Image-based quantitative determination of DNA damage signal reveals a threshold for G2 checkpoint activation in response to ionizing radiation.
Image-based quantitative determination of DNA damage signal reveals a threshold for G2 checkpoint activation in response to ionizing radiation.
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DOI：
10.1186/2041-9414-1-10
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发表时间：
2010-08-04
期刊：
影响因子：
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通讯作者：
Yamashita S
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文献类型：
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作者：
Ishikawa A;Yamauchi M;Suzuki K;Yamashita S
参与DNA损伤反应的蛋白质在染色质侧翼的DNA双链断裂（DSB）上积累为显微镜可见的核灶。由于电离辐射（IR）诱导的病灶的生长放大了ATM依赖的DNA损伤信号，离散病灶的形成在细胞周期检查点激活中起着至关重要的作用，特别是在暴露于低剂量IR的细胞中。然而，没有考虑数量和大小的病灶的定量参数。因此，我们已经开发了一种新的参数的DNA损伤信号的基础上的图像分析的焦点和量化的信号量足以G2期阻滞。我们在这里开发的参数被指定为SOID。SOID是积分密度之和的缩写，其表示一个细胞核内每个焦点的荧光之和。将单个细胞核的SOID计算为（每个焦点的面积（总像素数））X（每个焦点的每个像素的平均荧光强度）的总和。因此，SOID占的数量，大小和荧光密度的IR诱导的焦点，和参数反映的DNA损伤信号的通量更准确地比焦点数。使用非常低剂量的X射线，我们进行了“双向”比较的SOID的Ser 139-磷酸化组蛋白H2 AX焦点之间的G2-停滞的细胞和有丝分裂-进展的细胞，有丝分裂-进展的细胞之间的ATM或Chk 1/2抑制剂的存在或不存在，这两个废除IR诱导的G2/M检查点。分析表明，G2期阻滞存在一个DNA损伤信号阈值，约为4000~5000 SOID。SOID < 4000的G2细胞被G2/M检查点忽略，因此细胞可以进行有丝分裂。染色体分析显示，检查点忽略和有丝分裂进行的细胞平均约有两个染色单体断裂，表明4000~5000 SOID相当于少量DNA双链断裂。我们提出了一个新的定量分析DNA损伤信号的参数，并确定了IR诱导的G2期阻滞的DNA损伤信号阈值，即4000~5000 SOID。本研究强调，不仅要考虑灶的数量，而且还必须考虑灶的大小，以适当的定量DNA损伤信号。
Proteins involved in the DNA damage response accumulate as microscopically-visible nuclear foci on the chromatin flanking DNA double-strand breaks (DSBs). As growth of ionizing radiation (IR)-induced foci amplifies the ATM-dependent DNA damage signal, the formation of discrete foci plays a crucial role in cell cycle checkpoint activation, especially in cells exposed to lower doses of IR. However, there is no quantitative parameter for the foci which considers both the number and their size. Therefore, we have developed a novel parameter for DNA damage signal based on the image analysis of the foci and quantified the amount of the signal sufficient for G2 arrest. The parameter that we have developed here was designated as SOID. SOID is an abbreviation of Sum Of Integrated Density, which represents the sum of fluorescence of each focus within one nucleus. The SOID was calculated for individual nucleus as the sum of (area (total pixel numbers) of each focus) x (mean fluorescence intensity per pixel of each focus). Therefore, the SOID accounts for the number, size, and fluorescence density of IR-induced foci, and the parameter reflects the flux of DNA damage signal much more accurately than foci number. Using very low doses of X-rays, we performed a "two-way" comparison of SOID of Ser139-phosphorylated histone H2AX foci between G2-arrested cells and mitosis-progressing cells, and between mitosis-progressing cells in the presence or absence of ATM or Chk1/2 inhibitor, both of which abrogate IR-induced G2/M checkpoint. The analysis revealed that there was a threshold of DNA damage signal for G2 arrest, which was around 4000~5000 SOID. G2 cells with < 4000 SOID were neglected by G2/M checkpoint, and thus, the cells could progress to mitosis. Chromosome analysis revealed that the checkpoint-neglected and mitosis-progressing cells had approximately two chromatid breaks on average, indicating that 4000~5000 SOID was equivalent to a few DNA double strand breaks. We developed a novel parameter for quantitative analysis of DNA damage signal, and we determined the threshold of DNA damage signal for IR-induced G2 arrest, which was represented by 4000~5000 SOID. The present study emphasizes that not only the foci number but also the size of the foci must be taken into consideration for the proper quantification of DNA damage signal.