The Effects of Topoisomerase Inhibition on Dna Repair and Apoptosis in L929 Fibroblasts

Abstract

Objective: DNA repair pathways in cells are essential for the maintenance of genome integrity, and for countering the induction of tumorigenesis. Topoisomerase II is a nuclear enzyme that functions during both DNA replication and transcription. The topoisomerase II inhibitor etoposide is an antineoplastic drug that has been used to generate DNA damage and maintain apoptosis. Etoposide blocks cell division by interfering with the topoisomerase II and generates double strand breaks. Application of topoisomerase II inhibitors leads to the formation of double strand breaks that are rapidly repaired following removal of the drug. In the present study, we searched the apoptotic events and early double strand DNA repair process that prevent the apoptotic cell death of L929 fibroblasts in response to treatment with etoposide. Methods: Cytotoxicty of etoposide on L929 cells was determined in a time and dose dependent manner with MTT assay. The double strand DNA breaks were determined with comet assay. Acridin orange/Ethidium bromide fluorescence staining and Caspase 3/7 activity assays were performed in determined etoposide concentrations at 24 hour. Quantitative mRNA expressions of DNA repair genes (Ku70, Ku80, BRCA2, Rad51, XRCC4) were determined after etoposide treatment. Results: The levels of apoptotic cell markers and DNA double strand breaks were elevated in the increasing doses and time. The relative expression levels of Rad51, XRCC4 and BRCA2 were unstable in a time and dose dependent manner. Ku80 levels were generally decreased in etoposide treated groups when compared with controls. However, Ku70 was highly expressed at 9 and 12 hour with the increasing doses. Conclusion: According to the results of this study, etoposide activates apoptotic events. Also, low expression levels of DNA repair enzymes prevent cell survival from DNA damage.