Bouquet Fanny, Muller Catherine, Salles Bernard
Institut de Pharmacologie et de Biologie Structurale, CNRS/University Paul Sabatier Toulouse III UMR 5089, Toulouse, France.
Cell Cycle. 2006 May;5(10):1116-22. doi: 10.4161/cc.5.10.2799. Epub 2006 May 15.
The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to high toxicity and genetic instability. Various approaches have been undertaken to quantify the number of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation of the variant histone H2AX (named gammaH2AX), quantified by specific immunodetection approaches, has provided a valuable and highly sensitive method to monitor DSBs formation. Although it is admitted that the number of gammaH2AX foci reflected that of DSBs, contradictory reports leave open the question of a link between the disappearance of gammaH2AX signal and DSBs repair. We determined gammaH2AX expression (i) in cells either proficient or not in DSBs repair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin gamma1, a radiomimetic compound, (iii) and by three different immunodetection methods, foci numbering, flow cytometry or Western blotting. We showed here that gammaH2AX loss correlates with DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaks per genome are produced, independently of the method used. In addition, in DNA repair proficient cells, the early decrease in the number and intensity of gammaH2AX foci observed after a 2 Gy exposure was not associated with a significant change in the global gammaH2AX level as determined by Western blotting or flow cytometry. These results suggest that the dephosphorylation step of gammaH2AX may be limiting and that the loss of foci is mediated not only by gammaH2AX dephosphorylation but also through its redistribution towards the chromatin.
基因毒性处理诱导DNA双链断裂（DSB）会导致高毒性和基因不稳定。人们已采用多种方法来量化断裂数量并跟踪DSB修复的动力学过程。最近，通过特定免疫检测方法量化的变异组蛋白H2AX（称为γH2AX）的磷酸化，为监测DSB形成提供了一种有价值且高度灵敏的方法。尽管人们公认γH2AX焦点的数量反映了DSB的数量，但相互矛盾的报道使得γH2AX信号消失与DSB修复之间的联系问题仍未解决。我们通过以下方式确定γH2AX的表达：（i）在具有或不具有DSB修复能力的细胞中；（ii）在暴露于电离辐射（IR）或放射模拟化合物加利车霉素γ1之后；（iii）并采用三种不同的免疫检测方法，即焦点计数、流式细胞术或蛋白质免疫印迹法。我们在此表明，仅在低细胞毒性剂量下，即每个基因组产生少于100 - 150个DSB断裂时，γH2AX的丢失才与DSB修复活性相关，且与所使用的方法无关。此外，在DNA修复能力正常的细胞中，2 Gy照射后观察到的γH2AX焦点数量和强度的早期下降，与蛋白质免疫印迹法或流式细胞术测定的整体γH2AX水平的显著变化无关。这些结果表明，γH2AX的去磷酸化步骤可能具有局限性，并且焦点的丢失不仅由γH2AX去磷酸化介导，还通过其向染色质的重新分布介导。
