Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents

  • Tanaka T
  • Huang X
  • Halicka H
 et al. 
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Abstract

This review covers the topic of cytometric assessment of activation
of Ataxia telangiectasia mutated (ATM) protein kinase and histone
H2AX phosphorylation on Ser139 in response to DNA damage, particularly
the damage that involves formation of DNA double-strand breaks. Briefly
described are molecular mechanisms associated with activation of
ATM and the downstream events that lead to recruitment of DNA repair
machinery, engagement of cell cycle checkpoints, and activation of
apoptotic pathway. Examples of multiparameter analysis of ATM activation
and H2AX phosphorylation vis-a-vis cell cycle phase position and
induction of apoptosis that employ flow- and laser scanning-cytometry
are provided. They include cells treated with a variety of exogenous
genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase
I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric
oxide-releasing aspirin, DNA replication inhibitors (aphidicolin,
hydroxyurea, thymidine), and complex environmental carcinogens such
as present in tobacco smoke. Also presented is an approach to identify
DNA replicating (BrdU incorporating) cells based on selective photolysis
of DNA that triggers H2AX phosphorylation. Listed are strategies
to distinguish ATM activation and H2AX phosphorylation induced by
primary DNA damage by genotoxic agents from those effects triggered
by DNA fragmentation that takes place during apoptosis. While we
review most published data, recent new findings also are included.
Examples of multivariate analysis of ATM activation and H2AX phosphorylation
presented in this review illustrate the advantages of cytometric
flow- and image-analysis of these events in terms of offering a sensitive
and valuable tool in studies of factors that induce DNA damage and/or
affect DNA repair and allow one to explore the linkage between DNA
damage, cell cycle checkpoints and initiation of apoptosis.

Author-supplied keywords

  • Carcinogens
  • DNA double-strand breaks
  • DNA photolysis
  • DNA topoisomerase inhibitors
  • Genotoxins
  • Ionizing radiation
  • Replication stress
  • Tobacco smoke

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Authors

  • Toshiki Tanaka

  • Xuan Huang

  • H. Dorota Halicka

  • Hong Zhao

  • Frank Traganos

  • Anthony P. Albino

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