Sugimoto K, Yamada K, Egashira M, Yazaki Y, Hirai H, Kikuchi A, Oshimi K
Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
Blood. 1998 Feb 15;91(4):1407-17.
We related cellular content of DNA topoisomerase (topo) IIalpha and IIbeta with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIalpha increased especially in late S to G2/M phases, although the topo IIbeta level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIalpha but not the topo IIbeta level. A new G2/M population containing virtually no topo IIalpha appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIalpha or IIbeta staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIbeta epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIalpha epitope and another topo IIbeta epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIbeta protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIalpha remained of its original size, although both topo IIalpha and IIbeta left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIalpha and IIbeta in growing HL-60 cells. Although topo IIalpha and IIbeta were distributed throughout the cell during mitosis, only topo IIalpha was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.
我们使用二维流式细胞术，将DNA拓扑异构酶（topo）IIα和IIβ的细胞含量与增殖、分化和凋亡的HL-60细胞中的细胞周期位置相关联。在对数生长期的HL-60细胞中，topo IIα尤其在S期晚期至G2/M期增加，尽管topo IIβ水平在整个细胞周期中几乎保持恒定。全反式维甲酸诱导分化显著降低了topo IIα水平，但未降低topo IIβ水平。在分化过程中出现了一个几乎不含topo IIα的新G2/M群体，推测其为存活且非循环的细胞。topo IIα或IIβ染色的二维流式细胞术以及末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记分析表明，位于C末端的一个topo IIβ表位在经阿糖胞苷、依托泊苷和长春新碱处理的凋亡HL-60细胞中特异性降低。位于更中心部分的一个topo IIα表位和另一个topo IIβ表位的量在凋亡细胞和非凋亡细胞之间几乎相等。蛋白质印迹分析证实，topo IIβ蛋白在凋亡过程中完全降解为较小的片段并失去其C末端。相反，尽管凋亡细胞中topo IIα和IIβ均从核部分中消失，但大部分topo IIα仍保持其原始大小。共聚焦激光显微镜显示，在生长的HL-60细胞中topo IIα和IIβ定位于细胞核。虽然在有丝分裂期间topo IIα和IIβ分布于整个细胞，但只有topo IIα密集地集中在有丝分裂染色体中。即使在凋亡早期，这两种酶也与基因组DNA解离，并在晚期与DNA的碘化丙啶信号完全分离。因此，凋亡中的染色质浓缩过程完全不依赖于topo II，且明显不同于有丝分裂过程中的染色质浓缩。
