Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide.

ABSTRACT

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 γH2AX 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 γH2AX 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 γH2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of γH2AX foci predominantly in DNA replicating cells and close association and co-localization of γH2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the γH2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of γH2AX 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 (γH2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.