Kaufmann S H, Karp J E, Jones R J, Miller C B, Schneider E, Zwelling L A, Cowan K, Wendel K, Burke P J
Oncology Center, Johns Hopkins Hospital, Baltimore, MD.
Blood. 1994 Jan 15;83(2):517-30.
The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (< 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to-cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony-stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.
拓扑异构酶（topo）II靶向药物依托泊苷、柔红霉素（DNR）和安吖啶（m-AMSA）被广泛用于治疗急性髓性白血病（AML）。在本研究中，对成年AML患者骨髓中topo II介导的药物作用的多个方面进行了检测。集落形成试验显示，使白血病集落形成减少90%（LD90）所需的依托泊苷、DNR或m-AMSA剂量在不同预处理骨髓之间的变化范围超过20倍。在不存在和存在奎尼丁的情况下测量细胞核DNR积累，结果显示，在诊断时82个样本中有8个、首次复发时36个样本中有5个存在P-糖蛋白（Pgp）功能的证据，但奎尼丁诱导的DNR积累最大增幅（<2倍）太小，无法解释药物敏感性的差异。对耐药性最强的标本进行基于限制性内切酶的分析以及topo IIα和topo IIβ部分cDNA的测序，未能证明存在可解释耐药性的topo II基因突变。对含有超过80%原始细胞的骨髓样本进行蛋白质印迹分析显示，两种topo II同工酶的含量变化范围超过20倍，但与体外或体内的药物敏感性无关。此外，47个临床样本中有46个样本的topo IIα和topo IIβ水平低于人AML细胞系。免疫过氧化物酶染色显示，这些较低的topo II水平伴随着明显的细胞间异质性，topo IIα在一些原始细胞中丰富，而在其他原始细胞中减少或缺失。在含有更多S期细胞的预处理骨髓中，topo IIα阳性细胞的百分比有增加的趋势。与此观察结果一致，化疗前用粒细胞-巨噬细胞集落刺激因子治疗患者3天，导致topo IIα阳性细胞增加，同时细胞周期中穿梭的细胞数量增加。这些观察结果对AML中topo II的调节、topo II靶向化疗的应用以及未来将药物敏感性与临床材料中topo II水平相关联的尝试具有启示意义。
