Woynarowski J M, Sigmund R D, Beerman T A
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, NY 14263.
Biochim Biophys Acta. 1988 May 6;950(1):21-9. doi: 10.1016/0167-4781(88)90069-3.
This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.
本研究比较了表鬼臼毒素衍生物VM - 26和VP - 16以及9 - 苯胺吖啶衍生物m - AMSA和o - AMSA对新生DNA和成熟DNA的影响。在经3H/14C标记的L1210细胞经药物处理的细胞核中，分析了两种可能由拓扑异构酶II介导的损伤，即DNA - 蛋白质交联和DNA双链断裂。采用硫酸钾/十二烷基沉淀法评估成熟DNA和新生（1分钟龄）DNA中的DNA - 蛋白质交联。表鬼臼毒素和m - AMSA对新生DNA均表现出强烈的偏好。如中性洗脱技术所示，VM - 26和m - AMSA诱导的DNA双链切割也对新生DNA表现出偏好。在中性蔗糖梯度上的沉降显示，这些药物在新生DNA中产生高度降解片段（低于30 S）的速度比在成熟DNA中快得多。通过省略ATP或添加新生霉素，新生DNA中的损伤显著减少。在新生DNA中诱导损伤的能力与所研究药物的细胞毒性效力相关。结果表明，复制中的DNA可能是抗拓扑异构酶II药物的优先靶点。
