title: The cell death and DNA damages caused by the Tet-On regulating HSV-tk/GCV suicide gene system in MCF-7 cells.
abstract: Ganciclovir (GCV) affects the molecular mechanism of cell death and DNA damage by the rAAV (recombinant adeno-associated virus)-mediated Tet-On/HSV-tk/GCV suicide gene system in human breast cancer cell line MCF-7. A rAAV/TRE/Tet-On/HSV-tk combining a Tet-On regulating system and a suicide gene HSV-tk was used to transfect human breast cancer cell line MCF-7, and therapeutic effects on this system were studied. Afterwards, we used RT-PCR, western blotting, and a modified comet-assay to explore the potential mechanism of the HSV-tk/GCV suicide gene system in breast cancer treatments. MTT assay has shown that the cell number of GCV+rAAV+Dox group was significantly decreased compared with that of other groups after treatment and flow cytometric analysis detected that there was a substantial increase of S phase cells in this group, which means the HSV-tk/GCV suicide gene system probably works on the cell cycle. RT-PCR detected the expression level of p21 increased and PCNA had an opposite trend. Western blotting detected the protein expression of p21 and p53 increased and PCNA, CDK1, cyclin B decreased in GCV+rAAV+Dox group. The modified comet-assay shown that the very small extra fragments generated by the GCV+rAAV+Dox group treatment are visible as a small cloud extending from the comet in the direction of electrophoresis. The therapeutic mechanism of the HSV-tk/GCV suicide gene system on human breast cancer cell line MCF-7 is probably by upregulating the expression of p21 through a p53-dependent DNA damage signalling pathway, leading the decrease of protein expression of PCNA, cyclin B, CDK1 in MCF-7 cells and promoting the cell cycle arrest at G1/S phase. In summary, the HSV-tk/GCV suicide gene system arouses the death of MCF-7 cells from blocking the cell cycle and DNA damage.
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China; State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, PR China.
authors.forename: Zhao-Jun
authors.initials: ZJ
authors.lastname: Zeng
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Sheng-Guang
authors.initials: SG
authors.lastname: Xiang
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China; State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, PR China.
authors.forename: Wei-Wen
authors.initials: WW
authors.lastname: Xue
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Hong-De
authors.initials: HD
authors.lastname: Li
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Nan
authors.initials: N
authors.lastname: Ma
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Zi-Jing
authors.initials: ZJ
authors.lastname: Ren
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Zhu-Jun
authors.initials: ZJ
authors.lastname: Xu
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Chun-Hong
authors.initials: CH
authors.lastname: Jiao
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China.
authors.forename: Cui-Yun
authors.initials: CY
authors.lastname: Wang
authors.affiliation: Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078, PR China. Electronic address: weixinhu@aliyun.com.
authors.forename: Wei-Xin
authors.initials: WX
authors.lastname: Hu
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D016203:CDC2 Protein Kinase; D016923:Cell Death / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D056744:Cyclin B1 / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D000998:Antiviral Agents; C534699:CCNB1 protein, human; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; D015774:Ganciclovir
keywords: Ganciclovir; HSV-tk; Human breast cancer; Suicide gene system