Wittlinger Michael, Grabenbauer Gerhard G, Sprung Carl N, Sauer Rolf, Distel Luitpold V R
Department of Radiation Oncology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.
Int J Radiat Biol. 2007 Apr;83(4):245-57. doi: 10.1080/09553000701275432.
Proper detection of DNA damage and signal transduction to other proteins following irradiation (IR) is essential for cellular integrity. The serine 15 (Ser15) on p53 is crucial for p53 stabilization and a requirement for transient and permanent cell cycle arrest. Here, we sought to determine the relationship between p53 serine 15 phosphorylation (p53-p-Ser15) on cellular sensitivity and if this modification is associated with DNA double-strand break (DSB) repair.
Eight lymphoblastoid cell lines including ataxia-telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and radiosensitive patient derived cell lines were irradiated with 1 Gy, 2 Gy and 5 Gy. Then growth inhibition, p53 induction and phosphorylation on Ser15 as assessed by immunoblotting and DNA DSB repair as assessed by constant field gel electrophoresis were examined.
Phosphorylation of p53 at Ser15 in control cells rapidly increased, peaking at 3-6 hours and then sustained a low level of phosphorylation for up to 6 days following IR. For these cell lines, the amount of p53-p-Ser15 corresponded to the sensitivity of cells and the amount of DNA DSB. In A-T cells, p53-p-Ser15 was reduced in spite of increased DNA DSB. NBS cells had similar phosphorylation dynamics as the control cell line, which was not consistent with their increased sensitivity. Radiosensitive patients' cell lines differed only slightly from controls.
Cells that are competent in signal transduction have p53-p-Ser15 kinetics corresponding to cellular radiosensitivity as assessed by clonogenicity and DNA DSB repair, and cells impaired in signal transduction lack this correspondence. Therefore, using p53-p-Ser15 as a general marker of radiation sensitivity has confounding factors which may impair proper radiosensitivity prediction.
正确检测辐射（IR）后DNA损伤并将信号转导至其他蛋白质对于细胞完整性至关重要。p53上的丝氨酸15（Ser15）对于p53的稳定以及短暂和永久性细胞周期停滞的需求至关重要。在此，我们试图确定p53丝氨酸15磷酸化（p53-p-Ser15）与细胞敏感性之间的关系，以及这种修饰是否与DNA双链断裂（DSB）修复相关。
包括共济失调毛细血管扩张症（A-T）、尼曼-匹克氏症候群（NBS）和放射敏感患者来源的细胞系在内的8种淋巴母细胞系分别接受1 Gy、2 Gy和5 Gy的照射。然后通过免疫印迹法检测生长抑制、p53诱导和Ser15磷酸化情况，并通过恒定电场凝胶电泳检测DNA DSB修复情况。
对照细胞中p53在Ser15处的磷酸化迅速增加，在3 - 6小时达到峰值，然后在IR后长达6天维持低水平磷酸化。对于这些细胞系，p53-p-Ser15的量与细胞敏感性和DNA DSB的量相对应。在A-T细胞中，尽管DNA DSB增加，但p53-p-Ser15减少。NBS细胞具有与对照细胞系相似的磷酸化动力学，这与它们增加的敏感性不一致。放射敏感患者的细胞系与对照仅略有不同。
在信号转导方面有能力的细胞具有与通过克隆形成能力和DNA DSB修复评估的细胞放射敏感性相对应的p53-p-Ser15动力学，而在信号转导方面受损的细胞则缺乏这种对应关系。因此，将p53-p-Ser15用作辐射敏感性的通用标志物存在可能损害正确放射敏感性预测的混杂因素。
