Hwong C L, Wang C H, Chen Y J, Whang-Peng J, Hwang J L
Institute of Molecular Biology, Academia Sinica, Taipei, Republic of China.
Cancer Res. 1990 Sep 1;50(17 Suppl):5649S-5652S.
The carboxyl-terminal one-third of human topoisomerase II polypeptide expressed in Escherichia coli was used as antigen to generate polyclonal antibodies in rabbits. With the use of antiserum, DNA topoisomerase II levels of phytohemagglutinin-stimulated human lymphocytes were measured by immunoblotting. Our results showed that the increase in intracellular topoisomerase II level paralleled the entry of cells into proliferation. We also found that the increase in the topoisomerase II level resulted from an increase in the amount of topoisomerase II mRNA. The time course study indicated that the appearance of topoisomerase II mRNA was first observed at 36 h after phytohemagglutinin stimulation. The maximal level of topoisomerase II mRNA was seen at 45 h after stimulation. The same RNA blot was rehybridized with a thymidine kinase probe. The maximal level of thymidine kinase mRNA was observed at 39 h after phytohemagglutinin stimulation. In a comparison of the time course of topoisomerase II gene expression with that of [3H]thymidine incorporation and thymidine kinase gene expression, it was found that the expression of the topoisomerase II gene was later than the onset of DNA replication. Thus, this study suggests that topoisomerase I, which is constantly expressed throughout the cell cycle, might participate in the initiation of DNA replication, while topoisomerase II is involved in solving the DNA topological problems accompanying DNA strand separation during DNA replication.
以在大肠杆菌中表达的人拓扑异构酶II多肽的羧基末端三分之一作为抗原，在兔体内产生多克隆抗体。利用抗血清，通过免疫印迹法检测植物血凝素刺激的人淋巴细胞的DNA拓扑异构酶II水平。我们的结果表明，细胞内拓扑异构酶II水平的增加与细胞进入增殖阶段平行。我们还发现，拓扑异构酶II水平的增加是由于拓扑异构酶II mRNA量的增加所致。时间进程研究表明，在植物血凝素刺激后36小时首次观察到拓扑异构酶II mRNA的出现。刺激后45小时观察到拓扑异构酶II mRNA的最大水平。用胸苷激酶探针将同一RNA印迹重新杂交。在植物血凝素刺激后39小时观察到胸苷激酶mRNA的最大水平。在比较拓扑异构酶II基因表达的时间进程与[3H]胸苷掺入和胸苷激酶基因表达的时间进程时，发现拓扑异构酶II基因的表达晚于DNA复制的起始。因此，本研究表明，在整个细胞周期中持续表达的拓扑异构酶I可能参与DNA复制的起始，而拓扑异构酶II则参与解决DNA复制过程中伴随DNA链分离的DNA拓扑问题。
