ISOLATION OF GENETIC SUPPRESSOR ELEMENTS, INDUCING RESISTANCE TO TOPOISOMERASE-II-INTERACTIVE CYTOTOXIC DRUGS, FROM HUMAN TOPOISOMERASE-II CDNA
ISOLATION OF GENETIC SUPPRESSOR ELEMENTS, INDUCING RESISTANCE TO TOPOISOMERASE-II-INTERACTIVE CYTOTOXIC DRUGS, FROM HUMAN TOPOISOMERASE-II CDNA
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DOI：
10.1073/pnas.90.8.3231
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
RONINSON, IB
中科院分区：
文献类型：
--
作者：
GUDKOV, AV;ZELNICK, CR;RONINSON, IB
许多细胞毒性抗癌药物作用于拓扑异构酶II（topo II），通过稳定这种酶与DNA形成的可切割复合物。几种细胞系，选择耐拓扑异构酶II相互作用的药物，显示拓扑异构酶II的表达或活性降低，这表明这种减少可能是负责耐药性。在本研究中，依托泊苷抗性被用作选择策略，以分离遗传抑制元件（GSEs）从逆转录病毒库表达的随机片段的人拓扑异构酶II（α形式）的cDNA。分离出12个GSE，编码对应于topo II alpha分子短片段的肽（占蛋白质的2.4-6.5%）或163- 220 bp长的反义RNA序列。GSE编码反义RNA的表达导致Topo II α蛋白的细胞表达降低。这两种类型的GSE诱导耐药性的几个拓扑II毒药，但不是药物，不作用于拓扑II。这些结果提供了直接的证据表明，抑制拓扑异构酶II的结果在耐拓扑异构酶II相互作用的药物，表明拓扑异构酶II的结构域能够独立的功能相互作用，并证明表达选择的随机片段构成了一个有效的方法来产生GSE在哺乳动物细胞。
Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo IIalpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo IIalpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.