The Fanconi Anemia Pathway in Inflammatory Senescent Cells

我们在处理这一问题时侧重于两个问题。第一个是关于FANCD 2参与DDR的，FANCD 2是FA途径的中心蛋白。我们关于DDR的大部分信息来自于对双链断裂（DSB）的研究。一些蛋白质在DNA断裂附近被招募，而另一些蛋白质则位于数百至数千个碱基之外。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3个数量级。因此，我们研究了FANCD 2在由碱反应性化合物诱导的DDR中的参与，我们将免疫标签附于该碱反应性化合物。我们开发了一种新的实验方法，基于免疫标记，以区分DDR蛋白，接近DNA损伤的那些是遥远的。在两个空间上可分离的组群中，FANCD 2被招募到DNA损伤中。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DDR蛋白质相关，并依赖于DDR蛋白质位于距离碱基修饰。该队列未参与损伤的清除。很可能这部分参与了压力信号传导。我们现在正在研究它在炎症途径调节中的作用。 我们还确定了一种新的衰老培养细胞模型。我们将这些细胞暴露于DNA损伤剂，然后它们在7天后衰老。与衰老频率为60- 70%的其他细胞培养模型相反，这些细胞100%变得衰老。此外，再次与其他模型相反，没有抗衰老细胞的生长。相反，细胞保持活力和衰老长达6周。 先前的文献表明组蛋白变体H2 AX对衰老的发展有重要贡献。然而，在新系统中，衰老不依赖于H2 AX。也就是说，H2 AX敲除细胞与其中H2 AX已重新表达的相同敲除细胞一样易受DNA损伤诱导的衰老的影响。DNA损伤反应标志物在衰老细胞的病灶中可见，尽管不存在磷酸-H2 AX。FANCD 2是进入衰老过程中病灶的一种成分，但随着细胞完全衰老而消失。出乎意料的是，增殖的H2 AX敲除细胞表达干扰素应答的组分，这通常被认为是衰老细胞的特征。 我们的研究结果表明，衰老诱导和维持的替代模式。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.