title: Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.
abstract: There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of untargeted mustards.
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland, New Zealand. l.ferguson@auckland.ac.nz
authors.forename: L R
authors.initials: LR
authors.lastname: Ferguson
authors.forename: A P
authors.initials: AP
authors.lastname: Liu
authors.forename: W A
authors.initials: WA
authors.lastname: Denny
authors.forename: C
authors.initials: C
authors.lastname: Cullinane
authors.forename: T
authors.initials: T
authors.lastname: Talarico
authors.forename: D R
authors.initials: DR
authors.lastname: Phillips
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002474:Cell-Free System; D004247:DNA / Q000187:drug effects*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004247:DNA; D012321:DNA-Directed RNA Polymerases