TDP1 mediates the removal and repair of etoposide-stabilized Top2a cleavable complexes in HeLa cells

BORDA MA, PALMITELLI M, GONZÁLEZ CID M, DE CAMPOS NEBEL M.

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Topoisomerase II (Top2) poisons, such as Etoposide (ETO), are widely used chemotherapeutic agents that stabilize the Top2-DNA cleavable complexes, which may result in DNA double strand breaks (DSB) formation. The aim of this work was to analyze how tyrosyl-DNA-phosphodiesterase 1 (TDP1) influences the removal of ETO-stabilized Top2a cleavable complexes and the correct DNA damage signaling and repair in HeLa cells. Stable cell lines knocked down in TDP1 (HeLa TDP1kd) and a non silencing control (HeLa NS) were established by using shRNAmir sequences and further analyzed by qRT-PCR. After treatment with ETO, the removal of cleavable complexes was evaluated by the DRT (Differential Retention of Top2) assay by Confocal Microscopy. The activation of DNA damage signals, pS1981ATM and γH2AX, were analyzed by flow cytometry and the DNA repair efficiency by micronucleus (MN) formation. Our results show that Top2a cleavable complexes are processed with different kinetics in HeLa TDP1kd. In addition, while γH2AX signals were induced similarly in both HeLa TDP1kd and NS cells, pS1981ATM was not induced by ETO in HeLa TDP1kd. Moreover, the induction of MN was significantly increased in HeLa TDP1kd compared to NS cells. Thus, we conclude that TDP1 promotes the removal of ETO-stabilized Top2a cleavable complexes and participates in the repair of the induced DNA DSB in human cells.