Transcription inhibition by platinum-DNA cross-links in live mammalian cells.
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.
Ang
Wee Han
WH
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Myint
MyatNoeZin
M
Lippard
Stephen J
SJ
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D016190:Carboplatin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000187:drug effects; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D017930:Genes, Reporter; D006801:Humans; D009944:Organoplatinum Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000077150:Oxaliplatin; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D010984:Platinum / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; C034697:transplatin; D010984:Platinum; D004247:DNA; D016190:Carboplatin; D012321:DNA-Directed RNA Polymerases; D002945:Cisplatin