AP Endonuclease 2 in hematopoietic stem cell maintenance

描述(申请人提供)：这项提案将确定DNA修复蛋白AP核酸内切酶2(APE2)对于维持造血干细胞(HSC)功能和这些细胞对辐射的抗性是否重要。人们对静止的HSCs的DNA修复机制知之甚少，这些细胞在一生中保持着多谱系再繁殖的潜力，但保护它们的基因组对血细胞的终生供应至关重要。造血干细胞的基因组不稳定性与年龄、表型和白血病的发生有关。正常情况下，HSC相对耐辐射，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，放射治疗是一种广泛使用的癌症治疗方法，它会产生大量的活性氧物种，即使在未分裂的细胞中也会导致DNA断裂。APE2是一种新近发现的修复蛋白，对其在体内的功能知之甚少。我推测APE2有助于修复HSCs中的单链DNA断裂，这对于保护HSCs的基因组完整性和功能免受辐射损伤是重要的。此外，APE2还可能对HSC内源性氧化损伤起到保护作用。这种类型的损伤与HSC和所有细胞类型的衰老表型有关。在体外，APE2在去除阻止DNA断裂修复的3‘端封闭基团方面是有效的，许多辐射诱导的断裂具有被3’-磷酸乙醇酸部分封闭的DNA末端。我将通过将FACS纯化的造血干细胞暴露在增加剂量的电离辐射中，并分析DNA损伤、修复和HSC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实验和3H_2AX焦点的测量来确定，照射后不同时间。未经处理和辐射的HSC将在使用甲基纤维素培养的克隆存活分析中进行功能分析，也将通过移植到辐射受体小鼠中进行分析。我已经证明，APE2在发育过程中对骨髓中淋巴前体的快速扩张很重要，在骨髓从化疗耗竭中恢复的过程中尤其重要。这里提出的实验将确定APE2是否对保护HSCs的基因组稳定性免受内源性基因毒性应激和辐射暴露也是重要的。HSC对辐射的抵抗力是使接受放射治疗的患者的造血系统恢复的一个重要特征。然而，修复细胞内辐射损伤的相同因素也会导致HSC对旨在消除癌症干细胞的治疗产生抵抗力。因此，重要的是确定可能导致治疗耐药的蛋白质，并将其作为联合疗法提高疗效的靶点。

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.