Nuclear RAC1 is a modulator of the doxorubicin-induced DNA damage response

Abstract

Rho GTPases like RAC1 are localized on the inner side of the outer cell membrane where they act as molecular switches that can trigger signal transduction pathways in response to various extracellular stimuli. Nuclear functions of RAC1 were identified that are related to mitosis, cell cycle arrest and apoptosis. Previously, we showed that RAC1 plays a role in the doxorubicin (Dox)-induced DNA damage response (DDR). In this context it is still unknown whether cytosolic RAC1 modulates the Dox-induced DDR or if a nuclear fraction of RAC1 is involved. Here, we silenced RAC1 in mouse embryonic fibroblasts (MEF) pharmacologically with EHT1864 or by using siRNA against Rac1. Additionally, we transfected MEF with RAC1 mutants (wild-type, dominant-negative, constitutively active) containing a nuclear localization sequence (NLS). Afterwards, we analysed the Dox-induced DDR by evaluation of fluorescent nuclear γH2AX and 53BP1 foci formation, as well as by detection of activated proteins of the DDR by western blot to elucidate the role of nuclear RAC1 in the DDR. Treatment with EHT1864 as well as Rac1 knock-down reduced the Dox-induced DSB-formation to a similar extent. Enhanced nuclear localization of dominant-negative as well as constitutively active RAC1 mimicked these effects. Expression of the RAC1 mutants altered the Dox-induced amount of pP53 and pKAP1 protein. The observed effects were independent of S1981 ATM phosphorylation. We conclude that RAC1 is required for a substantial activation of the Dox-induced DDR and balanced levels of active/inactive RAC1 inside the nucleus are a prerequisite for this response.

Keywords: DNA double-strand breaks; Doxorubicin; Genotoxicity; Nucleus; RAC1; Small GTPases.