RESUMO
The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.
Assuntos
DNA Helicases/genética , Histonas , Proteínas Nucleares/genética , Homeostase do Telômero/genética , Telômero/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Correpressoras , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Instabilidade Genômica , Células HeLa , Histonas/genética , Histonas/metabolismo , Recombinação Homóloga , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Transdução de Sinais , Telomerase/genética , Telômero/metabolismo , Proteína Nuclear Ligada ao XRESUMO
In congenital and acquired long QT type 2, women are more vulnerable than men to torsade de pointes. In prepubertal rabbits (and children), the arrhythmia phenotype is reversed; however, females still have longer action potential durations than males. Thus, sex differences in K(+) channels and action potential durations alone cannot account for sex-dependent arrhythmia phenotypes. The L-type calcium current (I(Ca,L)) is another determinant of action potential duration, Ca(2+) overload, early afterdepolarizations (EADs), and torsade de pointes. Therefore, sex, age, and regional differences in I(Ca,L) density and in EAD susceptibility were analyzed in epicardial left ventricular myocytes isolated from the apex and base of prepubertal and adult rabbit hearts. In prepubertal rabbits, peak I(Ca,L) at the base was 22% higher in males than females (6.4+/-0.5 versus 5.0+/-0.2 pA/pF; P<0.03) and higher than at the apex (6.4+/-0.5 versus 5.0+/-0.3 pA/pF; P<0.02). Sex differences were reversed in adults: I(Ca,L) at the base was 32% higher in females than males (9.5+/-0.7 versus 6.4+/-0.6 pA/pF; P<0.002) and 28% higher than the apex (9.5+/-0.7 versus 6.9+/-0.5 pA/pF; P<0.01). Apex-base differences in I(Ca,L) were not significant in adult male and prepubertal female hearts. Western blot analysis showed that Ca(v)1.2alpha levels varied with sex, maturity, and apex-base, with differences similar to variations in I(Ca,L); optical mapping revealed that the earliest EADs fired at the base. Single myocyte experiments and Luo-Rudy simulations concur that I(Ca,L) elevation promotes EADs and is an important determinant of long QT type 2 arrhythmia phenotype, most likely by reducing repolarization reserve and by enhancing Ca(2+) overload and the propensity for I(Ca,L) reactivation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Síndrome do QT Longo/etiologia , Miócitos Cardíacos/metabolismo , Torsades de Pointes/etiologia , Potenciais de Ação , Fatores Etários , Animais , Canais de Cálcio Tipo L/genética , Simulação por Computador , Feminino , Ventrículos do Coração/metabolismo , Ativação do Canal Iônico , Síndrome do QT Longo/induzido quimicamente , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Masculino , Modelos Cardiovasculares , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Pericárdio/metabolismo , Fenótipo , Bloqueadores dos Canais de Potássio/toxicidade , Pirrolidinas/toxicidade , RNA Mensageiro/metabolismo , Coelhos , Projetos de Pesquisa , Fatores de Risco , Fatores Sexuais , Trocador de Sódio e Cálcio/metabolismo , Fatores de Tempo , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/metabolismo , Torsades de Pointes/fisiopatologiaRESUMO
Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis-deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease.
