Complex Cisplatin-Double Strand Break (DSB) Lesions Directly Impair Cellular Non-Homologous End-Joining (NHEJ) Independent of Downstream Damage Response (DDR) Pathways

The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro . Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment. Background: The biochemical mechanism of cisplatin-IR synergy is incompletely understood. Results: NHEJ of non-cisplatin damaged DNA substrates is unaltered by cellular cisplatin treatment while repair of cisplatin-DSB lesions is inhibited independent of cellular cisplatin treatment. Conclusion: Cisplatin-DSB compound lesions directly inhibit NHEJ while cisplatin-activated pathways do not impact NHEJ. Significance: The mechanism of cisplatin-IR synergy involves direct inhibition of NHEJ by compound cisplatin-DSB lesions.