Abstract
ObjectiveDNA double strand breaks (DNA-DSBs) are among the most lethal DNA lesions leading to genomic instability and repaired by either homologous recombination (HR) or the non-homologous end joining (NHEJ) mechanisms. The purpose of this study was to assess the importance and the level of activation of non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways in three cell lines, CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes following treatment with chemotherapy agent daunorubicin.ResultsThe Gamma histone H2AX (γH2AX) assay was used assess the effects of DNA-PK inhibitor NU7026 and RAD51 inhibitor RI-2 on repair of DNA-DSB following treatment with daunorubicin. In all cell lines, the NHEJ DNA repair pathway appeared more rapid and efficient. MOLT-4 and CCFR-CEM cells utilised both NHEJ and HR pathways for DNA-DSB repair. Whereas, SUP-B15 cells utilised only NHEJ for DSB repair, suggestive of a deficiency in HR repair pathways.
Highlights
DNA damage takes many forms including base deletion/ addition/substitution, DNA adducts, single strand DNA breaks and the most lethal of all DNA double strand breaks (DNA-DSB)
When both pathways were simultaneously blocked in daunorubicin treated MOLT-4 and CCRF-CEM cells with both RI-2 and NU7026 inhibitors, high levels of DNA-DSB were seen at all recovery times indicating incomplete DNA repair
We previously showed that DNA-DSB repair following daunorubicin treatment was lower in B-lymphocyte derived cells than in T-lymphocyte derived cells [15]
Summary
DNA repair following daunorubicin treatment was assessed by monitoring the disappearance of γH2AX over time (Fig. 1). The two T-lymphoblastic cell lines, MOLT-4 and CCRF-CEM (Fig. 1a, b), treated with daunorubicin alone showed increased DNA-DSB at 4 h and this declined after 12 h and further after 24 h recovery indicating DNA repair. Similar events were seen in both daunorubicin plus RI-2 (BRCA1 inhibitor) and daunorubicin plus NU7026 (DNA-PK inhibitor) groups When both pathways were simultaneously blocked in daunorubicin treated MOLT-4 and CCRF-CEM cells with both RI-2 and NU7026 inhibitors, high levels of DNA-DSB were seen at all recovery times indicating incomplete DNA repair. There was a significant increase in levels of DNA-DSB after 4 h when cells were treated with both NU7026 and RI-2 combined with daunorubicin, but no subsequent change in γH2AX expression, indicative of a lack of DNA-DSB repair. There was no significant increase in γH2AX expression compared to untreated cells (control) for each cell type (Fig. 1)
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