Gieseler F, Glasmacher A, Kämpfe D, Wandt H, Nuessler V, Valsamas S, Kunze J, Wilms K
Medizinische Poliklinik, Medical School, University of Wuerzburg, Germany.
Leukemia. 1996 Jul;10 Suppl 3:S46-S49.
We have developed a method to quantify topoisomerase (topo) II activities in partially purified nuclear extracts from human leukemia cells. By virtue of their different pH optima in the reaction buffer, two different topo II activities were found with activity optima at pH 7.9 and at pH 8.9 under high stringency conditions. The activities could be identified as topo II beta activity (pH 7.9) and topo II alpha activity (pH 8.9) by their different sensitivities to topo II alpha inhibitors, dephosphorylation experiments and immunoprecipitation with polyclonal antibodies. Seventy-two bone marrow or blood samples from patients with acute myeloid leukemias have been examined and their in vitro sensitivities to anthracyclines and epipodophyllotoxines correlated to the activities of topo II alpha and topo II beta. Although the topo II alpha activity could be directly inhibited by incubation of the cells with the mentioned drugs, no correlation between the topo II alpha activity and the sensitivity of the cells could be found. In contrast, the topo II beta activity which was not substantially inhibited by the drugs inversely correlated with the sensitivity of the cells. These findings were statistically significant for idarubicin (P=0.017) and daunorubicin (P=0.006). Vice versa, resistant cells (IC90 > median) had a higher topo II beta activity. Clinical relevance might be indicated by the finding that cells from patients that relapsed after initial treatment with anthracyclin-containing regiments had a significantly higher topo II alpha/beta activity ratio (P=0.0276). Obviously, the sensitivity of AML cells is substantially influenced by the activity of the resistant topo II (topo II beta) which gives evidence that the remaining topo II activity after treatment helps the cell to survive the DNA repair phase.
我们开发了一种方法来定量测定人白血病细胞部分纯化核提取物中的拓扑异构酶（topo）II活性。由于它们在反应缓冲液中的不同最适pH值，在高严格条件下发现了两种不同的topo II活性，其活性最适值分别在pH 7.9和pH 8.9。通过它们对topo IIα抑制剂的不同敏感性、去磷酸化实验以及用多克隆抗体进行免疫沉淀，可将这些活性鉴定为topo IIβ活性（pH 7.9）和topo IIα活性（pH 8.9）。对72例急性髓性白血病患者的骨髓或血液样本进行了检测，并将它们对蒽环类药物和表鬼臼毒素的体外敏感性与topo IIα和topo IIβ的活性相关联。尽管用上述药物孵育细胞可直接抑制topo IIα活性，但未发现topo IIα活性与细胞敏感性之间存在相关性。相反，未被药物显著抑制的topo IIβ活性与细胞敏感性呈负相关。这些发现对伊达比星（P = 0.017）和柔红霉素（P = 0.006）具有统计学意义。反之，耐药细胞（IC90>中位数）具有较高的topo IIβ活性。初始用含蒽环类药物方案治疗后复发患者的细胞具有显著更高的topo IIα/β活性比值（P = 0.0276），这一发现可能表明其具有临床相关性。显然，AML细胞的敏感性受到耐药性topo II（topo IIβ）活性的显著影响，这表明治疗后剩余的topo II活性有助于细胞在DNA修复阶段存活。
