Study Of Tumor Cell Apoptosis In HSV/TK Suicide System Induced By HTERT Core Promoter

With the development of modern industry and the serious pollution of environment,the incidence and death rate of lung cancer has been increasing in the world since 1970s. Lung cancer is the most common malignancy and the first leading cause of cancer-related deaths among people in the world. About 150～180 thousand people die with lung cancer every year. At present , more men can be effectively treated by surgery or radiation. Gene therapy is the best way for people to cure tumor in the 21st century. Especially, the rapid development of target gene therapy for malignant tumor has become one of the most tumor-curing field which can finally cure cancer.The human telomerase catalytic subunit (hTERT) is highly activated in 80～90% of cancer cells, but seldom expressed in normal cells, and it is considered as a broad-spectrum tumor marker. Tumor development requires oxygen and nutrients, which are supplied through neovascularization. The vascular endothelial growth factor (VEGF) plays an important role in tumor development and progression, supplying oxygen and nutrients through neovascularization. It has become a promising molecular target for lung cancer therypy. Herpes simplex virus thymidine kinase (TK) gene is a very important suicide gene with obvious bystander effect. So, in this experimentation we chose the lung cancer cell A549 and liver cancer cell SMMC-7721 to be the specimen. We used the cloned hTERT core promoter , VEGF enhancer and herpes simplex virus tk gene ,after linking the multiple regular sequence [VEGF]hTERT toHSV-TK gene ,clone it to prokaryote clone plasmid vector pMD18-T,construct the plasmid vector pMD-[VEGF]hTERT-tk. Then we utilized the enzyme cut site NruI and HindIII at the eukaryote pcDNA3.1(+) vector itself cutting its promoter PCMV. As a result , we can gain a vector long fragment without PCMV, at which we then linked a pEcoRI linker to form double overhang site of EcoRI and HindIII. We constructed a eukaryote expression vector pDNA3.1(+)-[VEGF]hTERT-tk with a multiple regular sequence by digesting the prokaryote clone vector pMD-[VEGF]hTERT-tk with EcoRI and HindIII and inserted it into eukaryote vector long fragment and linked them with T4 DNA ligase. Simultaneously, We constructed a eukaryote expression vector pcDNA3.1(+)-tk with a promoter of the vector pcDNA3.1(+) itself. Respectively, with the transfection of the two eukaryote expression vector to lung cancer cell A549 and liver cancer cell SMMC-7721, we screened four strain of resistant cloned cancer cell lineA549/[VEGF]hTERT-tk,A549/TKandSMMC-7721/[VEGF]hTERT-tk,SMMC-7721/TK by G418 .They formed stable cancer cell line after amplifying culture.To study the activity of multiple regular sequence, we utilized 10μg/ml GCV prodrug to treat the two kinds of resistant clone cancer cell A549/[VEGF]hTERT-tk,A549/TKandSMMC7721/[VEGF]hTERT-tk,SMMC-7721/TK .In the MTT shade selection assay ,we can evaluate the cancer cell growth repression ratio induced by multiple regular sequence mediating HSV-TK suicide system. We adopted a method that evaluated the cancer cell growth repression ratio after GCV treating for 12,24,36,48,72h.The result manifested that the longer the time of GCV prodrug treating, the higher the cell growth repression ratio may be. And moreover, the cancer cell growth repression which is induced by multiple regular sequence is higher than induced by PCMV promoter. With the statistical treat for the assay data, we can conclude the P<0.01, it suggested that there is significant differentiation between the two assay group. All of the result above suggested that thehTERT core promoter is greatly efficient and specific.To explain the conclusion further, we adopted PI stain the four strain resistant clone cancer cell line which is treated with GCV(10μg/ml) for different time. Then utilizing the FACS to survey the apoptosis ratio of the two assay group. Firstly, we respectively treated the two kinds of four strain cancer cell line contained the two style eukaryote expression vector with GCV(10μg/ml) for 24,48,72h ,gathering the different strain cancer cell line along the time gradient ,then stain the cell line with PI and use the FACS machine to survey the apoptosis of them. The result manifested that the apoptosis of lung cancer cell and liver cancer cell with transfection of eukaryote vector mediated with multiple regular sequence is higher than apoptosis with eukaryote vector universal promoter PCMV. The highest apoptosis ratio of A549 cell and SMMC-7721 cell at the GCV prodrug treated time of 72h is 17.79±1.81% and 15.96±1.28%, 13.31±1.22% and 13.19±1.34%. Adopting t evaluation for the data, the result is P<0.01, it suggested that there is greatly significant differentiation between the assay groups and control and the HSV/TK suicide system has obvious effect to the apoptosis of cancer cell. And with further comparison and analysis, we made a conclusion that the transcription activity of [VEGF]hTERT sequence in lung cancer cell A549 strain is prior to liver cancer cell line SMMC-7721.In the study, all of the data in the assay suggested us that hTERT promoter has a significant value in curing cancer with gene therapy.