The Study On The Mechanism Of Apoptotic And DNA Damage Caused By Tet-On Regulated HSV1-tk Gene Therapy System In MCF-7Cells

Breast cancer is seriouslly harmful to the health of women, which has gradually become a severe health problem of society. There is an urgent need to develop new effective and less toxic treatment systems to reduce breast cancer mortality. Cancer suicide gene therapy has good prospects of treatment. Herpes simplex virus type I thymidine kinase gene/GCV therapy system is the most widely used.In the previous study, we have successfully used Tet-On regulated system to precisely regulate the expression of herpes simplex virus type I thymidine kinase gene. By comet assay, we detected that MCF-7cells appeared obvious comet tail phenomenon after treated with the therapy system. We speculate that this system killing breast cancer cells may be mainly through participation DNA damage and repair or DNA synthesis process. In order to further reveal the molecular mechanism on HSVl-tκ/Tet-on/GCV under the treatment of breast cancer, we intend to use RT-PCR and Western blotting technique to detect the expression changes of many molecular associated with DNA damage and repair in mRNA and protein levels. The immunofluorescence was used to detect the localization about BRCA1and γ-H2AX in MCF-7cells; MTT and colony formation assay were used to measure the cell proliferation in vitro; DAPI stain and Flow cytometry were used to detect apoptosis. The results showed that,48hours after treatment, compared with other groups, the cells of treatment group were significantly decreased; The expression of the DNA double-strand breaks damage marker y-H2AX was up-regulated in the protein level; The expression of H2AX was up-regulated in the RNA level; The result of immunofluorescence displayed that y-H2AX locates in the nucleus and BRCA1mainly locates in the cytoplasm; Many proteins (ATM, P53, BRCA1, PNK, R2, TK1, CHK2, RB, p21, et al) related to DNA damage were increased in the treatment group; The phenomenon of shear of PARP1protein was detected by Western blotting, which probably generated apoptosis. Early apoptosis cells were detected in the treatment group by flow cytometry. After DAPI nuclear staining, the typical apoptotic morphology was observed too. We futher investigated the expression changes of the apoptosis-related molecule caspase-3. We found, compared with other control groups. caspase-3was up-regulated in both protein level and mRNA level in treatment group. The results showed that the rAAV/TRE/Tet-On/HSV1-tκ treatment system can induce DNA Double-strand breaks damage and one of mechanism of cell killing by rAAV/TRE/Tet-On/HSV1-tκ treatment system is promoting apoptosis.