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FANCD2的DDR参与，FANCD2是FA途径的中心蛋白。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。第二个相对较大的部分，与与碱基修饰距离较远的DDR蛋白相关，并依赖于该蛋白。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。 我们还发现了一种新的衰老培养细胞模型。我们将这些细胞暴露在DNA损伤剂中，之后它们在7天后变得衰老。与衰老频率为60%-70%的其他细胞培养模型相比，这些细胞100%会衰老。此外，与其他模型不同的是，抗衰老细胞不会生长。相反，这些细胞可以存活和衰老长达6周之久。 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，在新的体系中，衰老与H_2AX无关。也就是说，H_2AX基因敲除细胞和重新表达H_2AX基因的相同基因敲除细胞一样，对DNA损伤引起的衰老敏感。DNA损伤反应标记物在衰老细胞的病灶中可见，尽管没有磷酸-H_2AX。FANCD2是进入衰老过程中病灶的一个组成部分，但随着细胞完全衰老而消失。出乎意料的是，正在增殖的H2 AX基因敲除细胞表达干扰素反应的组成部分，这通常被认为是衰老细胞的特征。 我们的结果提示了另一种衰老诱导和维持模式。未来的研究将阐明这种区别的基础，并阐明FA途径在高衰老频率中的作用。

We focused on two issues in our approach to this question. The first was concerned with the participation in the DDR of FANCD2, the central protein of the FA pathway. 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, DDR 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 also identified a new cultured cell model of senescence. We expose these cells to a DNA damaging agent, after which they become senescent after 7 days. In contrast to other cell culture models with senescence frequencies of 60-70%, 100% of these cells become senescent. Furthermore, again in contrast to other models, there is no outgrowth of senescence resistant cells. Instead the cells remain viable and senescent for as long as 6 weeks. Previous literature indicates an important contribution of the histone variant H2AX towards development of senescence. However, in the new system, senescence is independent of H2AX. That is, H2AX knockout cells are as susceptible to DNA damage induced senescence as the same knockout cells in which H2AX has been re-expressed. DNA Damage Response markers are visible in foci in the senescent cells, despite the absence of phospho-H2AX. FANCD2 is a component of the foci during entry into senescence, but disappears as the cells become fully senescent. Unexpectedly, the proliferating H2AX knockout cells express components of the interferon response, typically considered a characteristic of senescent cells. Our results suggest an alternative mode of senescence induction and maintenance. Future studies will elucidate the basis of this distinction and clarify the role of the FA pathway in the high senescence frequency.