Kaufmann W K, Boyer J C, Estabrooks L L, Wilson S J
Department of Pathology, University of North Carolina, Chapel Hill 27599-7295.
Mol Cell Biol. 1991 Jul;11(7):3711-8. doi: 10.1128/mcb.11.7.3711-3718.1991.
Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.
将二倍体人成纤维细胞株用I型和II型DNA拓扑异构酶抑制剂处理10分钟，去除抑制剂后，测定复制子起始点处DNA合成的起始速率。通过碱性洗脱色谱法，显示DNA拓扑异构酶II抑制剂4'-(9-吖啶基氨基)甲磺酰间茴香胺（安吖啶）可产生DNA链断裂。这些链断裂被认为反映了药物诱导的拓扑异构酶-DNA可裂解复合物的稳定。去除药物导致复合物解离，从而使链断裂迅速重新封闭。速度沉降分析用于量化安吖啶处理对DNA复制的影响。结果表明，短暂暴露于低浓度的安吖啶可抑制复制子起始，但对正在运行的复制子内的DNA链延伸没有实质性影响。药物处理后20至30分钟发生复制子起始的最大抑制，30至90分钟后起始速率恢复。共济失调毛细血管扩张症细胞在可裂解复合物稳定期间显示出正常水平的安吖啶诱导的DNA链断裂，但在暴露于拓扑异构酶抑制剂后未能下调复制子起始。因此，响应DNA损伤对复制子起始的抑制似乎是一个活跃的过程，这需要共济失调毛细血管扩张症细胞中存在缺陷或缺失的基因产物。在正常人成纤维细胞中，喜树碱对DNA拓扑异构酶I的抑制产生可逆的DNA链断裂。短暂暴露于这种药物也会抑制复制子起始。这些结果表明，在遗传毒性损伤后下调复制子起始的细胞反应途径可能对伴随拓扑异构酶-DNA可裂解复合物稳定的染色质结构扰动作出反应。
