Wild Type P53 Produces Chemotherapy Resistance Related To Its Cell Location In Cancer Cell

Objects:In order to observe whether the cell proliferation,tolerance and apoptosis of cancer cell can be influencd by mutant type p53(mtp53)or wild tipe p53(wtp53),we constructed the recombinant p53 expressed in eukaryocyte to study the relationship between the location and function of mt or wt p53 in environment stress respectively.Methods:The different mtp53 gene obtaining by overlap PCR technique and wtp53 was cloned into eukaryoitc expression vector pcDNA3.1(+),and transfected into HepG2 and Hela cell respectively.MTT was used to evaluate the proliferation activity of HepG2 cells harbored different p53 gene in different environments.Subcellular localization of p53,colocalization of p53(or phosphor-serl5)protein with Hsp60 protein in cancer cell upon the environment stress were observed with a confocal microscope.Agarose gel shift assay was used to analyze the combination between p53 protein and mtND2 gene.Finally,wtp53-mediated chemotherapy resistance of tumor was experimented in nude mice,and chemotherapy-resistance-related factors in paraffin section were detected with immunohistochemistry.Results:1.We constructed different recombinant plasmids of pcDNA-mtp53(R175H、R273H、R282W)and pcDNA-GFP-mtND2 successfully and acquired stable transfection cell lines by G418 screening after transfected into HepG2 or Hela cells.2.HepG2 cells with over-expressing wtp53 exhibited stronger proliferation,anti-apoptosis activity and less sensibility to anticarcinogen than HpeG2 cells or cells with over-expressing mtp53 under stress conditions.3.The subcellular localization of p53 in HepG2 or Hela cells observed with confocal fluorescence microscope showed that the oxidative stress shifted not all the mt53 but wtp53 from nucleus to cytoplasm,and different mutation site exhibited different location in cells.4.Environment stress induced dephosphorylation of wtp53(P-ser15)protein and drove wtp53 fron nucleus to cytoplasm in HepG2 cells,while a similar phenomenon is not also observed in normal HepG2 cell and HepG2 cells with over-expressing mtp53.5.Confocal fluorescence of protein p53(GREEN)and protein Hsp60(RED)showed wtp53 existed in mitochondria when cells were under oxidative stress.6.EMSA in vitro showed wtp53 protein can bind mtND2 DNA directly.7.Over expression of mtND2 in HepG2 cells increased the degree of apoptosis,and also results in stonger drug sensitivity.Wtp53 in mitochondria inhibited the mtND2 expression.8.In nude mice,cancer cells with mtp53 grew better than cancer cells with wtp53 in normal condition.But cancer cells with wtp53 exhibited stronger chemotherapy resistance,and higher expression level of Ki-67,PCNA and TGF-βthan normal cancer cells.Conclusions:The cancer cells with over-expressing wtp53 stimulated a protective regulating mechanism to escape mortality under environment stress,and the chemotherapy-resistance was related to the subcellular localization of p53 in mitochondria.Specific direct interaction of p53 with DNA-binding domain in mitochondrial DNA(mtDNA)inhibited the mtND2 expression and blocked the pathway of apoptosis.