Bjornsti M A, Benedetti P, Viglianti G A, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Cancer Res. 1989 Nov 15;49(22):6318-23.
Yeast Saccharomyces cerevisiae strains that are permeable to the antitumor alkaloid camptothecin are killed by the drug if they express DNA topoisomerase I, the cellular target of the drug (J. Nitiss and J.C. Wang, Proc. Natl. Acad. Sci. USA, 85: 7501-7505, 1988). We show that in a yeast strain permeable to camptothecin but lacking DNA topoisomerase I, sensitivity to the drug was restored upon expression of human DNA topoisomerase I from a plasmid-borne human complementary DNA clone. When the human enzyme was expressed from a galactose-inducible, glucose-repressible yeast promoter, PGAL1, sensitivity to camptothecin was observed in the presence of galactose but not in the presence of glucose. Expression of human DNA topoisomerase I in Escherichia coli was also demonstrated by the complementation of a conditional lethal E. coli DNA topoisomerase I mutant. These systems can be used in the study of human DNA topoisomerase I-camptothecin interactions and in the screening of additional therapeutics targeting the enzyme.
如果可渗透抗肿瘤生物碱喜树碱的酿酒酵母菌株表达该药物的细胞靶点——DNA拓扑异构酶I，那么它们会被该药物杀死（J. 尼蒂斯和J.C. 王，《美国国家科学院院刊》，85: 7501 - 7505, 1988）。我们发现，在一个可渗透喜树碱但缺乏DNA拓扑异构酶I的酵母菌株中，当从携带质粒的人类互补DNA克隆中表达人类DNA拓扑异构酶I时，该菌株对药物的敏感性得以恢复。当人类酶从半乳糖诱导、葡萄糖抑制的酵母启动子PGAL1表达时，在半乳糖存在下观察到对喜树碱的敏感性，但在葡萄糖存在下则没有。通过对条件致死的大肠杆菌DNA拓扑异构酶I突变体的互补作用，也证明了人类DNA拓扑异构酶I在大肠杆菌中的表达。这些系统可用于研究人类DNA拓扑异构酶I - 喜树碱的相互作用，以及筛选针对该酶的其他治疗药物。
