| BackgroundThe nucleolus in the cell is the site of ribosomal DNA(rDNA)transcription and ribosomal biosynthesis.Disruption of Pol I-mediated rDNA transcription can trigger a unique cellular response called nucleolar stress.CX-5461 is a specific inhibitor of RNA polymerase I with high selectivity.It has a significant inhibitory effect on Pol I.Studies have confirmed that CX-5461 can inhibit the proliferation of tumor cells and displays a good therapeutic effect on leukemia and solid tumors.At present,it has entered phase II clinical trials as a new drug for the treatment of certain hematological tumors.We found that the application of CX-5461 to induce nucleolar stress in vascular smooth muscle cells can cause G2/M cell cycle arrest and upregulate the expression of certain cellular senescence markers,and CX-5461 can significantly inhibit neointimal hyperplasia resulting from arterial damage and reduce the formation of transplanted vascular neointima.In vitro experiments,we also found that distinct nucleolar protein NPM translocation occurred,and the levels of p53 phosphorylation and acetylation increased together with no accumulation of p53 protein after treating vascular smooth muscle cells with CX-5461.We confirmed that CX-5461 can activate the ATM/ATR pathway in primary vascular smooth muscle cells,thereby promoting p53 phosphorylation,and the ATR-p53 pathway is an important pathway that mediates the inhibitory effect of CX-5461 in vascular smooth muscle cellsObjectiveHow CX-5461 activates the ATM/ATR pathway in vascular smooth muscle cells to cause downstream p53 phosphorylation is still unclear.Therefore,we will further explore the pharmacological effects and possible mechanisms of CX-5461 activating ATM/ATR pathway in vascular smooth muscle cells.Research contents include:(1)Confirm the inhibitory effects on proliferation of vascular smooth muscle cells caused by CX-5461,and detect whether CX-5461 activates ATM/ATR-p53 pathway in vascular smooth muscle cells in a similar concentration-dependent manner;(2)Explore whether the activation of ATM/ATR-p53 pathway by CX-5461 is accompanied by DNA damage response and DNA strand breaks;(3)Identify whether activation of ATM/ATR pathway in vascular smooth muscle cells is related to increased G-quadruplex structures and transcription-replication collisionsMethods1.The effects of CX-5461 on cell proliferation and activation of ATM/ATR pathway:In vascular smooth muscle cells,different concentrations of CX-5461 were given for stimulation,and Cell Counting Kit-8 was used to detect cell proliferation;immunofluorescence was used to detect the degree of diffusion of NPM protein(nucleolar stress);Western Blot was used to detect the p-p53 levels2.After treating the cells with CX-5461 for 24 hours,immunofluorescence was utilized to detect p-ATM,p-ATR,and yH2AX levels;Comet assay to detect whether DNA strand break has occurred;immunofluorescence to detect whether there is a process of DNA damage repair:XPA(Nucleotide excision repair),MSH2(mismatch repair),DNA PKcs(non-homologous end joining),Rad51(homologous recombination);3.Research on the upstream mechanisms of ATM/ATR pathway Immunofluorescence and protein immunoprecipitation were used to detect replication stress:replication protein A(RPA),PCNA mono-ubiquitination level;In addition,use immunofluorescence to identify whether ATM/ATR activation process involves the formation of G-quadruplex and R-loop4.Lentiviral vector-mediated TIF-1A gene silencing:After silencing the TIF-1A gene in vascular smooth muscle cells,use immunofluorescence to detect the activation of yH2AX and Rad51 proteins.(TIF-1A protein is a key component of rDNA transcription initiation complex)Results1.In proliferating vascular smooth muscle cells,different concentrations of CX-5461(0.5μM,1μM,5μM,10μM)can cause cell proliferation inhibition in a concentration-dependent manner,and CX-5461 can induce nucleolar stress response and increase the p-p53 levels in a similar concentration-dependent manner.2.After treating cells with CX-5461 in proliferative state,the immunofluorescence results showed that p-ATM,p-ATR,and yH2AX levels increased,and the comet assay results showed that no obvious DNA strand breaks occurred;in addition,the immunofluorescence results showed that nucleotide excision repair,mismatch repair,and non-homologous end joining were not activated,but homologous recombination was activated.3.After treating cells with CX-5461 in proliferative state,the immunofluorescence results showed that the intracellular RPA foci increased,and the protein immunoprecipitation results showed that the PCNA mono-ubiquitination level increased;in addition,the immunofluorescence results showed that the intracellular G-quadruplex and R-loop structures did not increase significantly.4.We silenced the TIF-1A gene in the cell by the lentiviral vector,and the immunofluorescence results showed the activation of yH2AX and Rad51 proteinConclusionsIn vascular smooth muscle cells,CX-5461 can inhibit cell proliferation,induce nucleolar stress response in a concentration-dependent manner and activate ATM/ATR pathway.The process of activation ATM/ATR pathway by CX-5461 is not accompanied by the increase of G-quadruplex and R-loop structures,and does not cause obvious DNA strand breaks,but may be related to increased DNA replication stress. |
