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Histone H2AX phosphorylation induced by selective photolysis of BrdU‐labeled DNA with UV light: Relation to cell cycle phase
Author(s) -
Huang Xuan,
A. King Malcolm,
Halicka H. Dorota,
Traganos Frank,
Okafuji Masaki,
Darzynkiewicz Zbigniew
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20086
Subject(s) - microbiology and biotechnology , bromodeoxyuridine , histone , dna , biology , chromatin , biochemistry , cell growth
Background The induction of DNA double‐strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser ‐139) by ATM‐, ATR‐, or DNA‐dependent protein kinases (DNA‐PK). Phosphorylated H2AX, denoted as γH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser ‐139–phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring γH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of γH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. Methods HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU‐labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU‐labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for γH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6‐diamidino‐2‐phenylindole. The intensities of cellular far red (γH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. Results After a 1‐h pulse of BrdU followed by exposure to UV, nearly all cells with S‐phase DNA content had many‐fold higher γH2AX IF than G 1 or G 2 /M cells. The nonirradiated cells had minimal (“programmed”) expression of γH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of γH2AX IF independent of the cell cycle phase. Pulse‐chase experiments showed that the cohort of BrdU‐labeled (γH2AX‐positive) cells progressed through G 2 /M and into G 1 phase after 8 and 12 h of growth in BrdU‐free medium, respectively. Bivariate analysis of γH2AX versus cyclin A expression for the gated S‐phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. Conclusions Photolysis of BrdU‐labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of γH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics. © 2004 Wiley‐Liss, Inc.