Molecular events leading to CX-5461-induced DNA damage response in vascular smooth muscle cells

Background: Our previous studies have shown that the novel selective RNA polymerase I inhibitor CX-5461 suppresses proliferation of vascular smooth muscle cells, mainly via induction of activations of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) and p53. Currently, there is no information about what kinds of molecular events mediate such a DNA damage response (DDR) in vascular cells. Methods Primary aortic smooth muscle cells were isolated from normal adult Sprague Dawley rats and cultured in vitro . Immunofluorescence assays were conducted to assess the activation of various DDR pathways. Results We demonstrated that CX-5461-induced DDR was not associated with activations of the nucleotide excision repair, DNA mismatch repair, or the non-homologous end joining pathways, while the homologous recombination pathway was activated. However, the alkaline comet assay did not show massive DNA double strand breaks in CX-5461-treated cells. Instead, CX-5461-triggered DDR appeared to be related to induction of DNA replication stress, which was not attributable to increased formation of G-quadruplex or R-loop structures, but might be explained by increased replication-transcription conflict. CX-5461-induced DDR was not exclusively confined to rDNA within the nucleolar compartment; the extra-nucleolar DDR might represent a distinct secondary response related to the downregulated Rad51 expression in CX-5461-treated cells. Conclusions We suggest that DNA replication stress may be the primary molecular event leading to downstream ATM/ATR and p53 activations in CX-5461-treated vascular smooth muscle cells. Our results provide further insights into the molecular mechanisms underlying the reported beneficial effects of CX-5461 in the cardiovascular system.