Technologies that measure activation of components of the DNA damage response (DDR) have applications in exposure assessment and personalized medicine. The DDR and associated DNA repair pathways encompass hundreds of ...Technologies that measure activation of components of the DNA damage response (DDR) have applications in exposure assessment and personalized medicine. The DDR and associated DNA repair pathways encompass hundreds of proteins, making detailed measurement of activation technically challenging and laborious. The purpose of our study was to develop protein-specific assays for certain DDR components on a high-throughput electrochemiluminescence (ECL)-based platform. We developed five working assay pairs for ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), phosphorylated-ATM S1981, phosphorylated-CHK2 T68 and phosphorylated-tumor protein p53 (p53) S15. We validated the ECL results against traditional immunoblot and γ-H2AX foci measures in cell and cancer models. In an effort to test the ECL-based technology in a clinical setting, we utilized peripheral blood mononuclear cells (PBMCs) from patients undergoing computed tomography (CT) scans. CT scans represent both a valuable medical imaging diagnostic and a controlled environmental exposure to ionizing radiation for research studies, as they deliver ~2 to 31 millisieverts (mSv) and are known to activate DDR components. In this study, we show that ECL-based technology can measure the basal and damage-induced levels of DDR components in patient PBMC samples. Using a blinded study design and patient matched pre- and post CT scan samples, we show that ECL-derived data can consistently (94% of the time, 15/16 patients) identify PBMCs that have been exposed to low dose ionizing radiation associated with CT scans. Ultimately, the results of our pilot clinical study support the idea that ECL-based technology is applicable for use in clinical and population cohorts that study components of the DDR.展开更多
文摘Technologies that measure activation of components of the DNA damage response (DDR) have applications in exposure assessment and personalized medicine. The DDR and associated DNA repair pathways encompass hundreds of proteins, making detailed measurement of activation technically challenging and laborious. The purpose of our study was to develop protein-specific assays for certain DDR components on a high-throughput electrochemiluminescence (ECL)-based platform. We developed five working assay pairs for ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), phosphorylated-ATM S1981, phosphorylated-CHK2 T68 and phosphorylated-tumor protein p53 (p53) S15. We validated the ECL results against traditional immunoblot and γ-H2AX foci measures in cell and cancer models. In an effort to test the ECL-based technology in a clinical setting, we utilized peripheral blood mononuclear cells (PBMCs) from patients undergoing computed tomography (CT) scans. CT scans represent both a valuable medical imaging diagnostic and a controlled environmental exposure to ionizing radiation for research studies, as they deliver ~2 to 31 millisieverts (mSv) and are known to activate DDR components. In this study, we show that ECL-based technology can measure the basal and damage-induced levels of DDR components in patient PBMC samples. Using a blinded study design and patient matched pre- and post CT scan samples, we show that ECL-derived data can consistently (94% of the time, 15/16 patients) identify PBMCs that have been exposed to low dose ionizing radiation associated with CT scans. Ultimately, the results of our pilot clinical study support the idea that ECL-based technology is applicable for use in clinical and population cohorts that study components of the DDR.
