RESUMO
The 3D nuclear architecture is closely related to cellular functions and chromosomes are organized in distinct territories. Quantitative 3D telomere FISH analysis (3D Q-FISH) and 3D super-resolution imaging (3D-SIM) at a resolution up to 80 nm as well as the recently developed combined quantitative 3D TRF2-telomere immune FISH technique (3D TRF2/Telo-Q-FISH) have substantially contributed to elucidate molecular pathogenic mechanisms of hematological diseases. Here we report the methods we applied to uncover major molecular steps involved in the pathogenesis of EBV-associated Hodgkin's lymphoma. These methods allowed us to identify the EBV-encoded oncoprotein LMP1 as a key element in the formation of Hodgkin (H-cell) and multinucleated Reed-Sternberg cells (RS-cell), the diagnostic tumor cell of classical Hodgkin's lymphoma (cHL). LMP1 mediates multinuclearity through downregulation of shelterin proteins, in particular telomere repeat binding factor 2 (TRF2).
Assuntos
Núcleo Celular/genética , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4 , Doença de Hodgkin/etiologia , Doença de Hodgkin/patologia , Imageamento Tridimensional/métodos , Hibridização in Situ Fluorescente/métodos , Telômero/genética , Anticorpos/imunologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Infecções por Vírus Epstein-Barr/virologia , Doença de Hodgkin/diagnóstico , Humanos , Células de Reed-Sternberg/metabolismo , Células de Reed-Sternberg/patologia , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
Abnormal telomere attrition has been found to be closely related to patients with SAA in recent years. To identify the incidence of telomere attrition in SAA patients and investigate the relationship of telomere length with clinical parameters, SAA patients (n=27) and healthy controls (n=15) were enrolled in this study. Telomere length of PWBCs was significantly shorter in SAA patients than in controls. Analysis of gene expression of Shelterin complex revealed markedly low levels of POT1 expression in SAA groups relative to controls. No differences in the gene expression of the other Shelterin components-TRF1, TRF2, TIN2, TPP1, and RAP1-were identified. Addition of IFN-γ to culture media induced a similar fall in POT1 expression in bone marrow cells to that observed in cells cultured in the presence of SAA serum, suggesting IFN-γ is the agent responsible for this effect of SAA serum. Furthermore, ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were all found similarly increased in bone marrow cells exposed to SAA serum, TNF-α, or IFN-γ. In summary, SAA patients have short telomeres and decreased POT1 expression. TNF-α and IFN-γ are found at high concentrations in SAA patients and may be the effectors that trigger apoptosis through POT1 and ATR.
Assuntos
Anemia Aplástica/genética , Células da Medula Óssea/imunologia , Leucócitos Mononucleares/imunologia , Proteínas de Ligação a Telômeros/imunologia , Telômero/genética , Adolescente , Adulto , Idoso , Anemia Aplástica/imunologia , Anemia Aplástica/patologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Expressão Gênica , Humanos , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Índice de Gravidade de Doença , Complexo Shelterina , Telômero/patologia , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/imunologia , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Telomerase-negative immortalized human cells maintain telomeres by alternative lengthening of telomeres (ALT) pathway(s), which may involve homologous recombination. We find that endogenous BLM protein co-localizes with telomeric foci in ALT human cells but not telomerase positive immortal cell lines or primary cells. BLM interacts in vivo with the telomeric protein TRF2 in ALT cells, as detected by FRET and co-immunoprecipitation. Transient over-expression of green fluorescent protein (GFP)-BLM results in marked, ALT cell-specific increases in telomeric DNA. The association of BLM with telomeres and its effect on telomere DNA synthesis require a functional helicase domain. Our results identify BLM as the first protein found to affect telomeric DNA synthesis exclusively in human ALT cells and suggest that BLM facilitates recombination-driven amplification of telomeres in ALT cells.
