Abstract 578: Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress.

Abstract

Abstract Polo-like kinase 1 (Plk1) is a key regulator of mitosis and its over-expression/ deregulation is linked to cellular transformation. Recent studies highlight its role in resumption of cell cycle progression after DNA damage. Plk1 has also been shown to enhance DNA replication under stress of stalled replication forks. We reported Plk1 activation-induced G2/M checkpoint bypass in normal human lung cells (HLFs), in the presence of acute genotoxic stress. The present aim was to determine the effect of Plk1 on DNA double strand breaks (DSBs) during genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by Cr(VI), etoposide, and H2O2 after Plk1 activation using the comet assay and γH2AX immunofluorescence staining. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Transfection of a Plk1 constitutively active (c/a) mutant (T210D) abrogated both Cr(VI)- and etoposide-induced DNA DSBs as early as 30 min for the duration of treatment, in comparison to the vector-transfected cells. In sharp contrast, Plk1 had no effect on H2O2-induced DNA DSBs. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Moreover, Plk1 enhanced S phase progression during Cr(VI) treatment, which was accompanied by increased replication fork velocity and longer inter-origin distances as determined by using a single-molecule approach based on molecular combing. Expression of a Plk1 “kinase-dead” mutant (K82R) also abrogated DNA DSBs during Cr(VI) treatment, suggesting that this effect may be independent of Plk1 kinase activity. In summary, our data underline the ability of Plk1 to override DNA DSB-induced checkpoints, and to decrease DSBs after replication-stalling genotoxin exposure. We postulate these effects to be due to the replication-enhancing ability of Plk1, which may occur at the expense of genomic stability. This work was supported in part by NIH grants CA107972 and ES017334 (S.C.); ES05304 (S.R.P.); and a PhRMA Foundation predoctoral fellowship (G.C.K.). Citation Format: Daniel Gebreselassie, Gina Chun-Kost, Elisabetta Leo, Yves Pommier, Erika Saito, Steven Patierno, Susan M. Ceryak. Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2013-578