Cigarette smoke, sunlight, and diagnostic computerized tomography (CT) are some of the many environmental exposures that can cause DNA damage. Associated carcinogens include tobacco specific nitrosoamines, UV- and ionizing-radiation. Each exposure and DNA damaging agent can increase cancer incidence and fuel the question """"""""Why do some people get cancer after exposure while others do not?"""""""" Interindividual variability at the molecular level is believed to be a major determinant of exposure induced disease, with lower-than-average DNA repair capacity associated with increased cancer onset1-4. Measures of cellular DNA repair capacity could be useful tools to guide lifestyle or clinical procedure decisions, but assays that measure DNA repair capacity are technically challenging, low throughput, and expensive. DNA repair capacity is ultimately dictated by the DNA damage response (DDR), a hundred-plus protein network associated with damage-induced signal transduction, cell cycle, and DNA repair pathways2,5-6. The integrity, protein levels, and protein modification status of all components in this system ultimately dictate cellular DNA repair capacity. Defining and quantifying cellular DNA repair capacity is thus a complex task that requires detailed information on the levels, modification status, and integrity of hundreds of proteins associated with the DDR. We propose to further develop a multiplexed protein quantification (MPQ) assay to measure the levels and post translational modification status of 60 DDR protein targets en masse. In order to implement our assay we will use an existing high throughput platform available from MesoScale Discoveries (MSD). This technology is based on antibody capture and electrochemiluminescence detection of a specific protein. Unique features of the MSD technology include a wide dynamic range (six logs), the ability to measure femtogram quantities, the ability to expand to 1,564 targets, 5-minute readout for each plate, and ELISA-like technology that is easily transferable to diagnostic labs. We will validate antibodies specific to 60 DDR targets for use in a MPQ assay, with targets specific to DNA repair pathways that respond to ionizing radiation damage. This MPQ assay will be further validated using DDR compromised cells. We will also use the DDR specific MPQ assay in conjunction with phenotypic endpoints to score the DNA repair capacity of 8 similar yet genetically heterogeneous cell lines. The resulting data will be computationally analyzed to identify protein- based biomarker signatures of DNA repair capacity. As proof of clinical feasibility and assay sensitivity we will also perform a clinical test of our assay to analyze CT induced changes in DDR protein levels and to document the in vivo response to radiation. Ultimately we will us proposed studies to test our hypothesis that the MSD technology platform can be adapted to measure DNA repair capacity in laboratory and clinical systems.
PROJECT NARRATIVE Multiplexed Quantification of DNA Damage Response Proteins Deficiencies in the cellular DNA damage response have been implicated in the onset of environmentally induced cancers. Diagnostic assays to quantitate cellular DNA repair capacity would thus provide an important tool for clinical and population studies, as a robust assay to measure DNA repair capacity could be used to identify susceptible populations. We propose to further develop a multiplexed protein quantification tool that will measure the levels and modification status of many DNA repair proteins, and we will test this tool as a measure for cellular DNA repair capacity. Using leukocyte samples derived from patients before and after computerized tomography we will also demonstrate the clinical feasibility of our assay and identify the in vivo DNA repair pathways activated by this controlled exposure to ionizing radiation. Successful completion of this project would help provide a diagnostic tool for population and clinical studies, and provide a resource for cancer biology and chemotherapeutic development.
|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|