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的参与，FA途径的中心蛋白，在DDR。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，相关的，并依赖于，蛋白质位于距离基地修改。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。 我们已经确定了一个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天后衰老。与衰老细胞频率为60- 70%的其他细胞培养模型相反，这些细胞中的100%在暴露于辐射后不久变成FANCD 2的衰老病灶。然而，它们随着细胞衰老而消失。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，在该系统中，衰老不依赖于H2 AX。目前的研究针对衰老细胞中FANCD 2表达的检查。

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.