Mechanistic Study Of MTORC1 Dependent DNA Replication Stress Induced By P53 Deficiency

Background:DNA replication stress is an important cause of genomic instability and a“hallmark” of many cancers.About 2/3 of the mutations in tumors are caused by errors in the replication process,so it is important to reduce the replication stress and protect the accuracy of DNA replication to prevent the occurrence of tumor.TP53 is the most important tumor suppressor in mammals,which can prevent DNA replication stress to maintain the stability of the genome.However,the mechanisms still need to be elucidated.In this study,we use p53 knock out cell line as a model to explore the underlying molecular mechanism on how hyper-activation of m TORC1 leads to replication stress in p53 deficient cells.This study will renew our understanding on how p53 functions as a tumor suppressor and have implications in cancer prevention and treatment as well.Methods:HCT116 WT cell line and HCT116 p53 knockout cell line were used in this study.DNA damage markers were detected by immunofluorescence staining and Western Blot;Gene mutation rate was evaluated by HPRT reporter assay;Cell proliferation was measured by CCK8 assay;p53 rescue experiment was done by constructed a pc DNA3.1-p53 expression plasmid and transfected in HCT116 p53 KO cell line.Result:1.Compared with HCT116 WT cells,p53 knockout cells have heterogeneous nucleus,huge nucleus can be seen;p53 knockout cells have more γH2AX foci and increased HPRT mutation rate.2.Compared with HCT116 WT cells,m TORC1 pathway was over activated in p53 knockout cells.3.Rapamycin can partial rescue p53 deficiency caused γH2AX and genome instability.4.Re-express p53 in p53 KO cells can rescue DNA damage and inhibit m TORC1 overactivation.5.DNA replication stress in p53 knockout cells may be related with the increased level of transcription-replication conflicts.Conclusions:Our results suggest that deletion of p53 gene increases DNA replication stress;p53 deletion induces m TORC1 pathway dependent DNA replication stress;Activation of m TORC1 induced DNA replication stress in p53 knockout cells,which may be related with the increased level of transcription-replication conflicts.This study provides a new idea for the treatment of p53-deficient cancers.