Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells.
Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells.
复制标题
人类癌症 KB 细胞的依托泊苷抗性突变体中 DNA 拓扑异构酶 II 的磷酸化增加。
作者：
H. Takano;K. Kohno;M. Ono;Yuzo Uchida;M. Kuwano
我们已经分离出两个依托泊苷（VP 16）的耐药细胞系，KB/VP-1和KB/VP-2，从人癌KB细胞逐步暴露后，逐步增加剂量的VP 16。KB/VP-1和KB/VP-2对VP 16的抗性分别是亲本细胞的30倍和50倍，对替尼泊苷的抗性分别是亲本细胞的20倍和30倍。此外，两种耐药细胞系对阿霉素和柔红霉素的交叉耐药性均高于KB细胞2倍以上。放射性VP 16的蓄积和向外转运水平在KB/VP-1、KB/VP-2和KB中相似。用动质体DNA脱连环法测定的KB/VP-1和KB/VP-2的DNA拓扑异构酶II的核提取物的活性与KB的一致。然而，在用特异性抗拓扑异构酶II抗体进行的免疫印迹分析和用特异性人DNA拓扑异构酶II互补DNA进行的北方印迹分析中，两种耐药细胞系中的拓扑异构酶II的细胞水平均小于KB中水平的十分之一。然而，DNA拓扑异构酶I的细胞水平在突变体和它们的亲本之间是相似的。共价DNA-拓扑异构酶II复合物的定量沉淀分析表明，与KB细胞相比，KB/VP-1或KB/VP-2细胞的核提取物大大降低了VP 16诱导的3 '-32 P-DNA切割。DNA拓扑异构酶II的相对特异性磷酸化在突变体中比在亲本细胞中高约14至18倍。DNA拓扑异构酶II的磷酸化氨基酸分析表明，丝氨酸是所有三种细胞系KB、KB/VP-1和KB/VP-2中的磷酸化氨基酸。这些数据表明，DNA-拓扑异构酶II的表达减少可能是获得性VP 16抗性和两种VP 16抗性变体中VP 16诱导的DNA-拓扑异构酶II复合物裂解减少的原因。
We have isolated two etoposide (VP16)-resistant cell lines, KB/VP-1 and KB/VP-2, from human cancer KB cells after stepwise exposure to increasing doses of VP16. KB/VP-1 and KB/VP-2 showed 30- and 50-fold higher resistance to VP16 and also 20- and 30-fold higher resistance to teniposide than the parent cell line. Furthermore, both resistant cell lines showed more than 2-fold cross-resistance to Adriamycin and daunomycin than KB cells. The levels of accumulation and outward transport of radioactive VP16 were similar in KB/VP-1, KB/VP-2, and KB. The activity of nuclear extracts of DNA topoisomerase II for both KB/VP-1 and KB/VP-2 assayed by decatenation of kinetoplast DNA was consistently similar to that of KB. However, in both immunoblot assay with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA, cellular levels of topoisomerase II in both resistant cell lines were less than one-tenth the level in KB. The cellular levels of DNA topoisomerase I, however, were similar between the mutants and their parent. A quantitative precipitation assay of covalent DNA-topoisomerase II complexes showed greatly reduced VP16-induced cleavages of 3'-32P-DNA by nuclear extracts of KB/VP-1 or KB/VP-2 cells in comparison with KB cells. The relative specific phosphorylation of DNA topoisomerase II was about 14- to 18-fold higher in the mutants than in the parental cells. Phosphoamino acid analysis of DNA topoisomerase II showed that serine was the phosphorylated amino acid in all three cell lines, KB, KB/VP-1, and KB/VP-2. These data suggest that reduced expression of DNA-topoisomerase II might account for the acquired VP16 resistance and reduced VP16-induced cleavages of DNA-topoisomerase II complexes in both VP16-resistant variants.
M.Saijo：“从小鼠 FM3A 细胞中纯化和表征 II 型拓扑异构酶：拓扑异构酶 II 的磷酸化及其活性的修饰”生物化学。
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