Molecular basis of CX-5461-induced DNA damage response in primary vascular smooth muscle cells

Our previous studies have shown that the novel selective RNA polymerase I inhibitor CX-5461 suppresses proliferation of vascular smooth muscle cells, mainly by inducing DNA damage response (DDR), including activations of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) and p53. Currently, there is no information about the molecular mechanism(s) underlying CX-5461-induced DDR in vascular cells, while the results obtained in Cancer cells and immortalized cell lines are controversial. In this study, we examined the responses of various DDR pathways to CX-5461 treatment in primary aortic smooth muscle cells isolated from normal adult Sprague Dawley rats. 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-induced 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 the 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. In summary, 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 basis of the beneficial effects of CX-5461 in proliferative vascular diseases.

ATM/ATR; CX-5461; DNA damage response; Nucleolus; Replication stress; Vascular smooth muscle cell; p53.