Recombinant Replication-defective Adenovirus Ad-p53AIP1 And Its Effects To Tumor Cells

Tumor is a serious threat to human health.In China and many developing countries,cancer is still in the rising trend.Up to now,the traditional therapeutical measures for tumor mainly include surgery,chemotherapy and radiotherapy.Over the last decade,gene therapy was considered that it has a bright application prospect.Already scholars predicted that the tumors of human gene therapy will be the fundamental way to conquer cancer.At present,a representative drug of cancer gene therapy is Gendicine-a recombinant human p53 adenovirus injection which is the first formal approval of gene therapy drugs in the word.It indeed brings hope for some cancer patients.However,although transfer of wild-type p53 kills cancer cells in some cases (p53-sensitive cancers),it does not do so in others(p53-resistant cancers).Therefore,searching for the more effective gene on tumor cells also has long been one of the hot spots of cancer gene therapy research.p53AIP1 gene,a p53 downstream target,which is discovered by a Japanese research team in 2000 induced apoptosis effectively in both p53-sensitive and p53-resistant cancer cell lines.p53AIP1 gene can trigger apoptosis directly and might be more widely applicable for treatment of cancer than p53 gene itself.AdMax Adenoviral Vector System is used to produce the recombination replication-defective adenovirus.At first,we inserted the exogenous p53AIP1 gene into the shuttle vectors downstream the MCMV promoter.Then the shuttle vector was cotransfected with Ad genomic vector plasmid pBHGlox A E1,3Cre into HEK293 cells through the Cre-loxP system and produced the recombination replication-defective adenoviruses which carried exogenous p53AIP1 gene:Ad-p53AIP1.Human hepatocellular carcinoma HepG₂ cell line and cervical cancer cell HeLa which contain wild-type p53 were infected with recombination adenovirus Ad-p53AIP1,and cell growth was inhibited remarkably by MTT test.Transfection of p53AIP1 gene resulted in cell cycle arrest and induced the apoptosis of tumor cells,which was confirmed by Western blotting,flow cytometry,rhodamine staining,Wright-Giemsa staining,Hoechst 33258 nuclear staining and observation of electron microscopy.The in vivo preliminary test showed that Ad-p53AIP1 could obviously inhibit the tumorigenesis of mouse breast cell line 4T1 and inhibit the growth of tumors to a certain extent in vivo.Furthermore,Ad-p53AIP1 could up-regulate p53 protein reversely and down-regulate MDM2 gene expression.In addition,Ad-p53AIP1 could regulate cell cycle-related proteins and apoptosis-related proteins such as p21.These results indicate that Ad-p53AIP1 could inhibit growth of tumor cells through regulating the cell cycle process.Then,Ad-p53AIP1 could induce the apoptosis of these cells which contain wild-type p53.In addition,p53AIP1 could induce p53 gene expression reversely that we did not find any paper reported so far.We presume that the possible mechanism is because the downregulating MDM2 gene which is inhibited by Ad-p53AIP1 will not degradate and inhibit p53 gene expression.Ectogenic p53 gene could induce p53AIP1 gene expression,otherwise,ectogenic p53AIP1 gene could induce p53 gene expression in tumor cells.So,both are regulating the cell cycle process and induce the apoptosis of tumor cells by the pattern of reciprocal induction.This maybe a new strategy for clinic therapy of tumor in the future.