Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Namur Research Institute for Life Science (NARILIS - LARN), University of Namur, 5000 Namur, Belgium.
Radiat Res. 2021 Jan 1;195(1):47-59. doi: 10.1667/RADE-20-00165.1.
Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double-strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1 (53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of their proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 72 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ∼10-fold decrease from low to full confluence that is independent of mouse strain. On the other hand, the baseline level of spontaneous 53BP1+ foci in a fully confluent cell population was strain-dependent, suggesting genomic associations, and correlated with radiation sensitivity based on previous measurements in the same cell lines. Finally, we have developed an online open-access tool to correct for the effect of cell confluence on 53BP1+ foci-based quantification of DSB. This tool provides guidelines for the number of cells required to reach statistical significance for the detection of DSB induced by low doses of ionizing radiation as a function of confluence and time postirradiation.
由于高代谢活性，增殖细胞不断产生自由基，诱导 DNA 双链断裂（DSB）。荧光标记的 DNA 修复蛋白 53 结合蛋白 1（53BP1）核焦点被用作测量基线和环境刺激（如辐射）下 DSB 形成的标准指标。在这里，我们证明自发的 53BP1+焦点形成的背景水平可以用细胞汇合度的函数来进行数学建模，细胞汇合度是其增殖率的一种度量。该模型使用来自 15 种不同品系的小鼠的 72 个原代皮肤成纤维细胞培养物的自发 53BP1+焦点数据进行了验证，显示从低到全汇合度的自发 53BP1+焦点减少了约 10 倍，这与小鼠品系无关。另一方面，在完全汇合的细胞群体中，自发的 53BP1+焦点的基线水平是依赖于品系的，这表明存在基因组关联，并与同一细胞系中之前的辐射敏感性测量结果相关。最后，我们开发了一种在线开放访问工具，用于校正细胞汇合度对基于 53BP1+焦点的 DSB 定量的影响。该工具提供了根据汇合度和辐照后时间，检测低剂量电离辐射诱导的 DSB 所需的细胞数量的指导方针，以达到统计学意义。
