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Transcription Inhibition by Platinum−DNA Cross-Links in Live Mammalian Cells

2010; American Chemical Society; Volume: 132; Issue: 21 Linguagem: Inglês

10.1021/ja101495v

1943-2984

Wee Han Ang, MyatNoeZin Myint, Stephen J. Lippard,

Metal complexes synthesis and properties

We have investigated the processing of site-specific Pt−DNA cross-links in live mammalian cells to enhance our understanding of the mechanism of action of platinum-based anticancer drugs. The activity of platinum drugs against cancer is mediated by a combination of processes including cell entry, drug activation, DNA-binding, and transcription inhibition. These drugs bind nuclear DNA to form Pt−DNA cross-links, which arrest key cellular functions, including transcription, and trigger a variety of responses, such as repair. Mechanistic investigations into the processing of specific Pt−DNA cross-links are critical for understanding the effects of platinum−DNA damage, but conventional in vitro techniques do not adequately account for the complex and intricate environment within a live cell. With this limitation in mind, we developed a strategy to study platinum cross-links on plasmid DNAs transfected into live mammalian cells based on luciferase reporter vectors containing defined platinum−DNA lesions that are either globally or site-specifically incorporated. Using cells with either competent or deficient nucleotide excision repair systems, we demonstrate that Pt−DNA cross-links impede transcription by blocking passage of the RNA polymerase complex and that nucleotide excision repair can remove the block and restore transcription. Results are presented for ∼3800-base pair plasmids that are either globally platinated or carry a single 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link formed by either cis-{Pt(NH3)2}2+ or cis-{Pt(R,R-dach)}2+, where {Pt(NH3)2}2+ is the platinum unit conveyed by cisplatin and carboplatin and R,R-dach is the oxaliplatin ligand, R,R-1,2-diaminocyclohexane.