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Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

ABSTRACT: Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.

SUBMITTER: Soubeyrand S

PROVIDER: S-EPMC5527959 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

Soubeyrand Sébastien S Pope Louise L Haché Robert J G RJ

Molecular oncology 20091009 1

Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damag ...[more]

PMID: 19858003

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Project description:DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient ("cleavable") complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as ?H2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of ?H2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of ?H2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of ?H2AX foci predominantly in DNA replicating cells and close association and co-localization of ?H2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the ?H2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of ?H2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1-DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (?H2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.

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Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

Publications

Topoisomerase IIalpha-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.

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