Department of Radiation Oncology, University of Pittsburgh, 5117 Centre Avenue, Pittsburgh, PA, 15213-1863, United States; Department of Pharmacology and Chemical Biology, University of Pittsburgh, 5117 Centre Avenue, Pittsburgh, PA, 15213-1863, United States.
Department of Medicine, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA, 15213-1863, United States.
DNA Repair (Amst). 2019 Aug;80:1-7. doi: 10.1016/j.dnarep.2019.06.002. Epub 2019 Jun 4.
Since many anticancer therapies target DNA and DNA damage response pathways, biomarkers of DNA damage endpoints may prove valuable in basic and clinical cancer research. Ataxia telangiectasia-mutated (ATM) kinase is the principal regulator of cellular responses to DNA double-strand breaks (DSBs). In humans, ATM autophosphorylation at serine 1981 (p-S1981) is an immediate molecular response to nascent DSBs and ionizing radiation (IR). Here we describe the analytical characteristics and fit-for-purpose validation of a quantitative dual-labeled immunoblot that simultaneously measures p-S1981-ATM and pan-ATM in human peripheral blood mononuclear cells (PBMCs) following ex vivo exposure to 2 Gy IR, facilitating the calculation of %p-ATM. To validate our assay, we isolated PBMCs from 41 volunteers. We report that the median basal level of p-S1981-ATM and pan-ATM was 2.4 and 49.5 ng/10 PBMCs, respectively, resulting in %p-ATM of 4%. Following exposure of PBMCs to 2 Gy IR, p-S1981-ATM levels increased 12-fold to 29.8 ng/10 PBMCs resulting in %p-ATM of 63%. Interestingly, we show that PBMCs from women have a 2.6-fold greater median p-S1981-ATM level following IR exposure than men (44.4 versus 16.9 ng/10 cells; p < 0.01). This results in a significantly greater %p-ATM for women (68% versus 49%; p < 0.01). Our rigorous description of the analytical characteristics and reproducibility of phosphoprotein immunoblotting, along with our finding that the ATM DNA damage response is greater in women, has far reaching implications for biomedical researchers.
由于许多抗癌疗法针对 DNA 和 DNA 损伤反应途径，因此 DNA 损伤终点的生物标志物在基础和临床癌症研究中可能具有重要价值。共济失调毛细血管扩张突变（ATM）激酶是细胞对 DNA 双链断裂（DSB）反应的主要调节剂。在人类中，ATM 丝氨酸 1981 位的自身磷酸化（p-S1981）是对新形成的 DSB 和电离辐射（IR）的即时分子反应。在这里，我们描述了一种定量双标记免疫印迹的分析特性和适用性验证，该方法可同时测量人类外周血单核细胞（PBMC）在离体暴露于 2GyIR 后 p-S1981-ATM 和泛 ATM 的含量，从而计算出 %p-ATM。为了验证我们的测定方法，我们从 41 名志愿者中分离出 PBMC。我们报告说，p-S1981-ATM 和泛 ATM 的中位基础水平分别为 2.4 和 49.5ng/10 PBMC，导致 %p-ATM 为 4%。在 PBMC 暴露于 2GyIR 后，p-S1981-ATM 水平增加了 12 倍，达到 29.8ng/10 PBMC，导致 %p-ATM 为 63%。有趣的是，我们发现，与男性相比，女性在接受 IR 暴露后 PBMC 中的 p-S1981-ATM 水平高 2.6 倍（44.4 与 16.9ng/10 个细胞；p<0.01）。这导致女性的 %p-ATM 显著更高（68%比 49%；p<0.01）。我们对磷酸化蛋白免疫印迹的分析特性和可重复性进行了严格描述，并且发现 ATM DNA 损伤反应在女性中更强，这对生物医学研究人员具有深远的意义。
