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目的:确定DNA拓扑异构酶Ⅱ(Topo Ⅱ)是否为salvicine在酿酒酵母细胞内的主要作用靶点及其作用方式。方法:用Topo Ⅱ介导的超螺旋pBR322解族反应检测salvicine在无细胞体系中对Topo Ⅱ催化活力的影响;用克隆形成实验检测salvicine对四种酵母细胞系生长的作用。结果:salvicine能明显抑制无细胞体系中Topo Ⅱ对超螺旋pBR322的解旋作用。salvicine对JN394母系细胞及TOP1缺失的JN394top1~-细胞毒作用相似,验证了Topo Ⅰ不是其作用靶点。在25℃时,salvicine对温度敏感型JN394t2-1细胞具有良好的细胞毒作用;但在30℃时,Topo Ⅱ的活力大为降低,在有意义的浓度范围内salvicine对此类细胞的生长无明显抑制作用。另外,Topo Ⅱ发生突变的JN394t2-5细胞显示出对salvicine和etoposide(VP16)高度的耐受性。结论:Topo Ⅱ是salvicine细胞内王要作用靶点;salvicine通过捕获Topo Ⅱ-DNA断裂复合物而杀死细胞。此外,salvicine与VP16在Topo Ⅱ上有相似的作用位点。
OBJECTIVE: To determine whether Topo Ⅱ is the main target of salvicine in Saccharomyces cerevisiae cells and its mode of action. Methods: The effect of salvicine on the viability of Topo Ⅱ in cell-free system was tested by Topo Ⅱ-mediated supercoiled pBR322 decay reaction. The effect of salvicine on the growth of four yeast cell lines was tested by clonogenic assay. Results: salvicine significantly inhibited the unmotivated effect of Topo Ⅱ on supercoiled pBR322 in a cell-free system. salvicine on JN394 maternal cells and TOP1 deletion JN394top1 ~ - cytotoxicity similar to verify that Topo Ⅰ is not its target. At 25 ℃, salvicine has a good cytotoxic effect on the temperature-sensitive JN394t2-1 cells; however, the viability of Topo Ⅱ is greatly reduced at 30 ℃. The salvicine has no effect on the growth of such cells at a significant concentration range Obvious inhibitory effect. In addition, JN394t2-5 cells mutated in Topo II showed high tolerance to salvicine and etoposide (VP16). Conclusion: Topo Ⅱ is the target of salvicine intracellular. Salvicine kills cells by capturing Topo Ⅱ-DNA cleavage complex. In addition, salvicine has a similar site of action as VP16 on Topo II.