Cellular Recognition and Repair of Monofunctional-Intercalative Platinum--DNA Adducts.

Abstract

The cellular recognition and processing of monofunctional-intercalative DNA adducts formed by [PtCl(en)(L)](NO3)2 (P1-A1; en = ethane-1,2-diamine; L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine, acridinium cation), a cytotoxic hybrid agent with potent anticancer activity, was studied. Excision of these adducts and subsequent DNA repair synthesis were monitored in plasmids modified with platinum using incubations with mammalian cell-free extract. On the basis of the levels of [α-(32)P]-dCTP incorporation, P1-A1-DNA adducts were rapidly repaired with a rate approximately 8 times faster (t1/2 ≈ 18 min at 30 °C) than the adducts (cross-links) formed by the drug cisplatin. Cellular responses to P1-A1 and cisplatin were also studied in NCI-H460 lung cancer cells using immunocytochemistry in conjunction with confocal fluorescence microscopy. At the same dose, P1-A1, but not cisplatin, elicited a distinct requirement for DNA double-strand break repair and stalled replication fork repair, which caused nuclear fluorescent staining related to high levels of MUS81, a specialized repair endonuclease, and phosphorylated histone protein γ-H2AX. The results confirm previous observations in yeast-based chemical genomics assays. γ-H2AX fluorescence is observed as a large number of discrete foci signaling DNA double-strand breaks, pan-nuclear preapoptotic staining, and unique circularly shaped staining around the nucleoli and nuclear rim. DNA cleavage assays indicate that P1-A1 does not act as a typical topoisomerase poison, suggesting the high level of DNA double-strand breaks in cells is more likely a result of topoisomerase-independent replication fork collapse. Overall, the cellular response to platinum-acridines shares striking similarities with that reported for DNA adduct-forming derivatives of the drug doxorubicin. The results of this study are discussed in light of the cellular mechanism of action of platinum-acridines and their ability to overcome resistance to cisplatin.