| Breast cancer is a major malignancy tumor in women all over the world. Suicide gene therapy is one of the important choices in human gene therapies. After transferred into the tumor cells, a suicide gene could catalyze the non-toxic prodrug into toxic metabolic product that influences the biosynthesis and causes death of the cells. In our study, we treated human breast cancer cell line MCF-7 with a recombinant adeno-associated virus combined with a Tet-On regulation element and a suicide gene HSVtk. Furthermore, we used comet assay, Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) to explore primarily the influences and the molecular mechanism of this suicide gene therapy system in breast cancer treatment. Chapter 1 The DNA damage influence of HSVtk/GCV suicide genetherapy system mediated by adeno-associated virus vector under theregulation of Tet-On in breast cancer treatmentHEK293T cells were cotransfected with plasmids pAAV/TRE/ Tet-On/HSVtk, pAAV-Helper and pAAV-RC by calcium phosphate co-precipitation method, and then the rAAV/TRE/Tet-On/HSVtks were extracted. After condensed and purified, Dot blot hybridization was performed to determine the titer of rAAVs, and its concentration was up to 2.0×1011/ml. After infecting human breast cancer cell line MCF-7 with rAAVs, we treated the cells with Dox and GCV, There were five treatments:rAAV+Dox+GCV group, rAAV+Dox group, rAAV +GCVgroup, rAAV group and negative control (NC) group. We used comet assay to determine the influence of DNA damage on MCF-7 cells in the suicide gene therapy, The results showed that,48 hours after treatment, the comet tail of rAAV+Dox+GCV group was more obvious, compared with that of other groups. We used statistical analysis of the comet assay and the percentage of comet tail area to determine the DNA damage degree of the HSVtk suicide gene therapy system on breast cancer cells. The results, which revealed that the degree of DNA migration in rAAV+Dox+GCV group is greater than that of others (P<0.05), demonstrated that the cell DNA damage level was up-regulated after interfered by HSVtk suicide gene therapy system.Chapter 2 The primary study on the molecular mechanism of DNA damage in the treatment of HSVtk/GCV suicide gene therapy system mediated by adeno- associated virus vector under the regulation of Tet-On on breast cancer cellsWe used genechip to determine the gene expression profolio by treating MCF-7 cells with HSVtk/GCV suicide gene therapy system. The results showed that, there were 246 up-regulated genes and 64 down-regulated genes, and many of them are associated with DNA damage, repair and DNA replication in rAAV+Dox+GCV group. And there are also many zinc finger proteins. Combining with the results, A set of active genes related with DNA damage response were determined by RT-PCR. The results of RT-PCR about expression level of ATM, p53,p27,CyclinE and CDK2 genes between the rAAV+Dox+GCV and the other groups showed, that the expression levels of ATM,p53 and p27 was obviously upregulated, but CDK2 and CyclinE didn't change. The protein expression levels of ATM,p53,p27,CyclinE and CDK2 were investigated by Western blotting, the result shows that the expression levels of ATM,p53 and p27 protein was upregulated, whereas CDK2 and CyclinE was not changed obviously in the rAAV+Dox+GCV group as compared with other groups. We hypothesize that the DNA damage mechanism of HSVtk/GCV suicide gene system on breast cancer cells may be a p53-dependent singal pathway, which upregulating the expression level of p27 and inhibitting the activity of CyclinE-CDK2 complex in cell cycle regulation, it can result in cell cycle arrest and cell death. |
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