Study On The Effect Of Antisense HTOP3 On The Endogenous Expression Of HTOP3, DNA Damage Repair And Apoptosis Of A-T Cells

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by a pleiotropic phenotype that includes progressive cerebellar degeneration, radiation hypersensitivity, genomic instability. Cultured A-T cells lack radiation-induced cell cycle checkpoints and express cellular defects that correlate with the clinical phenotype, such as the defect of DNA damage repair capacity and a high rate of apoptosis. A-T is caused by functional inactivation of the ATM gene on 11q22-23. DNA hTOP3 (Human Topoisomerase III, hTOP3) is a Ribozyme that are able to break and reseal the backbone of DNA and thereby adjust the topological states of DNA, adjust the DNA damage repair progress. hTOP3 is expressed up-regulate in AT cells which may contribute to the defection of its phenotype.Objective: In this work, we specifically down regulate the endogenous hTOP3 activity in AT cells. After gene transfection of antisense hTOP3 constructed ourselves, we analyze the expression of endogenous hTOP3 effected by antisense hTOP3. We therefore investigate the DNA damage repair capacity and apoptosis of A-T Cells induced by irradiation using SCGE and FACS method respectively, to find whether the process of DNA damage repair and apoptosis regulated by hTOP3 is ATM dependent.Methods: (1) Construction of antisense hTOP3 mammalian expression vector pcDNA3.0-antihTOP3; (2) Gene Transfection: Electroporation was used to transfect pcDNA3.0-antihTOP3, pcDNA3.0 vector into AT fibroblasts AT5BIVA, respectively. RT-PCR analysis was performed to test the expression of hTOP3mRNA in AT and transfected AT cells; (3) Using SCGE analysis and Annexin V/PI stain following FACS method respectively, we investigate the DNA damage repair capacity and apoptosis of A-T cells induced by irradiation,Results: (1)Expression of the endogenous hTOPS in AT cells was down regulated after transfection with pcDNAS.O-antihTOPS, while there was no such effect of the hTOP3 expression in pcDNA3.0-transfected AT cells; (2)Using SCGE assay, we identified that the DNA damage repair capacity of AT cells post-irradiation has improved after transfection of pcDNAS.O-antihTOPS. However, there is no difference of DNA damage repair in AT cells transfected with pcDNAS.O; (3)pcDNA3.0-antihTOP3-transfected A-T cells clearly showed a reduction in their frequency of apoptotic cells induced by irradiation. In contrast, pcDNAS.O-transfected A-T cells had no such effect.Conclusion: Rather than playing a downstream role in an ATM dependent signal transduction network, hTOPS may function independently of ATM in the processing and repair of DNA damage and apoptosis in AT cells. Perhaps by facilitating access to spontaneous and induced DNA lesions by repair complexes or otherwise promoting the correct repair of damaged DNA by ATM-independent means.