Sugimoto Y, Tsukahara S, Oh-hara T, Isoe T, Tsuruo T
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo.
Cancer Res. 1990 Nov 1;50(21):6925-30.
DNA topoisomerase I (topo I) has been identified as a principal target of a plant alkaloid camptothecin (CPT) and its derivative (CPT-11). The latter compound is expected to be a clinically useful antitumor agent. Three human tumor cell lines resistant to CPT (A549/CPT, HT-29/CPT, St-4/CPT) were isolated in vitro, and a murine tumor cell line resistant to CPT-11 (P388/CPT) was isolated in vivo by continuous exposure of the drugs. A549/CPT, HT-29/CPT, and St-4/CPT showed 1.8-, 6.9-, and 8.8-fold more resistance to CPT, and P388/CPT showed 45-fold more resistance to CPT than did the parental line. To examine the possible involvement of topo I in drug-resistant mechanisms, a monoclonal antibody was developed by using purified human topo I as antigen. The antibody T14C (immunoglobulin G1) recognized both human and murine topo I, as shown by Western blot analysis. By using this monoclonal antibody, cellular contents of topo I were examined in CPT-resistant tumor lines. Respective contents of topo I in HT-29/CPT, St-4/CPT, and P388/CPT were approximately 8-, 4-, and 3-fold less than those in their parental cell lines. A549/CPT, a weak CPT-resistant line, possessed amounts of topo I similar to those of the parental line. HT-29/CPT showed lower topo I activity than did the parental HT-29 in the nuclear extracts and in the hydroxylapatite column-eluted fractions. Purified topo I from HT-29 and HT-29/CPT showed similar catalytic activity when the same amounts of protein were used. These results indicate that the quantitative reduction of topo I content seems to be the most frequently occurring event in the development of resistance to camptothecin.
DNA拓扑异构酶I（拓扑异构酶I）已被确定为植物生物碱喜树碱（CPT）及其衍生物（CPT - 11）的主要靶点。后一种化合物有望成为临床上有用的抗肿瘤药物。通过体外连续暴露于药物，分离出三种对CPT耐药的人肿瘤细胞系（A549/CPT、HT - 29/CPT、St - 4/CPT），并通过体内连续暴露于药物分离出一种对CPT - 11耐药的小鼠肿瘤细胞系（P388/CPT）。A549/CPT、HT - 29/CPT和St - 4/CPT对CPT的耐药性分别比亲代细胞系高1.8倍、6.9倍和8.8倍，而P388/CPT对CPT的耐药性比亲代细胞系高45倍。为了研究拓扑异构酶I在耐药机制中可能的作用，以纯化的人拓扑异构酶I为抗原制备了一种单克隆抗体。如蛋白质印迹分析所示，抗体T14C（免疫球蛋白G1）可识别人和小鼠的拓扑异构酶I。利用这种单克隆抗体，检测了CPT耐药肿瘤细胞系中拓扑异构酶I的细胞含量。HT - 29/CPT、St - 4/CPT和P388/CPT中拓扑异构酶I的含量分别比其亲代细胞系少约8倍、4倍和3倍。A549/CPT是一种对CPT耐药性较弱的细胞系，其拓扑异构酶I的含量与亲代细胞系相似。在核提取物和羟基磷灰石柱洗脱组分中，HT - 29/CPT的拓扑异构酶I活性低于亲代HT - 29。当使用相同量的蛋白质时，从HT - 29和HT - 29/CPT纯化得到的拓扑异构酶I表现出相似的催化活性。这些结果表明，拓扑异构酶I含量的定量减少似乎是喜树碱耐药发展过程中最常见的事件。
