Danks M K, Garrett K E, Marion R C, Whipple D O
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Cancer Res. 1996 Apr 1;56(7):1664-73.
DNA topoisomerase I is the cytotoxic target for chemotherapeutic agents of the camptothecin class. The cytotoxicity of these drugs is thought to be mediated by a dose-dependent increase in topoisomerase I molecules bound to DNA, resulting in DNA damage and cell death. We observed that in SJ-G5 human anaplastic astrocytoma cells growing in culture, the maximum number of topoisomerase I-DNA complexes occurred 5-15 min after the addition of 0.25-25 microM 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan; TPT) or 5 microM 7-ethyl-10-hydroxycamptothecin (SN-38). We postulated that the decline in number of complexes seen after 15 min might be due to decreases in the amount of topoisomerase I or the redistribution of this enzyme such that it could no longer bind to DNA. To investigate these possibilities, we incubated SJ-G5 cells for 20-60 min with 0.25-5 microM TPT and analyzed the cells for amount and localization of topoisomerase I by indirect immunofluorescence staining and fluorescence digital imaging microscopy. We verified the results obtained with fluorescence digital imaging microscopy by rapid fractionation of nuclear and cytoplasmic proteins, separation of these proteins by polyacrylamide gel electrophoresis, and densitometric scanning of immunoblots. Results showed that topoisomerase I dissociated from nucleoli within 60 min after treatment with 1-5 microM TPT. A small (25%) but significant (P < 0.05) decrease in the amount of nuclear topoisomerase I was also observed during this time course. Simultaneously, the cytoplasmic content of the Mr 67,000 form of topoisomerase I increased 50-100%. Preincubation of cells with 10 microM cycloheximide for 10 min prevented the increase of topoisomerase I in the cytoplasm, indicating that the increase was due, at least in part, to de novo protein synthesis. Interestingly, chemotherapeutic agents other than camptothecins were also found to dissociate topoisomerase I from nucleoli. These agents included m-AMSA (4'-(9-acridinylamino)methanesulfon-m-anisidide), mitoxantrone, actinomycin D, and daunorubicin. Drugs such as 1-beta-D-arabinofuranosylcytosine or hydroxyurea, which have no effect on RNA synthesis, did not induce the translocation. The biological significance of the ability of camptothecins to redistribute their own drug target is under investigation.
DNA拓扑异构酶I是喜树碱类化疗药物的细胞毒性靶点。这些药物的细胞毒性被认为是由与DNA结合的拓扑异构酶I分子剂量依赖性增加介导的，导致DNA损伤和细胞死亡。我们观察到，在培养的SJ-G5人间变性星形细胞瘤细胞中，添加0.25 - 25 microM 9-二甲基氨基甲基-10-羟基喜树碱（拓扑替康；TPT）或5 microM 7-乙基-10-羟基喜树碱（SN-38）后5 - 15分钟，拓扑异构酶I-DNA复合物的数量达到最大值。我们推测，15分钟后复合物数量的下降可能是由于拓扑异构酶I数量的减少或该酶的重新分布，使其不再能与DNA结合。为了研究这些可能性，我们用0.25 - 5 microM TPT将SJ-G5细胞孵育20 - 60分钟，并通过间接免疫荧光染色和荧光数字成像显微镜分析细胞中拓扑异构酶I的数量和定位。我们通过快速分离核蛋白和细胞质蛋白、聚丙烯酰胺凝胶电泳分离这些蛋白以及免疫印迹的光密度扫描，验证了荧光数字成像显微镜获得的结果。结果显示，用1 - 5 microM TPT处理后60分钟内，拓扑异构酶I从核仁中解离。在此时间进程中，还观察到核内拓扑异构酶I的量有小幅（25%）但显著（P < 0.05）的减少。同时，分子量为67,000形式的拓扑异构酶I的细胞质含量增加了50 - 100%。用10 microM放线菌酮预孵育细胞10分钟可阻止细胞质中拓扑异构酶I的增加，表明这种增加至少部分是由于从头合成蛋白质。有趣的是，还发现除喜树碱外的其他化疗药物也能使拓扑异构酶I从核仁中解离。这些药物包括m-AMSA（4'-(9-吖啶基氨基)甲磺酰基间茴香胺）、米托蒽醌、放线菌素D和柔红霉素。对RNA合成无影响的药物，如1-β-D-阿拉伯呋喃糖基胞嘧啶或羟基脲，不会诱导这种转位。喜树碱重新分布其自身药物靶点能力的生物学意义正在研究中。
