Abstract
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.
Highlights
The biochemical mechanism of cisplatin-IR synergy is incompletely understood
These experiments represent the first determination of the downstream effects of cisplatin on NHEJcatalyzed DSB repair independent of cisplatin lesions on the DNA being rejoined
This is the first evidence in live cells that the presence of a cisplatin lesion in close proximity to a DSB impairs NHEJ-catalyzed DSB repair
Summary
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 cisplatinDSB lesions is inhibited independent of cellular cisplatin treatment. DSB repair of DNA containing cisplatin terminal lesions has never been directly investigated intracellularly This manuscript explores the relationship between cisplatin treatment and DSB repair by separately evaluating the effects of cisplatin-DNA adduct formation and activation of downstream signaling pathways on NHEJ-dependent DSB repair in a NSCLC cell line. These data support the hypothesis that the synergistic cell death observed with combined cisplatin-IR treatment is a function of a cisplatin-DNA lesion near the site of a DSB and independent of DNA damage signaling
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