Abstract LB-336: High-content, high-throughput imaging cytometry for fully automated DNA damage analysis
Authors: Holden Elena , Newmark Judith , Wong Connie , François Maxime , Fenech Michael , Leifert Wayne , Darzynkiewicz Zbigniew 
Published: July 2012

Abstract
Assessment of DNA damage is a vital part of oncology research and cancer drug development. In this study, we present a novel method using laser scanning cytometry (LSC) to automate a panel of DNA damage assays that are traditionally scored manually. The comet assay is one of the simplest and most widely-used methods to evaluate DNA damage. In order to fully automate this assay, we successfully developed the first algorithm that can reliably segment and quantify the amount of DNA present in the comet head versus the tail separately, providing a standardized and fully automated measurement of DNA damage. In the micronucleus (MN) assay, measurement of micronuclei formed from chromosome breakage and/or malsegregation provides an assessment of DNA damage and serves as a biomarker of genotoxicity and mutagenicity. Using simultaneous measurements by LSC of cellular morphology, nuclear and MN counts, precise DNA content, and specific molecular biomarker intensity, we developed a protocol to identify and quantify different cell sub-types within human buccal mucosa tissue consisting of basal cells, differentiated cells and bi-nucleated cells, as well as scoring cells which contain MN. The automated high-throughput generation of this data allows a high content analysis “cytome” assay of DNA damage, chromosome instability, cell death and the regenerative potential of human buccal mucosal tissue in response to exogenous and endogenous insults.
The multiplexed DNA damage response assay combines DNA content measurement with assessment of ATM and H2AX phosphorylation. The integrated signal of each of the above markers allows correlation of the extent of DNA damage with the cell cycle phase. We employed LSC's automated imaging features to extend the dynamic range of measurable damage induced by various genotoxic agents, allowing identification and enumeration of DNA damage-reporting foci and analysis of expression and degree of overlap of these marker phospho-proteins in individual foci.
Induction of cell senescence combined with reproductive cell death is the goal of chemotherapy and radiotherapy of solid tumors. The morphometric analytical capabilities of LSC were used to develop new biomarkers that reveal the degree (depth) of cell senescence based on senescence-characteristic morphological features. Combining these biomarkers with markers of DNA damage response, we assessed several factors that enhanced or attenuated senescence induced by anticancer drugs.
In conclusion, we have developed a panel of LSC algorithms that allow previously unavailable automation and standardization of high-content, high-throughput DNA damage assays to be performed on a single platform.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-336. doi:10.1158/1538-7445.AM2011-LB-336