Abstract
Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.
MeSH terms
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Animals
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Biological Assay / methods*
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Cell Line, Tumor
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Cisplatin / pharmacology
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DNA Damage / drug effects
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DNA Damage / genetics*
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Enzyme-Linked Immunosorbent Assay
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Female
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Histones / genetics
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Histones / metabolism*
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Humans
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Mice
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Mice, Nude
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Mutation
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Phosphorylation / physiology*
Grants and funding
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was also supported by the Intramural Research Program at the Center for Cancer Research at National Cancer Institute [Z01BC006150] and by the Radiation and Nuclear Countermeasures Program at the National Institute of Allergy and Infectious Diseases. The Frederick National Laboratory for Cancer Research, which is funded by the federal government and operated by Leidos Biomedical Research, Inc., provided support in the form of salaries for authors J.J., Y.Z., and R.E.P. Leidos Biomedical Research, Inc., is prime contractor for the operation of Frederick National Laboratory and is therefore precluded from conducting any business that represents a conflict of interest with this contract. L.K.F., who is a government contractor at the National Cancer Institute, received support in the form of a salary from Kelly Government Solutions, a division of Kelly Services. The specific roles of all authors are articulated in the 'author contributions' section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.