The Molecular Mechanism Of P53^N236S In Elongating The Life Span Of Werner Syndrome Mice

The aging of population has become a challenge that many countries have to face.Meanwhile,diseases associated with aging,such as tumors,are still major health issues of the society.Aging is a natural process of life.How to slow down the aging process while reducing the occurrence of aging-related diseases has become a hot topic of people’s research.The occurrence of some diseases during premature aging and the animal model of premature aging has become an important means for people to study the mechanism of aging.Werner Syndrome is an autosomal recessive genetic disease,which is caused by the mutation of the WRN gene of the RECQ helicase family.The clinical manifestations of WS include premature aging and tumor susceptibility.Due to the long telomere length and telomerase activity in mouse cells,both Wrn gene and telomerase gene need to be knocked out in order to construct the WS mouse model.When the mice with Wrn gene and telomerase gene knocked out were bred to the fifth generation（G5DM）,the mice showed the progeria phenotypes similar to human WS.The previous researchers found that some cells could escape senescence and become immortalized during the culture of Mouse embryo fibroblast of G5DM,and some immortalized cells could form tumors in SCID mice.The same site mutation of the tumor suppressor gene p53 was found in these tumor-forming cells in SCID mice(p53^N236S).Since these tumors had no telomerase activity,telomeres were maintained by the alternative lengthening of telomere.p53 is called the guardian of the genome by responding to stress signals（DNA damage,etc.）,p53 is activated,triggering transient cell cycle arrest cell death（apoptosis）or permanent cell cycle arrest（cell senescence）.Both apoptosis and senescence are effective tumor suppressor mechanisms,which can effectively prevent tumor transformation in damaged cells.But both processes also deplete proliferating progenitor cells,or stem cells,and damage tissue structure and function.Although apoptotic cells can be eliminated from the tissue,senescent cells can persist,thereby altering the microenvironment of the tissue in a senescent phenotype manner,and promoting the development of cancer and age-related diseases.p53,a tumor suppressor gene,is mutated in more than 50%of human tumors.The p53^N236Smutation concerned in this study is a point mutation occurring in thep53 DNA binding region,and the p53 protein amino acid at position 236 is mutated from asparagine to serine.Our previous study found that p53^N236S not only lost the function of wild-type p53,but also gained the new function of some oncogenes.In order to further study the mutation of p53^N236S,transgenic mice of p53^N236S were constructed in the laboratory in the early stage,and the mice showed the phenotype of high tumor incidence and strong metastasis.Since p53^N236S mutation is filtered the WS escape of aging cells,in order to study p53^N236S mutation to the aging,and aging with the interactions of tumor,we introduce the p53^N236S mutations through hybridization with Wrn and telomerase RNA template（Terc）Double knockout WS（Double mutant）mice,formed the Wrn,Terc,p53^N236Sthree mice with the mutation（Triple mutant）.To study the effect of p53^N236S on the survival of progerated mice with Wrn syndrome,the previous researchers plotted the survival curves of（G1,G2,G3）TM mice and（G1,G2,G3）DM mice,and found that the average life span of G5TM mice（165±19days）was longer than that of G5DM mice（120±6 days）.Later,it was further found that p53^N236S induced aging of Wrn syndrome depended on the length of telomeres.The telomere signals of cells were detected by TRF-Southern technology.The experimental results showed that the telomere signals gradually weakened in G1DM,G3DM and G5DM cells;however,the telomere signals did not significantly weaken in G1TM,G3TM and G5TM cells.In DM mice,telomeres gradually shortened with the increase of algebra,and life span decreased with the increase of mouse algebra,indicating the correlation between telomere length and life span,which is consistent with the literature reports.However,in TM cells,the telomere signal did not show significant algebraic shortening,suggesting that p53^N236S may protect the telomere and reduce the rate of telomere shortening.Meanwhile,p53^N236S may participate in the telomere lengthening mechanism to maintain telomere length and extend the lifespan of mice.Previous studies have found that mice with three gene mutations to the fifth generation（G5TM）live longer than G5DM mice,and so far,we have not found G5TM mice that form spontaneous tumors.These data suggest that the combination of p53^N236Sand WS to form G5TM mice may delay the aging phenotype of WS mice and inhibit the tumor phenotype of p53^N236S mice,but the molecular mechanism is unclear.In order to study the molecular mechanism of extending the life of WS mice with p53^N236S,we made G1,G3,G5 DM/TM,WT,p53^-/-,and p53^S/S MEFs.The results showed that during cell culture,G5DM cells aged severely from the most to the sixth generation and could not be passed on,while G5TM cells maintained a good state of proliferation,similar to immortalized cells,and could be passed on normally for 60generations and still maintain rapid proliferation.We observed the senescence status of G5DM and G5TM cells by SA-β-gal staining.G5DM cells showed positive senescent cells from the third generation,while G5TM cells from the 60th generation did not show positive senescent cells.The results of fluorescence in situ probe hybridization showed that the telomeres of G5TM cells were longer than those of G5DM cells.Further karyotype analysis of G5TM cells in the sixtieth generation and telomere labeling by telomerase fluorescence probes showed that telomere heterogeneity was strong,with some telomere longer and some telomere shorter or