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

Abstract

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.

Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

“…It has been shown that DNA damage induced by platinum-based agents may block transcription at both the initiation and elongation stages (31,45,46). Thus, aside from its effect on transcription elongation observed in this study, it would be interesting to investigate, in the future, whether S 6 mG also affects transcription initiation by altering the binding of transcription factors to DNA.…”

“…Thus, transcription inhibitors are thought to selectively impede the expression of these antiapoptosis factors and induce the apoptosis of cancer cells (29,43,44). Many transcriptional inhibitors have been clinically tested as anticancer agents, including cisplatin and other platinum-based drugs (31,(45)(46)(47). Given that S 6 mG, but not S G, exhibits a strong inhibitory effect on transcription in our experimental systems, it is therefore possible that S G in human genomic DNA has little or no direct role, at the level of transcription, in the cytotoxic effects of thiopurine drugs; however, S 6 mG, a methylation damage derived from S G in DNA, may contribute, at least in part, to the cytotoxicity of thiopurine drugs through serving as a transcriptional inhibitor.…”

Photochemistry and DNA photocleavage by a new unsupported dirhodium(II,II) complex

“…DFT calculations were performed on the cation [Rh 2 (phen) 2 (CH 3 CN) 6 ] 4+ (1a) in a polarizable continuum model that mimicked the solvation effects of CH 3 CN. The bond lengths and angles from the optimized structure agree well with those from the single-crystal X-ray diffraction structure (see the electronic supplementary material, shown in figure 3.…”

A DNA Repair Protein BRCA1 as a Potentially Molecular Target for the Anticancer Platinum Drug Cisplatin

“…This indicated that cisplatin inhibited the transcription of the p21 WAF1 gene (Ljungman et al, 1999). Recently, the processing of site-specific Pt-DNA crosslinks in mammalian cells was investigated (Ang et al, 2010). Site-specific platinated oligonucleotides, containing 1,2-d(GpG) and 1,3-d(GpTpG) adducts, were inserted into an expression vector between its promoter and a luciferase reporter gene.…”