Abstract 2841: A tool for genome-wide induction of 8-oxo-guanine to study DNA biology and genome stability
Authors: Aninda S. Dey, Natalia Cestari Moreno, Ryan P. Barnes
Journal: Cancer Research (2025)

Abstract Oxidative stress is when cells experience more reactive oxygen species (ROS) than their antioxidant defenses can handle. ROS are generated by metabolic processes, environmental toxins, and radiation. ROS can react with guanine nucleotides and produce one of the most common and physiologic forms of DNA damage, 8-oxo-guanine (8oxoG). The increased level of ROS is highly linked to the development of cancer due to DNA oxidation. ROS reaction with DNA can produce a variety of DNA lesions, such as base alterations, strand breaks, and cross-linking, which, if not corrected, can result in mutations and genomic instability. The body's capacity to resist oxidative stress via antioxidant defenses is critical for preserving genomic integrity; yet, when these defenses are overwhelmed, the risk of cancer development increases. Understanding the processes underlying oxidative DNA damage is crucial for developing targeted cancer prevention and treatment. Current methods to study this damage involve harsh chemical treatments (Hydrogen Peroxide, Potassium Bromate etc.) that affect overall DNA and cell integrity and are not specific in the type of DNA damage generated. To overcome this, our group developed chemoptogenetic tools (FAP) which produce singlet oxygen throughout the genome, to study the impact of 8-oxo-guanine on DNA damage. Singlet oxygen is a great model ROS because when it reacts with DNA, it only produces 8oxoG. The main objective of this project is to develop a system for genome-wide induction of 8oxoG. To achieve this, we produced U2OS and RPE cells that express the H2B-mCER-FAP and sorted them based on mCER expression to derive subpopulations. Expression of H2B-FAP does not impair cell growth, nor disrupt histone chromatin loading. These cells were treated with a high-intensity 660 nm LED light at 100 mW/cm2 for a set period to excite the FAP-bound MG2I dye and produce singlet oxygen genome-wide. We confirmed the induction of 8oxoG by pulsed-field gel electrophoresis and are in the process of confirming by mass spectrometry. Currently, we are conducting DNA damage and survival experiments using dye and light to study how the activation impacts cell viability. Four-day growth experiments were performed on U2OS H2B-FAP cells, with increasing dye concentration and brightness of the light. The experiments successfully demonstrated the activation of the system. In addition, we performed clonogenic survival assays with U2OS/RPE H2B-FAP cells where a decreasing number of viable colonies with the increasing dye concentration was observed. Our goal is to characterize the new histone FAP system in terms of lesion production, cell viability, and genome stability, and compare it with other tools that produce different ROS. As the tool produces targeted 8oxoG it will be extremely helpful to study oxidative DNA damage in depth which might be helpful in developing cancer prevention and treatment. Citation Format: Aninda S. Dey, Natalia Cestari Moreno, Ryan P. Barnes. A tool for genome-wide induction of 8-oxo-guanine to study DNA biology and genome stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2841.