Animais , Carcinoma , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.
Assuntos
Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Ubiquitina/química , Ubiquitinação , Plaquetas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/química , Espectrometria de Massas , Selectina-P/metabolismo , Ativação Plaquetária , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
BACKGROUND/AIM: ß-Catenin is one of the key players in colonic carcinogenesis. Being part of the E-cadherin complex, it regulates cell-cell adhesion and the migratory ability of cells. However, the role of nuclear ß-catenin in the cell migration process is poorly understood. Gelsolin is one of the most abundant actin-binding proteins, and is implicated in tumour cell motility and invasiveness. The aim of the present study was to evaluate the potential association between expression of ß-catenin and gelsolin, and their influence on the migration ability of colon adenocarcinoma LS180 cells. MATERIALS AND METHODS: The colonic adenocarcinoma cell line LS180, its more motile sublines (EB3, 3LNLN, 5W) and ß-catenin-knockdown LS180 cells were used to investigate the expression levels and subcellular localization of ß-catenin and gelsolin. RESULTS: Increased motility of colonic cancer cells was accompanied by a reduction of ß-catenin and up-regulation of gelsolin. CONCLUSION: ß-Catenin seems to be involved in the regulation of gelsolin expression, which in turn affects the migratory ability of colonic cancer cells. Our results could have important implications for the design of new anticancer therapies.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Gelsolina/metabolismo , beta Catenina/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Interferência de RNA , beta Catenina/genéticaRESUMO
Activation of oxidative stress-responses and downregulation of insulin-like signaling (ILS) is seen in Nucleotide Excision Repair (NER) deficient segmental progeroid mice. Evidence suggests that this is a survival response to persistent transcription-blocking DNA damage, although the relevant lesions have not been identified. Here we show that loss of NTH-1, the only Base Excision Repair (BER) enzyme known to initiate repair of oxidative DNA damage inC. elegans, restores normal lifespan of the short-lived NER deficient xpa-1 mutant. Loss of NTH-1 leads to oxidative stress and global expression profile changes that involve upregulation of genes responding to endogenous stress and downregulation of ILS. A similar, but more extensive, transcriptomic shift is observed in the xpa-1 mutant whereas loss of both NTH-1 and XPA-1 elicits a different profile with downregulation of Aurora-B and Polo-like kinase 1 signaling networks as well as DNA repair and DNA damage response genes. The restoration of normal lifespan and absence oxidative stress responses in nth-1;xpa-1 indicate that BER contributes to generate transcription blocking lesions from oxidative DNA damage. Hence, our data strongly suggests that the DNA lesions relevant for aging are repair intermediates resulting from aberrant or attempted processing by BER of lesions normally repaired by NER.