A Preliminary Study About The Regulation Of P53 In Ultraviolet B-induced Premature Senescent Human Skin Fibroblasts And Function Of Tumor Suppression

Solar ultraviolet B irradiation can induce skin photocarcinogenesis and it also can induce stress-induced premature senescence in human skin fibroblasts with the key regulation factor-p53.In this study(including four parts),we explored the regulation of p53 in Ultraviolet B-induced premature senescent human skin fibroblasts with function of tumor suppression.Objective:In partⅠ,we developed a model of UVB-induced premature senescence in human skin fibroblast(HSF)to facilitate further study about relationship among stress-induced premature senescence and tumorigenesis induced by UVB.In partⅡ,to explore the effects of UVB-induced SIPS on cell cycle regulation in HSF.To explore the effects of on mRNA expression of p53-related genes involved in growth arrest,apoptosis and tumorigenesis.To further elucidate the role of UVB-induced SIPS in molecular mechanisms of tumorigenesis.In partⅢ,to transfect p53 short hairpin RNA(shRNA)plasmid into human skin fibroblasts for establishing a cell line with repressed expression of p53.To explore the effect of repressesd expression of p53 on UVB-induced SIPS and tumor suppression activity in HSF.In partⅣ,to establish a cell line that exogenous wild-type p53(wtp53)gene transfected human skin fibroblasts.To explore the effect of over expression of p53 on UVB-induced SIPS and tumor suppression activity in HSF.Methods:1.We chosed a subcytotoxic dose from different dosages of UVB and radiated HSF five times at this level to induce senescence.2.SAβ-gal activity was investigated.3.Cell viability was determined by MTT assay.4. Three senescence- associated genes,fibronectin(FN),osteonectin(ON)and smooth muscle 22(SM22)mRNA expression was determined by RT-PCR.5.In UVB-stressed premature HSF,cell cycle assay was analyzed with fluorescence-activated cell sorting(FACS)by flow cytometer.6.Protein expression of p53,p21 and p16 was determined by western blot(WB).7.A real time PCR array was performed to investigate mRNA level of a number of genes involved in growth arrest,apoptosis and tumorigenesis induced by UVB. 8.Eukaryotic expressing plasmids pGCsi-p53 containing short hairpin RNA of p53 gene and pCMV-p53 containing wild type p53 gene were introduced by lipofectamine-mediated gene transfection into human skin fibroblasts.After transfection,the cells were selected by G418.Then resistant clones were chosen and expanded in DMEM culture medium.RT-PCR, real-time quantitative PCR,WB were used to determine the expression of p53 gene and p53 protein.9.SAβ-gal activity,MTT assay and PCR array were investingated in UVB-stressed HSF lines which repressesd expression of p53 and overexpresssion of p53.Results: PartⅠ:We found suitable subcytotoxic dosage and radiation times for inducing premature senescence.After repetitive five subcytotoxic exposures to UVB at the dose of 10mJ/cm~2,biomarkers of senescence were markedly established:cell growth arrest,senescence-associatedβ-galactosidase(SAβ-gal)activity,,and overexpression of senescence-associated genes.First,there was a loss of replicative potential as assessed by MTT assay.Second,there was an increase in the proportion of cells positive for SAβ-gal activity.Third,the levels of the mRNA of three senescence-associated genes,FN,ON and SM22, were also upregulated.PartⅡ:FACS analysis showed that UVB-stressed HSF are blocked mostly in G1 phase of the cell cycle.Protein expressions of p53,p21 and p16 in senescence pathways were increased significantly.PCR array indicated some genes are differentially expressed in UVB-induced SIPS.First,anti-oncogenes involved in growth arrest p53,p21,p16,p19 were all overexpressed.Second, among genes involved in p53-dependent apoptosis,bax was downregulated while bcl-2 was up-regulated slightly.Third,among genes involved in tumorgensis induced by UVB,HIF-1α,VEGF and hdm2 were down-regulated.PartⅢ:A repressed expression of p53 HSF cell clone was established successfully.In the transfected HSF cell p53 mRNA and p53 protein expression were repressesd sigmficantly.SAβ-gal activity was reduced and growth arrest was inhibited in UVB-treated HSF-pGCsi-p53 cells.Repressed expression of p53 downregulated senescence associated genes without influencing p16 pathway under UVB.mRNA expression of genes involved in apoptosis and tumorigenesis in HSF-pGCsi-p53 cells showed up-regulation of bcl-2,downregulation of bax,up-regulation of HIF-1α,VEGF and hdm2. Repressed expression of p53 inhibitored p53-related apoptosis and tumor repression activity without influence of UVB.PartⅣ:An anti-G418 cell clone was established and expanded in culture. The transfected wtp53 gene HSF-p53 cells showed overexpression of p53 mRNA and p53 protein.Overexpression of p53 in UVB-treated HSF-wtp53 cells couldn't induce SAβ-gal activity and growth arrest.Expression of genes involved in tumorigenesis showed overexpression of p53 couldn't up-regulated senescence associated genes and function of p16 pathway under UVB was. negligible.mRNA expression of genes involved in apoptosis and tumorigenesis in HSF-pCMV-p53 cells showed downregulation of bcl-2, up-regulation of bax,downregulation of HIF-1α,VEGF and hdm2. Overexpression of p53 enhanced p53-related apoptosis and tumor repression activity and UVB radiation could even promoted them.Conclusions:PartⅠ:SIPS could be induced by repeated subcytotoxic UVB in HSF,which shared many features with replicative senescence(RS).We could use this UVB-induced SIPS model in further related studies.PartⅡ:G1 stage arrest and expression of proteins involved in senescence pathways further confirm UVB-induced SIPS in HSF.Expression of p53-related genes suggest that UVB-induced SIPS may play an important role in p53-related apoptosis resistance and tumor suppression activity.The role of p53 pathwys in related mechanisms should be further explored.PartⅢ:RS and UVB-stessed SIPS in HSF could be reserved or partly inhibitored by repressed expression of p53.Repressed expression of p53 inhibitor p53-related apoptosis and tumor repression activity with a promoting tumorigenesis function,which confirmed tumorigenesis regulation of p53 in UVB-induced SIPS.PartⅣ:RS and UVB-stessed SIPS in HSF couldn't be induced by overexpression of p53.Overexpression of p53 can enhance p53-related apoptosis and tumor repression activity with a tumorigenesis repression function,and UVB can even promote it.Overexpression of p53 may prefer to induce apotosis but not senescence under UVB radiation...