Danks M K, Qiu J, Catapano C V, Schmidt C A, Beck W T, Fernandes D J
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Biochem Pharmacol. 1994 Nov 1;48(9):1785-95. doi: 10.1016/0006-2952(94)90465-0.
Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
(a) 拓扑异构酶II的α（170 kDa）和β（180 kDa）同工酶的亚细胞分布，以及 (b) 在有或无VM - 26的情况下孵育的肿瘤细胞中，每种同工酶与DNA形成复合物的程度。蛋白质印迹法显示，拓扑异构酶IIβ在细胞分级分离过程中高度不稳定。然而，将人CEM白血病细胞与5 - 100 microM VM - 26预孵育30分钟，可通过逐渐增加与DNA结合的该同工型的量来保护β同工酶不被降解。用25 microM VM - 26孵育30分钟的CEM细胞核中检测到的拓扑异构酶IIβ的量比未处理的对照细胞核中的量高7倍。VM - 26对HL - 60白血病细胞和WiDR结肠癌细胞中的拓扑异构酶IIβ也有保护作用。相比之下，嵌入剂米托蒽醌和m - AMSA在细胞分级分离过程中不能保护拓扑异构酶IIβ不被降解。VM - 26对拓扑异构酶IIβ的稳定作用使得后续能够研究拓扑异构酶II同工酶的亚细胞分布。在CEM细胞核的非基质（高盐可溶性）部分检测到了两种同工酶，但只有拓扑异构酶IIα存在于核基质中。VM - 26稳定了α和β拓扑异构酶II同工酶与非基质DNA的结合以及拓扑异构酶IIα与基质DNA的结合。在拓扑异构酶II同工酶的亚核分布和DNA结合模式中观察到的差异支持了以下假设：每种同工酶具有独特的细胞功能，并且α和β同工酶在完整细胞中都是VM - 26的潜在靶点。此外，结果表明，用VM - 26对各种细胞系进行预处理是在细胞分级分离过程中稳定拓扑异构酶IIβ的一种有用方法。
