RESUMO
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.