even completely lost in the same cell.There was an exchange of telomeres in the cells and a fusion of chromosomes due to the shorter telomeres,suggesting that the G5TM cells extended telomeres through the ALT mechanism,and the cells could be passed on normally without aging.Wrn,as a DNA helicase,plays an important role in DNA replication,which is an important part of the cell cycle.Therefore,we used flow detection to detect the cell cycles of G5DM and G5TM,and found that more cells with p53^N236S have the presence of chromosome polyploids.Further experiments revealed that all cells with p53^N236Sbecame polyploid after a certain number of passages.These data suggest that p53^N236Smutation can induce spontaneous polyploid formation in cells.Studies have shown that plants can increase stress resistance by forming polyploids,so we wonder whether the p53^N236S mutation can also cope with DNA damage and replication pressure by forming polyploids.To test this hypothesis,we measured the tolerance of p53^S/S cells and WT cells to the spindle toxicity drugs nocodazole and taxol,drug half maximal inhibitory concentration,drug half maximal inhibitory concentration experimental and Apoptosis experiment results show that p53^S/S cells to take an examination of nocodazole and taxol has stronger resistance.Since polyploid formation often induces tumor formation,we verified the tumorigenesis of p53^S/S polyploid cells and TM polyploid cells.These results showed that p53^S/S polyploid cells could form tumors in SCID mice,while TM polyploid cells did not form tumors,indicating that the tumorigenesis of p53^N236S mutation was not strong.So are there polyploid cells in G5TM mice?In order to further study in mice,we for TM and cell cycle detection of DM mice bone marrow cells and karyotype analysis,found that 5.8%of TM in mice bone marrow cell DNA content of 8 N（diploid mouse cells in G0/G1 phase DNA quantity of 2 N）,and article TM cells exist in the mice bone marrow karyotype chromosome number is 80.It indicated that there were tetraploid cells in the bone marrow of TM mice.These results suggest that G5TM mice may prolong the lifespan of progeria mice by promoting polyploid formation in response to DNA damage caused by the absence of Wrn and telomerase.To investigate the regulatory effect of p53^N236S on cell cycle and telomeremaintenance,we examined the expression of cyclin-dependent kinase inhibitor（CKI）,DNA repair related genes and proteins related to telomere maintenance in G5TM and G5DM cells.RNA Seq data showed that several CKI（p16/Cdkn2a,p15/Cdkn2b,p18/Cdkn2c,p19/Cdkn2d,p27/Cdkn1b,p57/Cdkn1c）except p21/Cdkn1a were all highly expressed in G5TM cells.We verified the protein expression of p16,p19,p21and p27 in G5TM cells by western blot assay.Except for the low expression of p21 in G5TM cells,p16,p19 and p27 were all highly expressed in G5TM cells.The results agree with RNA-seq data,and G5TM cells did not show obvious cycle arrest,may be due to p21 is much more important role in cell cycle arrest,or the several CKI protein mutation or modify lose its cycle inhibition function relationship.Real-time quantitative PCR data showed that,compared with G5DM,Fanconi anemia related gene:Fanca,Fancb,Fancc,Fancd1（Brca2）,,Fancd2,Fanci,Fancj（Bach1）,,Fancm,Fanco（Rad51c）,Fancr（Rad51）and Recql4,Timeless,Blm,which are related to telomeres maintenance and DNA repair,were highly expressed in G5TM cells.Among them,Brca2 and Rad51 are related to homologous recombination repair,and their high expression promotes the repair of DNA,especially telomeres,and may initiate the ALT pathway to lengthen telomere length.Recql4,Blm and Wrn belong to RECQ helicase.Their increased expression may compensate for part of the function of Wrn,slowing down the rate of telomere shortening and extending the lifespan of mice.Literature has shown that the expression of the above-mentioned fankone and telomere maintenance as well as DNA repair related genes is regulated by DREAM complex.Previous experimental results showed that p53^N236S lost the regulatory ability of p21/Cdkn1a,and p21/CDKN1A was poorly expressed in TM cells,while p21/CDKN1A promoted the transformation of DREAM complex into a transcriptional inhibitor.Combined with RNA-seq,we found that B-myb（Mybl2）,and Foxm1 high expression in G5TM cells,promote the DREAM compounds by the transcription factor is turning to transcription factor,may promote the numerous DNA repair and high telomere maintenance related gene expression,promote cell repair ability.We through the protein immunoblot Foxm1protein is verified by the experiments,and is regulated by the DREAM pathways Gins2,Pol E,Timeless,Bubr1,Bub1,Rad51,PCNA and Mcm7,Mcm2 protein expression,such as data showed that compared with G5DM cells,these proteins expressed in high G5TM cells and expression after the formation of polyploid quantity increased further.We verified the expression of Foxm1 protein and other proteins regulated by the DREAM pathway,such as Gins2,Pol E,Timeless,Bubr1,Bub1,Rad51,PCNA and Mcm7,Mcm2,by western blot assay.The data showed that these proteins were highly expressed in G5TM cells and further increased after polyploid formation compared with G5DM cellsIn summary,p53^N236S mutation induced polyploids and improve the stress resistance of cells.Meanwhile,p53^N236Spromotes the expression of DNA repair and telomere maintenance proteins by regulating DREAM complex,and improves the DNA damage repair ability and initiated ALT mechanism.Together,these regulation might contribute to rescue the aging phenotypes of WS.p53 maintains a delicate balance in the cells,which is closely related to tumorigenesis and aging,and alteration of p53 can lead to an imbalance between tumorigenesis and aging.Our study revelaed that the p53^N236S could prolong life span of G5 WS mice without increasing tumorigenesis.This might provides a new idea in tumor-free anti-aging strategy.