Zini N, Santi S, Ognibene A, Bavelloni A, Neri L M, Valmori A, Mariani E, Negri C, Astaldi-Ricotti G C, Maraldi N M
Istituto di Citomorfologia Normale e Patologica, CNR, Bologna, Italy.
Exp Cell Res. 1994 Feb;210(2):336-48. doi: 10.1006/excr.1994.1046.
Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.
针对DNA拓扑异构酶I以及拓扑异构酶IIα和β亚型产生的单克隆抗体，先前已证明它们具有高度特异性，能够检测拓扑异构酶II亚型与细胞周期相关的变化（Negri等人，1992年，《实验细胞研究》200, 452 - 459），已被用于精细的亚细胞定位。通过共聚焦显微镜和电子显微镜进行的免疫细胞化学已用于对HeLa和K562细胞中不同拓扑异构酶的精细分布进行拓扑学和定量评估。拓扑异构酶I和拓扑异构酶IIα均存在于核质和核仁中，尽管相对比例不同，而拓扑异构酶IIβ仅存在于核仁水平。从K562细胞获得的纯化核基质组分上进行的免疫印迹和免疫细胞化学定量评估进一步证实了这一点。事实上，全细胞核中存在的拓扑异构酶I和拓扑异构酶IIα的量在分离的细胞核中部分丢失，但是，虽然拓扑异构酶I在核基质制剂中进一步显著减少，但拓扑异构酶IIα的含量仅略有下降。另一方面，绝大多数拓扑异构酶IIβ保留在核基质中，并且只能与核仁残余物相关联才能检测到。这些结果与针对不同拓扑异构酶类型假设的特定功能作用一致。
