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Updated: Jun 5, 2026
Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay
Published on: August 23, 2024
An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer
Wenge Zhu1, Chrissie Y Lee, Ronald L Johnson
1National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892-2753, USA.
Abstract:
Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.
Insights
Scientists developed a new imaging assay to detect excess DNA replication in cancer cells. This assay aids in discovering novel anticancer therapeutics by identifying compounds that selectively target cancer cell proliferation.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- DNA re-replication, a process leading to cell death, can be induced in human cancer cells but not normal cells.
- This differential response presents a therapeutic strategy for developing anticancer drugs.
- Existing methods for detecting excess DNA replication lack sensitivity.
Purpose of the Study:
- To develop a high-throughput imaging assay for detecting DNA re-replication in intact cells.
- To identify novel compounds with potential anticancer therapeutic applications.
- To investigate the regulation of genome duplication in cancer cells.
Main Methods:
- Development of a novel imaging assay to quantify DNA replication exceeding four genomic equivalents.
- High-throughput screening of 1,280 bioactive molecules using the developed assay.
- Validation of the assay's sensitivity by comparing it with existing methods on normal and tumor-derived cells.
Main Results:
- The assay successfully identified known inducers of excess DNA replication, including microtubule dynamics inhibitors.
- Novel compounds inducing excess DNA replication in both normal and cancer cells were discovered.
- Two compounds selectively induced excess DNA replication in cancer cells, and one induced endocycles selectively in cancer cells.
Conclusions:
- The developed imaging assay is a sensitive tool for high-throughput screening of potential anticancer therapeutics.
- This assay facilitates the discovery of compounds targeting cancer-specific DNA replication.
- The findings offer a new approach for cancer treatment research and genome duplication studies.
