Related Experiment Video
Updated: Jun 5, 2026
A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage
Published on: May 10, 2022
Development of a high-content screening method for chemicals modulating DNA damage response
Sunshin Kim1, Dong Hwa Jun, Hye Jin Kim
1Carcinogenesis Branch, Division of Cancer Biology, National Cancer Center, Goyang, Gyeonggi, Korea.
Abstract:
The cellular response to DNA damage is emerging as a promising target for cancer therapy. In the present study, the authors exploited the relationship between the level of the phosphorylated form of histone H2AX (γH2AX) and the extent of DNA damage and developed a quantitative, cell-based, high-content screening system for measuring the DNA damage response (DDR). In this system, the authors quantified the level of γH2AX by measuring DNA damage-induced γH2AX nuclear foci using an automated cell imager. They found that the total area of γH2AX foci per cell exhibited a good correlation with the concentration of DNA damage-inducing agents, including etoposide. The effects of 2 well-known inhibitors of DNA damage could be quantified using this system, suggesting the suitability of the γH2AX-foci quantification method for large-scale screening applications. This was confirmed by using this method to screen a chemical library; the resulting "hits" included compounds that inhibited early signaling events in DDR, as well those that inhibited subsequent DNA damage repair processes. Overall, this γH2AX foci-measuring system may be an effective screening method for identifying DNA damage response inhibitors that could eventually be used to develop novel anticancer drugs.
Insights
Researchers developed a new system to measure DNA damage response (DDR) by quantifying histone H2AX (γH2AX) foci. This high-content screening method shows promise for identifying novel anticancer drug candidates targeting DDR.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The cellular DNA damage response (DDR) is a critical target for cancer therapy.
- Histone H2AX phosphorylation (γH2AX) serves as a sensitive biomarker for DNA damage.
- Quantitative assessment of DDR is crucial for drug discovery.
Purpose of the Study:
- To develop a quantitative, cell-based, high-content screening system for measuring the DNA damage response (DDR).
- To validate the utility of γH2AX foci quantification for large-scale screening applications.
- To identify novel inhibitors of DDR for potential anticancer drug development.
Main Methods:
- Developed a high-content screening system to quantify DNA damage-induced γH2AX nuclear foci using an automated cell imager.
- Correlated the total area of γH2AX foci per cell with the concentration of DNA damage-inducing agents.
- Validated the system by quantifying known DNA damage inhibitors and screening a chemical library.
Main Results:
- The total area of γH2AX foci per cell showed a strong correlation with DNA damage agent concentration.
- The system successfully quantified the effects of known DNA damage inhibitors.
- Screening a chemical library identified compounds inhibiting both early DDR signaling and later DNA repair.
Conclusions:
- The developed γH2AX foci-measuring system is a suitable method for quantitative, high-content screening of DDR.
- This system can effectively identify inhibitors of DNA damage response pathways.
- The approach holds potential for the discovery of novel anticancer therapeutics targeting DDR.
