Matsuo K, Kohno K, Takano H, Sato S, Kiue A, Kuwano M
Department of Biochemistry, Oita Medical School, Japan.
Cancer Res. 1990 Sep 15;50(18):5819-24.
We have isolated stable teniposide (VM26)-resistant cell lines from human cancer KB cells by stepwise exposure to increasing doses of the drug. At each step, we have purified VM26-resistant cell lines. KB/VM-a, KB/VM-b, KB/VM-1, KB/VM-2, KB/VM-3, and KB/VM-4 showed 3-, 6-, 12-, 16-, 74-, and 95-fold higher resistance to VM26 than did KB. We have further characterized KB/VM-2 and KB/VM-4 which showed about 15- and 100-fold higher resistance to VM26 or etoposide (VP16) than did KB. Both VM26-resistant cell lines showed 4- to 11-fold higher relative resistance to daunomycin and Adriamycin than did KB. Steady-state levels of the cellular accumulation of radioactive VP16 in KB/VM-2 and KB/VM-4 cells were about 40% of that of KB cells, whereas similar levels of radioactive daunomycin accumulation were observed in KB/VM-2 and KB/VM-4 cells as KB cells. Topoisomerase II activity of nuclear extracts of both KB/VM-2 and KB/VM-4 assayed by decatenation of kinetoplast DNA was consistently two-thirds or less the activity of KB cells. A similar reduction was seen in both immunoblot assays with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA. DNA topoisomerase I activity, however, was similar between the mutants and their parent. Furthermore, cell growth of KB/VM-2 and KB/VM-4 was more thermolabile than that of KB, while KB/VM-b already showed temperature-sensitive growth. KB/VM-1 did show reduced accumulation of VP16 as in KB/VM-2 or KB/VM-4, but it had a normal level of topoisomerase II content as in KB cells. These data suggest that the reduced expression of DNA topoisomerase II, possibly combined with decreased permeability to the drugs, can account for the acquired VM26 resistance of KB/VM-2 and KB/VM-4 cells and also that the temperature-sensitive phenotype might not be obligatorily coupled with the reduced expression of topoisomerase II or the decreased permeability.
我们通过逐步增加药物剂量处理人源癌细胞KB细胞，分离出了稳定的替尼泊苷（VM26）耐药细胞系。在每一步骤中，我们都对VM26耐药细胞系进行了纯化。KB/VM-a、KB/VM-b、KB/VM-1、KB/VM-2、KB/VM-3和KB/VM-4对VM26的耐药性比KB细胞分别高3倍、6倍、12倍、16倍、74倍和95倍。我们进一步对KB/VM-2和KB/VM-4进行了特性分析，它们对VM26或依托泊苷（VP16）的耐药性比KB细胞分别高约15倍和100倍。这两种VM26耐药细胞系对柔红霉素和阿霉素的相对耐药性比KB细胞高4至11倍。KB/VM-2和KB/VM-4细胞中放射性VP16的细胞内稳态积累水平约为KB细胞的40%，而KB/VM-2和KB/VM-4细胞中放射性柔红霉素的积累水平与KB细胞相似。通过动质体DNA解连环反应测定，KB/VM-2和KB/VM-4细胞核提取物的拓扑异构酶II活性始终是KB细胞活性的三分之二或更低。在使用特异性抗拓扑异构酶II抗体的免疫印迹分析和使用特异性人DNA拓扑异构酶II互补DNA的Northern印迹分析中也观察到了类似的降低。然而，突变体与其亲本之间的DNA拓扑异构酶I活性相似。此外，KB/VM-2和KB/VM-4的细胞生长比KB细胞对温度更敏感，而KB/VM-b已经表现出温度敏感生长。KB/VM-1确实如KB/VM-2或KB/VM-4一样表现出VP16积累减少，但它的拓扑异构酶II含量水平与KB细胞一样正常。这些数据表明，DNA拓扑异构酶II表达降低，可能与药物通透性降低相结合，可以解释KB/VM-2和KB/VM-4细胞获得性VM26耐药性，并且温度敏感表型可能不一定与拓扑异构酶II表达降低或通透性降低相关联。
