ATM participates in the homologous recombination repair of DNA damage induced by etoposide in human cells treated in G2 phase

PALMITELLI M; DE CAMPOS NEBEL M; GONZÁLEZ CID M

Montevideo

Congreso; X Congreso Internacional Asociación Latinoamericana Mutagénesis, Carcinogénesis y Teratogénesis Ambiental; 2016

Asociación Latinoamericana Mutagénesis, Carcinogénesis y Teratogénesis Ambiental

Ataxia-telangiectasia-mutated (ATM) is activated by DNA double-stranded breaks (DSB) and promotes DSB repair by homologous recombination (HR) pathway. The DNA-Topoisomerase II poison Etoposide (ETO) is an antitumor drug that induces persistent DSB and also genome instability. Our aim was to analyze the DSB repair process and the progression of the DNA damage-induced by ETO in G2 phase in human cells. HeLa ATMkd, Rad21kd (deficient in cohesin that facilitates HR) and non-silencing control NS cell lines were generated by transfection of specific shRNAmir. Cells were treated with ETO 2μg/ml in the presence or not of ATM chemical inhibitor (KU55933 10µM). In ATMkd cells, Rad51 foci were evaluated by immunofluorescence and sister chromatid exchanges (SCE) by cytogenetic analysis at 5-6hs post-treatment. In Rad21kd cells, the progression of γH2AX foci (biomarker for DSB) was examined post-mitotically in G1 phase on micronuclei (MN) and the main nuclei of binucleated cells (BN) 10hs post-treatment. ETO increased the number of Rad51 foci/cell (9.9±7.1 vs. 5.7±4.5) and reduced the frequency of SCE/chromosome (0.13±0.07 vs. 0.18±0 05) in ATMkd compared to NS. In these cells, in the presence of KU55933-ETO combined treatment, an increase (p20 γH2AX foci (20.6±3.9% vs. 7.7±3.5%) was obtained compared to ETO treatment alone. Instead, in Rad21kd cells, KU55933-ETO did not cause an additional increase in MN containing γH2AX foci (37.1±14.9% vs. 34.2±12.2%) or nuclei with >20 γH2AX foci (38.6±0.2% vs. 41.6±13.3%) compared to cells treated with ETO. The combined treatment did not decrease the percentage of BN cells in both Rad21kd and NS cells. In conclusion, ATM regulates the initial and the final steps of HR. Moreover, under an impaired HR, ATM does not affect the progression of the DSB generated by ETO.