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

Abstract 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.