Abstract 3600: Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening

Abstract

Abstract The mechanism of action of many classes of chemotherapeutic agents involves either the repression of DNA repair or an increase in DNA damage. However the measurement of DNA damage levels within a cell has been notoriously difficult and current methods to asses DNA damage potential of new chemotherapeutics have major technical flaws. The single cell gel electrophoresis (SCGE) assay is a long-standing method for measuring levels of DNA damage within a cell. The principle of SCGE is that DNA damage can cause DNA strand breaks in cells. These breaks cause the relaxation of the compact highly supercoiled DNA. The application of an electric field while the cells are embedded in agarose allows damaged DNA to migrate faster than intact DNA. The more breaks in the DNA, the further it will travel in the agarose resulting in the formation of a “comet” tail, the size and length of which directly correlates to the amount of DNA damage. The SCGE method benefits from both technical simplicity and high sensitivity. The major drawback is the assay is extremely laborious, lacks appropriate controls and has poor reproducibility. We have recently overcome these drawbacks by developing a 96-well plate format of the SCGE, aptly named “CometChip”. The CometChip uses micro-pillar technology to create an agarose 96-well chip where each well has approximately 300 micro-wells used to capture individual cells. Using this technology we can incorporate multiple treatments, controls and time points on a single CometChip which can then be rapidly analyzed using a fluorescence based imaging instrument. The utility of the new technology was tested using 75 different chemical compounds considered either genotoxic, non-genotoxic or unknown. The compounds were tested on two lymphocyte cell lines with different p53 status to compare the accumulation and repair of DNA damage. We report that the CometChip gives highly reproducible and accurate results without loss of sensitivity. In the high throughput screening approach using multiple CometChip apparatus, we estimate the throughput of the assay to be approximately 10,000% greater than doing traditional slide based comet analysis. The massive increase in processivity brings new opportunity for large-scale compound screening. The increased sensitivity coupled with large sample sizes will allow researchers the option to measure minor changes in DNA damage with unparalleled accuracy. Citation Format: Peter Sykora, Sandra Woodgate, Jay George, Robert W. Sobol. Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3600.