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. 2010 Jun 2;132(21):7429-35.
doi: 10.1021/ja101495v.

Transcription inhibition by platinum-DNA cross-links in live mammalian cells

Wee Han Ang  1 , MyatNoeZin Myint, Stephen J Lippard
Affiliations

Transcription inhibition by platinum-DNA cross-links in live mammalian cells

Wee Han Ang et al. J Am Chem Soc. .

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 approximately 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(NH(3))(2)}(2+) or cis-{Pt(R,R-dach)}(2+), where {Pt(NH(3))(2)}(2+) is the platinum unit conveyed by cisplatin and carboplatin and R,R-dach is the oxaliplatin ligand, R,R-1,2-diaminocyclohexane.

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Figures

Figure 1
Figure 1
Chemical structures of platinum anticancer drugs.
Figure 2
Figure 2
Scheme for preparing site-specifically platinated probes.
Figure 3
Figure 3
Site-specifically platinated probes containing Pt-DNA cross-links of cisplatin and oxaliplatin; platination sites are highlighted in bold.
Figure 4
Figure 4
Transcription profile of globally platinated probes in XPF (left) and XPFcorr (right) cells.
Figure 5
Figure 5
Comparison of transcription profiles of globally platinated probes in XPF and XPFcorr cells 8 h (left) or 48 h (right) after transfection.
Figure 6
Figure 6
Transcription of site-specifically platinated probes containing 1,2-d(G*pG*)-Pt (pGLuc4temGG, left) and 1,3-d(G*pTpG*)-Pt (pGLuc5temGTG, right) cross-links 24 h after transfection.
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