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The Mechanism of the Therapeutic Effects of rAAV-mediated HSVtk Suicide Gene System on Breast Cancer Cells

Author: XiangShengGuang
Tutor: HuWeiXin
School: Central South University
Course: Biochemistry and Molecular Biology
Keywords: breast cancer suicide gene therapy rAAV HSVtk/GCV p21
CLC: R737.9
Type: Master's thesis
Year: 2009
Downloads: 16
Quote: 1
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Abstract

Breast cancer is a common malignancy in human, especially in women all over the world. Surgery, radiotherapy, and chemotherapy are the three major cancer therapies. With the development of molecular biology and biotechnology, gene therapies are becoming more and more important in cancer treatment. Suicide gene therapy is one of the best choices and highlights of human gene therapies. When transferred into the mammalian cells, a suicide gene could catalyse the non-toxic predrug into toxic metabolic product that influences the biosynthesis or causes apoptosis of the cells. In this study, we used a recombinant adeno-associated virus rAAV/TRE/Tet-On/HSVtk combined with a Tet-On regulation system and a suicide gene HSVtk, which was transfected to human breast cancer cell line MCF-7, and studied the therapeutic effects of this system. Furthermore, we used gene chip method, reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting to explore the potential mechanism of the HSVtk suicide gene system in breast cancer treatment.Chapter 1 The therapeutic effects of rAAV-mediated HSVtk suicide gene system on breast cancer cellsHEK293T cells were cotransfected with plasmids pAAV/TRE/Tet-On/HSVtk, pAAV-RC, and pAAV-Helper using calcium phosphate co-precipitation method, and the rAAVs were purified before experiment. Dot blot hybridization was performed to determine the titer of rAAV, and the result demonstrated its concentration was up to 3.49×10¹¹/ml. By infecting human breast cancer cell line MCF-7 wiith rAAV, we compared the effects of ganciclovior （GCV） with the induction by Dox or not on MCF-7 cells by MTT assay. There were five treatments: rAAV+Dox+GCV group, rAAV+GCVgroup, rAAV+Dox group, rAAV group and negative control （NC） group. The results showed that, 72 hours after treatment, the relative cell mass of rAAV+Dox+GCV group was significantly decreased （P<0.05）, compared with that of the other groups. We used flow cytometry to determine the influence of the HSVtk suicide gene therapy system on cell cycle. The results revealed that the S phase cells were significantly increased only in rAAV+Dox+GCV group （P<0.05）. The data suggest the S phase of the cells could be blocked by the HSVtk suicide gene system.Chapter 2 The mechanism of the therapeutic effects of rAAV-mediated HSVtk suicide gene system on breast cancer cellsWe treated MCF-7 cells with the drugs for 48 hours and extracted the whole RNA of the cells, then used gene chip to determine the gene expression profolio. The results showed that, there were 246 genes up-regulated and 64 genes down-regulated in rAAV+Dox+GCV group, and many of them are associated with DNA replication, DNA damage, and repair etc, and there are also many zinc finger proteins. A set of interesting genes were confirmed by RT-PCR. The expression patterns of p21 and PCNA gene between the rAAV+Dox+GCV and the NC group were consitent with the gene chip assay. We used Western blotting to invetigate the protein expression patterns of p21, PCNA, CDK1, CyclinB, p53 and phosphorylated p53 （Ser6） in rAAV+Dox+GCV group and NC group. We found that the expression of p21 protein was upregulated, whereas the expression of PCNA, CDK1, and CyclinB were downregulated in the rAAV+Dox+GCV group as compared with NC group. However, the expression level of p53 and phosphorylated p53 （Ser6） had no significant changes between the two groups. We hypothesize that the therapeutic effects of the HSVtk suicide gene system on breast cancer is through a p53-independent way. By up-regulating the expression of p21, the system could down-regulate the expression of PCNA, Cyclin B and CDK1, and then block cell cycle and lead breast cancer cells to death.

The Mechanism of the Therapeutic Effects of rAAV-mediated HSVtk Suicide Gene System on Breast Cancer Cells

Abstract

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