Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate development of preventive strategies for a variety of diseases - either in standalone epidemiological studies, or to complement molecularly-based association studies. The proposed approach builds on a current fully-automated high-throughput biodosimetry system, the RABIT (Rapid Automated BIodosimetry Tool), which measures yields of 3-H2AX foci in lymphocytes, using fingerstick-based blood samples and an in-situ multi-well plate platform. The RABIT system currently assays only one DNA repair protein (3-H2AX), and at only one time for each sample;the system will be adapted to characterize DNA repair kinetics by A) adding an automated irradiator, to introduce DNA double strand breaks into the fingerstick sample, B) extending the robotically-based analysis system to allow automated measurements of a function of time after the irradiation challenge, C) extending the system to allow simultaneous measurements of more than one DNA repair protein. The system will be able to characterize, with high throughput (e1,000 samples / day) global DSB repair kinetics, as assayed by fully automated immunohistochemical measurements of 3-H2AX, ATM[ser1981], 53BP1, and Mdc1 repair proteins - chosen as representative of a range of nodal points in the main DSB repair pathways. Our goals are as follows: 1. Design and incorporate into the RABIT an innovative compact automated capillary irradiator, to introduce DNA DSBs into the fingerstick blood sample. Because we irradiate only short capillary tubes, and on a one- by-one sequential basis, the irradiator can be very compact, so low-activity 90Sr/90Y radioactive seeds can be used. Use of these radioactive seeds minimizes the shielding requirements, both because of the low activity and because these seeds emit short-ranged beta particles. 2. Adapt the RABIT so that it automatically assays each fingerstick sample for 3-H2AX, ATM[ser1981], 53BP1, and Mdc1, and at multiple times (0.5, 2, 4, 8, 24 hrs) after the automated irradiation. 3. Having already optimized the biological protocol for the 3-H2AX assay for use in our fully automated in-situ system, the same will be done for the other three repair proteins. 4. Establish a quantitative approach for comparing the measured DNA repair kinetics of different biomarkers / different samples, and to be able to identify outliers. 5. Demonstrate the practicality and scalability of the system by measuring, for the first time, global DSB repair kinetics in a healthy human population. Fingerstick capillary blood samples will be collected from 150 healthy volunteers, and automatically analyzed using the modified RABIT system. The four repair proteins whose kinetics will be analyzed represent a range of nodal points in the main DSB repair pathways.
Project Narrative Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate the development of preventive strategies for a variety of diseases.
|Nagel, Zachary D; Engelward, Bevin P; Brenner, David J et al. (2017) Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease. Mutat Res 800-802:14-28|
|Sharma, Preety M; Ponnaiya, Brian; Taveras, Maria et al. (2015) High throughput measurement of ?H2AX DSB repair kinetics in a healthy human population. PLoS One 10:e0121083|
|Turner, Helen C; Sharma, P; Perrier, J R et al. (2014) The RABiT: high-throughput technology for assessing global DSB repair. Radiat Environ Biophys 53:265-72|