AP Endonuclease 2 in hematopoietic stem cell maintenance

描述（由申请人提供）：该提案将确定DNA修复蛋白AP内切酶2 （APE2）对维持造血干细胞（HSC）功能和这些细胞对辐射的抗性是否重要。我们对静止造血干细胞的DNA修复机制知之甚少，这些造血干细胞在整个生命过程中都保持着多系再繁殖的潜力，但保护它们的基因组对于血细胞的终身供应至关重要。造血干细胞的基因组不稳定性与衰老表型和白血病发生有关。正常情况下，造血干细胞是相对耐辐射的，部分原因是它们处于静止状态，这有助于保护它们免受DNA损伤。然而，放射治疗，一种广泛使用的癌症治疗，会产生大量的活性氧，甚至在非分裂细胞中也会导致DNA断裂。APE2是最近发现的一种修复蛋白，对其在体内的功能知之甚少。我假设APE2有助于修复hsc中的单链DNA断裂，这对于保护hsc的基因组完整性和功能免受辐射暴露后的损伤是重要的。此外，APE2也可能保护HSC免受内源性获得性氧化损伤。这种类型的损伤与HSC和所有细胞类型的衰老表型相关。在体外，APE2可以有效去除阻止DNA断裂修复的3‘端阻断基团，并且许多辐射诱导的断裂具有被3’-磷酸乙醇酸部分阻断的DNA末端。我将通过增加电离辐射剂量暴露facs纯化的造血干细胞并分析DNA损伤、修复和造血干细胞功能来验证这一假设。单链和双链断裂的细胞百分比将通过彗星试验和测量3H2AX焦点，照射后的不同时间来确定。未经处理和辐照的HSC将在使用甲基纤维素培养和移植到辐照受体小鼠的克隆生存试验中进行功能分析。我已经证明，在骨髓发育过程中，APE2对于淋巴样前体的快速扩张是重要的，而且在骨髓从化疗耗竭中恢复时，它尤其重要。本文提出的实验将确定APE2是否对保护hsc免受内源性基因毒性应激和辐射暴露的基因组稳定性也很重要。HSC对辐射的抵抗力是放疗患者造血系统恢复的重要特征。然而，修复细胞辐射损伤的相同因素也可能导致HSC对旨在消除癌症干细胞的治疗产生耐药性。因此，确定可能导致对治疗产生耐药性的蛋白质是很重要的，这些蛋白质可能成为联合治疗提高疗效的目标。

DESCRIPTION (provided by applicant): This proposal will determine whether the DNA repair protein AP endonuclease 2 (APE2) is important for the maintenance of hematopoietic stem cell (HSC) function and for the resistance of these cells to radiation. Very little is known about DNA repair mechanisms in the quiescent HSCs that retain multi-lineage repopulation potential throughout life, but protection of their genome is essential for the lifelong supply of blood cells. Genomic instability in HSCs is associated with aging phenotypes and leukemogenesis. Normally, HSC are relatively radioresistant, in part due to their quiescent state, which helps to protect them from DNA damage. However, radiation treatment, a widely used cancer therapy, generates tremendous amounts of reactive oxygen species that can cause breaks in DNA, even in non-dividing cells. APE2 is a recently identified repair protein, and very little is known about its in vivo function. I hypothesize that APE2 helps to repair single- strand DNA breaks in HSCs, and that this is important to protect the genomic integrity and function of HSCs from damage following radiation exposure. In addition, APE2 might also provide protection to HSC from endogenously acquired oxidative damage. This type of damage is associated with aging phenotypes in HSC and all cell types. In vitro, APE2 is efficient at removal of 3'-end blocking groups that prevent repair of DNA breaks, and many radiation-induced breaks have DNA ends that are blocked by 3'-phosphoglycolate moieties. I will test this hypothesis by exposing FACS-purified hematopoietic stem cells to increasing doses of ionizing radiation and assaying DNA damage, repair, and HSC function. The percent of cells that have single- strand and double-strand breaks will be determined by comet assay and by measuring 3H2AX foci, various times after irradiation. Untreated and irradiated HSC will be analyzed functionally in clonogenic survival assays using methylcellulose cultures and also by transplantation into irradiated recipient mice. I have already shown that APE2 is important for the rapid expansion of lymphoid precursors in the bone marrow during development, and that it is especially important during recovery of the bone marrow from chemotherapeutic depletion. The experiments proposed here will determine if APE2 is also important to protect the genomic stability of HSCs from endogenous genotoxic stress and from radiation exposure. Resistance of HSC to radiation is an important feature that allows recovery of the hematopoietic system in patients treated with radiation. However, the same factors that repair radiation damage in cells can also cause the resistance of HSC to treatments intended to eliminate cancer stem cells. Therefore, it is important to identify proteins that could cause resistance to treatment and that could be a target for combination therapies to increase efficacy.