title: Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways.
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.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Catherine R
authors.initials: CR
authors.lastname: Sears
authors.forename: John J
authors.initials: JJ
authors.lastname: Turchi
mesh_terms: D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D002945:Cisplatin