Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.
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.
Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Jiuping
J
Ji
Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Yiping
Y
Zhang
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
Christophe E
CE
Redon
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
William C
WC
Reinhold
Early Clinical Trials Development Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
Alice P
AP
Chen
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
Laura K
LK
Fogli
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
Susan L
SL
Holbeck
Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Ralph E
RE
Parchment
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
Melinda
M
Hollingshead
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
Joseph E
JE
Tomaszewski
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
Quentin
Q
Dudon
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
Yves
Y
Pommier
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.|Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland, United States of America.
James H
JH
Doroshow
Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
William M
WM
Bonner
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D010766:Phosphorylation / Q000502:physiology*
D006657:Histones; D002945:Cisplatin