Aoyama M, Grabowski D R, Isaacs R J, Krivacic K A, Rybicki L A, Bukowski R M, Ganapathi M K, Hickson I D, Ganapathi R
Experimental Therapeutics Program, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH, USA.
Blood. 1998 Oct 15;92(8):2863-70.
Regulation of topoisomerase II (TOPO II) isozymes alpha and beta is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIalpha and TOPO IIbeta proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (alpha and beta) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIbeta protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIbeta protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 micromol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 micromol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 micromol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIbeta protein levels are posttranscriptionally regulated and that degradation of TOPO IIbeta is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (alpha + beta) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIbeta may have a specific role in transcription of genes involved in differentiation with RA treatment.
拓扑异构酶II（TOPO II）α和β同工酶的调节受细胞生长和转化状态的影响。利用经全反式维甲酸（RA）诱导分化的HL-60细胞，我们研究了TOPO II同工酶的表达和调节以及拓扑异构酶I（TOPO I）的水平。在RA诱导人白血病HL-60细胞分化过程中，TOPO I的水平保持不变，而TOPO IIα和TOPO IIβ蛋白的水平及磷酸化分别增加了两倍至四倍和四倍至八倍。在用RA处理48小时时，TOPO II（α和β）蛋白水平及磷酸化明显升高，并持续至96小时。在随后于无RA培养基中培养96小时的分化细胞中也检测到TOPO IIβ蛋白水平升高。用35S-甲硫氨酸标记细胞的脉冲追踪实验表明，对照细胞中TOPO IIβ蛋白的降解速率比经RA处理的分化细胞快约两倍。对照或经RA处理的细胞的核提取物中，线粒体DNA（kDNA）的解连环活性水平相当。虽然依托泊苷（1至10 μmol/L）诱导的DNA切割没有显著差异，但在暴露于10 μmol/L依托泊苷后，经RA处理的细胞与对照细胞相比，凋亡显著降低（P = 0.012）。与TOPO I水平未改变一致，喜树碱（CPT）诱导的DNA切割在对照或经RA处理的细胞中相似。然而，在暴露于1至10 μmol/L CPT后，经RA处理的细胞与对照细胞相比，凋亡显著降低（P = 0.003至P < 0.001）。结果表明，TOPO IIβ蛋白水平在转录后受到调节，并且在RA诱导的分化过程中TOPO IIβ的降解减少。此外，虽然TOPO II（α + β）的总水平随RA增加，但TOPO II催化活性和依托泊苷稳定的DNA切割活性水平保持不变。因此，TOPO IIβ可能在RA处理诱导的分化相关基因的转录中具有特定作用。
