Smith P J, Debenham P G, Watson J V
MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, Great Britain.
Mutat Res. 1989 Mar;217(2):163-72. doi: 10.1016/0921-8777(89)90068-2.
The biological effects of a number of DNA ligands which interact with the minor groove of B-form DNA (e.g. netropsin, distamycin and Hoechst 33258) are thought to arise from the direct disturbance of the processes of DNA replication and transcription. Although ligand binding appears to be an important factor in cytotoxicity, the pathways by which drug molecules can be actively dissociated from nuclear DNA are unknown. Recent evidence suggests that minor groove ligands can distort the manner in which DNA associates with nucleosomal core particles and we have hypothesized that in an intact cell such imposed torsional stress could be subject to the action of cellular topoisomerases. We have used flow cytometry to study the effects of various inhibitors (including topoisomerase-interactive drugs) on the responses of a mutant cell line (HoeR415) to Hoechst 33342, given that the mutant shows resistance to the cytotoxicity of this DNA-specific dye due to an enhanced capacity to dissociate nuclear DNA-dye complexes. Ligand-DNA dissociation in the mutant was found to be energy-dependent but not specifically, affected by the drug-efflux blocker verapamil or by inhibitors of DNA synthesis. The topoisomerase II inhibitors novobiocin, VP16, nalidixic acid and the topoisomerase I-interactive drug camptothecin inhibited ligand-DNA dissociation to various extents with novobiocin being the most effective (100% inhibition at 1 mM). Both novobiocin and camptothecin were without effect on the nuclear loss of a DNA intercalator, adriamycin. We conclude that efficient topoisomerase activity is required for the active dissociation of DNA minor groove-ligand complexes.
一些与B型DNA小沟相互作用的DNA配体（如纺锤菌素、偏端霉素和Hoechst 33258）的生物学效应被认为源于DNA复制和转录过程的直接干扰。尽管配体结合似乎是细胞毒性的一个重要因素，但药物分子从核DNA上主动解离的途径尚不清楚。最近的证据表明，小沟配体可以扭曲DNA与核小体核心颗粒结合的方式，我们推测在完整细胞中，这种施加的扭转应力可能会受到细胞拓扑异构酶的作用。鉴于该突变体由于解离核DNA-染料复合物的能力增强而对这种DNA特异性染料的细胞毒性具有抗性，我们使用流式细胞术研究了各种抑制剂（包括拓扑异构酶相互作用药物）对突变细胞系（HoeR415）对Hoechst 33342反应的影响。发现突变体中的配体-DNA解离是能量依赖性的，但不受药物外排阻滞剂维拉帕米或DNA合成抑制剂的特异性影响。拓扑异构酶II抑制剂新生霉素、VP16、萘啶酸和拓扑异构酶I相互作用药物喜树碱在不同程度上抑制配体-DNA解离，其中新生霉素最有效（1 mM时100%抑制）。新生霉素和喜树碱对DNA嵌入剂阿霉素的核丢失均无影响。我们得出结论，DNA小沟-配体复合物的主动解离需要有效的拓扑异构酶活性。
