Loss of Atrx Sensitizes Cells to DNA Damaging Agents through p53-Mediated Death Pathways

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrxf/f mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors.

在前脑和塞尔托利细胞（Sertoli cell）特异性敲除Atrx的小鼠中出现广泛的细胞死亡，表明Atrx对细胞存活至关重要。然而，在其他组织中进行条件性敲除并不会导致细胞死亡增加，这说明不同细胞类型对该染色质重塑蛋白的缺失会产生不同的应答反应。本研究中，我们从Atrxf/f小鼠中分离原代巨噬细胞，用表达Cre重组酶（Cre recombinase）或β-半乳糖苷酶（β-galactosidase）的腺病毒进行感染，并在不同实验条件下检测细胞存活情况。巨噬细胞在缺失Atrx的情况下仍可存活，但在脂多糖（lipopolysaccharide, LPS）激活后会快速发生凋亡，这表明细胞响应外界刺激时的染色质重组过程受到了损伤。利用该实验体系，我们进一步检测了不同凋亡刺激因素对细胞存活的影响。我们观察到，在血清剥夺、抗Fas抗体、C2神经酰胺或地塞米松处理的情况下，Atrx基因敲除型（Atrx-null）细胞的存活情况与野生型细胞相似，但对5-氟尿嘧啶（5-fluorouracil, 5-FU）更为敏感。通过重新导入Atrx或敲除p53可挽救细胞存活，这证明该效应具有细胞自主性，且依赖于p53通路。最后，我们证实多种原代细胞类型——即成肌细胞（myoblasts）、胚胎成纤维细胞（embryonic fibroblasts）及神经球（neurospheres）——对5-FU、顺铂（cisplatin）及紫外线（UV）照射处理均表现出敏感性。综合来看，我们的研究结果表明，缺失Atrx的细胞对DNA损伤剂更为敏感，这可能会在细胞处于增殖高峰期的发育过程中加剧细胞死亡。此外，本研究为携带ATRX突变的癌症（包括胶质母细胞瘤（glioblastoma）和胰腺神经内分泌肿瘤（pancreatic neuroendocrine tumors））提供了潜在的治疗策略。