RESUMO
Heating induces histone H2AX phosphorylation at serine 139 (gammaH2AX). Phosphorylated H2AX subsequently forms foci in numerous mammalian cell lines. The aim of this study was to clarify details in the mechanisms involved in the phosphorylation of H2AX after heating. The cell lines used were DNA-PKcs knockout cells, ATM knockout cells, and their parental cell lines. To elucidate mechanisms of induction of phosphorylation of H2AX after heating, ATM/ATR inhibitor (CGK733) and DNA-PK inhibitor (NU7026) were used. The intensity of gammaH2AX signals was assayed with flow cytometry. The thermal dose-response curve for the fluorescence intensity of gammaH2AX appearance in DNA-PKcs-/- cells during the heating period was similar to that observed in DNA-PKcs+/+ cells. On the other hand, the slope of thermal dose-response curve for them in ATM-/- cells was lower than that in ATM+/+ cells. Phosphorylation of H2AX after heating was suppressed by a combination of CGK733 and NU7026 in the culture medium in DNA-PKcs-/- cells, ATM-/- cells and in their parental cells. Although the phosphorylation of H2AX after heating was not suppressed by NU7026 in their parental cells, such phosphorylation was suppressed by CGK733 in their parental cells. These results indicate that ATM is the predominant protein which is active in the phosphorylation of histone H2AX after heating.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Benzenoacetamidas/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Histonas/química , Temperatura Alta , Humanos , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Serina/química , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
A characteristic hot-filament type X-ray generator was constructed for irradiation of cultured cells. The source provides copper K, iron K, chromium K, molybdenum L, aluminium K and carbon K shell characteristic X-rays. When cultured mouse m5S cells were irradiated and frequencies of dicentrics were fitted to a linear-quadratic model, Y = alphaD + betaD2, the chromosomal effectiveness was not a simple function of photon energy. The alpha-terms increased with the decrease of the photon energy and then decreased with further decrease of the energy with an inflection point at around 10 keV. The beta-terms stayed constant for the photon energy down to 10 keV and then increased with further decrease of energy. Below 10 keV, the relative biological effectiveness (RBE) at low doses was proportional to the photon energy, which contrasted to that for high energy X- or gamma-rays where the RBE was inversely related with the photon energy. The reversion of the energy dependency occurred at around 1-2 Gy, where the RBE of soft X-rays was insensitive to X-ray energy. The reversion of energy-RBE relation at a moderate dose may shed light on the controversy on energy dependency of RBE of ultrasoft X-rays in cell survival experiments.
Assuntos
Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular/efeitos da radiação , Aberrações Cromossômicas , Raios X , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Relação Dose-Resposta à Radiação , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Doses de Radiação , Radiometria , Eficiência Biológica Relativa , Espalhamento de RadiaçãoRESUMO
Exon shuffling, the juxtaposition and new combinations of exons from different genes, facilitates evolutionary changes by increasing protein diversity or by generating new function. Exon shuffling is generated as a consequence of segmental duplications. Long interspersed element (LINE)-1 (L1)-mediated 3' transduction is a potential pathway for exon shuffling by which L1 associates 3' flanking DNA in cis as a read-through transcript and carries the DNA to a new genomic location. In this pathway, however, the targets are limited to the regions located 3' to L1s. Here we propose that the genomic DNA distant from L1 may be mobilized by an alternative (trans) action of L1. A partial ATM sequence containing a single exon and flanking introns has been retrotransposed to a new genomic location on chromosome 7. There was no L1 around the exon of the authentic ATM locus. An unusual feature that the poly(A) tail tagged to the transposed sequence oriented oppositely to the ATM's transcriptional orientation suggests that a trans action of reverse transcriptase on antisense transcript has driven the duplication of genomic DNA without removing introns. Taking account of similar duplication events in previous studies, a certain class of segmental duplications in the human genome may be accounted for by the trans action of retrotransposon machinery.
Assuntos
Éxons/genética , Proteínas Serina-Treonina Quinases/genética , Retroelementos/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular , Cromossomos Humanos Par 7 , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA/genética , Proteínas de Ligação a DNA , Duplicação Gênica , Genoma Humano , Humanos , Elementos Nucleotídeos Longos e Dispersos , Dados de Sequência Molecular , Primatas/genética , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Transdução Genética , Proteínas Supressoras de TumorRESUMO
Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.
