Alternative end-joining repair pathway induces chromosomal rearrangements in human cells treated with etoposide

PALMITELLI M; GONZÁLEZ CID M; DE CAMPOS NEBEL M

Mar del Plata

Congreso; Reunión Conjunta. LXIII Reunión Científica de la Sociedad Argentina de Investigación Clínica; 2018

Sociedad Argentina de Investigación Clínica y Sociedad Argentina de Inmunología

Chromosomal rearrangements (CR) involving the MLL (mixed-lineage leukemia) gene cause secondary malignancies associated with the treatment of human tumors with etoposide (ETO). The incorrect repair of DNA double-strand breaks (DSB) by alternative end-joining (alt-EJ) pathway generates CR, genomic instability and tumorigenesis. The role of alt-EJ in the generation of CR induced by ETO in human cells deficient in the cohesion subunit Rad21 (homologous recombination defective) and in DNA-PKcs (one of the main factors of classical nonhomologous end-joining) was evaluated. HeLa Rad21kd cells and their non-silencing NS control cells were treated with ETO 2µg/ml for 1-2h in the presence or absence of the chemical inhibitor of DNA-PKcs, NU7026 10µM. After 2h of treatment, an increase in the percentage of G2 cells with DSB (88.2%-93.4%), analyzed by flow cytometry, was observed. At 10h post-treatment (PT), the immunofluorescence analysis of nuclei with more than 20 γH2AX foci (DSB biomarker) in G1 post-mitotic binucleated cells showed a significant increment in HeLa Rad21kd and HeLa NS exposed to NU7026-ETO (82.8±2.1% vs. 21.7±3.2%, p=0.0001) compared to cells treated with ETO alone (Rad21kd =36.4±5.9% vs. NS=7.7±1.8%). Abnormal repair of ETO-induced DSB led to inter-chromosomal exchanges and mainly, dicentric chromosomes at second metaphases (28h PT), being this frequency 3.1-times higher in NU7026-ETO-treated HeLa Rad21kd cells than in NS (2.42±0.33 vs. 0.78±0.16, p=0.0001). Moreover, MLL gene rearrangements at band 11q23 using fluorescence in situ hybridization procedure were found in 7.2% and 4.9% interphase nuclei of Rad21kd and NS cells exposure to the combination of NU7026-ETO, respectively. Meanwhile, the percentage of MLL gene rearrangements after ETO treatment was similar in both cell lines (NS= 6.3% and Rad21kd= 5.9%). These results indicate that ETO-induced DSB go through the successive cell division and that alt-EJ plays an important role in the CR formation involving MLL gene.