The Fanconi Anemia Pathway in Inflammatory Senescent Cells

我们在处理这个问题时重点关注了两个问题。第一个涉及FA途径的中心蛋白FANCD2在DDR中的参与。我们关于DDR的大部分信息来自对双链断裂(DSB)的研究。一些蛋白质在接近DNA断裂的地方被招募，而另一些蛋白质位于数百到数千个碱基之外。然而，DSB在培养细胞中相对罕见，在循环淋巴细胞中更不常见。相比之下，DNA碱基损伤和丢失发生的频率至少高出3个数量级。因此，我们研究了FANCD2在碱性反应化合物诱导的DDR中的参与，我们将免疫标签附加到该化合物上。我们开发了一种新的实验方法，基于免疫标记，来区分接近DNA损伤的DDR蛋白和远离DNA损伤的DDR蛋白。在两个空间可分离的队列中，FANCD2被招募到DNA损伤中。其中一个位于DNA损伤附近，有助于损伤的修复。第二个相对较大的组分，与与碱基修饰距离较远的蛋白质相关，并依赖于这些蛋白质。这群人没有参与清除损坏的工作。这一部分很可能参与了压力信号的传递。我们现在正在研究它在调节炎症途径中所起的作用。 我们已经确定了一种合适的衰老细胞模型。我们将细胞暴露在高剂量的电离辐射下。这些细胞在7-10天后变得衰老。与其他衰老细胞率为60-70%的细胞培养模型相比，这些细胞100%成为衰老细胞，FANCD2的焦点在辐射后不久可见。然而，随着细胞的衰老，它们会消失。以往的文献表明，组蛋白变异体H_2AX对衰老的发生有重要贡献。然而，在这个系统中，衰老不受H_2AX的影响。目前的研究主要针对FANCD2在衰老细胞中的表达进行检测。

We focused on two issues in our approach to this question. The first was concerned with the participation of FANCD2, the central protein of the FA pathway, in the DDR. Most of our information about the DDR comes from studies of double strand breaks (DSBs). Some proteins are recruited close to DNA breaks, while others are located hundreds to thousands of bases away. However, DSBs are relatively rare in cultured cells and even less common in circulating lymphocytes. In contrast, DNA base damage and loss occurs at least 3 orders of magnitude more frequently. Consequently, we examined the participation of FANCD2 in the DDR induced by a base reactive compound, to which we attached an immunotag. We developed a new experimental approach, based on the immunotag, to differentiate DDR proteins that were close to the DNA damage from those that were distant. FANCD2 was recruited to the DNA damage in two spatially separable cohorts. One was located in close proximity to the DNA lesion and contributed to the repair of the lesion. A second, somewhat larger fraction, associated with, and was dependent on, proteins located at a distance from the base modification. This cohort had no involvement in removal of the damage. It is likely that this fraction is involved in stress signaling. We are now examining the role it plays in the regulation of inflammatory pathways. We have identified a suitable senescent cell model. We expose cells to high doses of ionizing radiation. These cells become senescent after 7-10 days. In contrast to other cell culture models with senescent cell frequencies of 60-70%, 100% of these cells become senescence Foci of FANCD2 are visible shortly after exposure to the radiation. However, they disappear as the cells become senescent. Previous literature indicates an important contribution of the histone variant H2AX towards development of senescence. However, in this system senescence is independent of H2AX. Current studies are directed towards an examination of FANCD2 expression in the senescent cells.