RESUMO
Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.
Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Interferon Tipo I/imunologia , Proteína Homóloga a MRE11/imunologia , Sequência de Aminoácidos , Animais , Carpas/genética , Carpas/virologia , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Resistência à Doença/genética , Resistência à Doença/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/classificação , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteína Homóloga a MRE11/classificação , Proteína Homóloga a MRE11/genética , Orthoreovirus de Mamíferos/imunologia , Orthoreovirus de Mamíferos/fisiologia , Filogenia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Ubiquitinas/genética , Ubiquitinas/imunologia , Ubiquitinas/metabolismoRESUMO
We derived a mouse model in which a mutant form of Nbn/Nbs1mid8 (hereafter Nbnmid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. The Nbnmid8 allele was expressed exclusively in hematopoietic lineages (in Nbn-/mid8vav mice). Unlike Nbnflox/floxvav mice with Nbn deficiency in the bone marrow, Nbn-/mid8vav mice were viable. Nbn-/mid8vav mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn-/mid8 mice developed highly penetrant T cell leukemias. Nbn-/mid8vav leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbnmid8 mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn-/mid8vav malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1 We propose that overexpression of MRE11 compensates for the metastable Mre11-Nbnmid8 interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.
Assuntos
Hidrolases Anidrido Ácido/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Proteína Homóloga a MRE11/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfócitos T/imunologia , Hidrolases Anidrido Ácido/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/patologia , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/imunologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Instabilidade Genômica/imunologia , Hematopoese/genética , Hematopoese/imunologia , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/imunologia , Proteína Homóloga a MRE11/imunologia , Camundongos , Camundongos Knockout , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/prevenção & controle , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.
