The Fanconi Anemia Pathway in Inflammatory Senescent Cells

在解决这个问题的方法中，我们主要关注两个问题。第一个与FANCD2的DDR参与有关，FANCD2是FA通路的中心蛋白。我们关于DDR的大部分信息来自对双链断裂（DSBs）的研究。一些蛋白质是在DNA断裂附近招募的，而另一些则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碱基损伤和丢失的发生频率至少高出3个数量级。因此，我们检测了FANCD2在碱基反应性化合物诱导的DDR中的参与，并在其上附加了免疫标记。我们开发了一种新的实验方法，基于免疫标记，区分DNA损伤附近和远处的DDR蛋白。FANCD2被招募到两个空间可分离的DNA损伤队列中。其中一个位于DNA损伤附近，有助于损伤的修复。第二个，稍大的部分，与位于距离碱基修饰一段距离的DDR蛋白相关，并依赖于DDR蛋白。这组患者没有参与损伤的清除。这部分很可能与应激信号有关。我们现在正在研究它在调节炎症途径中所起的作用。

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 model of cell senescence. We expose cells to a DNA damaging agent. 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 senescent. Furthermore, 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. Our results suggest an alternative mode of senescence induction and maintenance. Future studies will elucidate the basis of this distinction and extend these observations to an in vivo model.