TOPISOMERASE II--GENE REGULATION AND DRUG RESISTANCE

拓扑异构酶 II——基因调控和耐药性

• 批准号: 2092789
• 负责人: DALE P SUTTLE
• 金额: $ 13.26万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-10 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2092789
• 关键词: DNA; DNA replication; DNA topoisomerases; antineoplastics; chloramphenicol acetyltransferase; complementary DNA; drug metabolism; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; gene expression; gene mutation; genetic mapping; genetic promoter element; genetic transcription; isomerase; messenger RNA; multidrug resistance; neoplasm /cancer genetics; neoplastic cell; neoplastic transformation; nucleic acid sequence; polymerase chain reaction; transcription factor; transfection /expression vector; vinca alkaloids; western blottings

Topoisomerase II (topo II) is a ubiquitous enzyme that is required for many essential cell functions such as DNA replication, recombination and repair and chromosome segregation. Acting as a dimer, topoisomerase II passes a double stranded DNA segment through a transient double strand break in a second DNA strand to modify the topology of the DNA molecule. Topo II is also a major structural protein in the nuclear scaffold. There are two topo II genes: one expressing a 170 kDa topo IIalpha form, and the other coding for a 180 kDa topo IIbeta form. The crucial role of topo II in DNA replication and repair would indicate that the topo II genes are highly and specifically regulated. Several classes of antitumor drugs inhibit topoisomerase II through the stabilization of cleavable DNA-enzyme complexes. Cells that are selected for resistant to a single topo II- interacting drug often exhibit cross-resistance to many topo II inhibitors resulting in an altered-topoisomerase multidrug resistant phenotype (at- MDR). The first major objective of this project is to identify and characterize mutations in the topo II enzyme that result in the at-MDR phenotype. A first step in meeting this objective will be to confirm the direct correlation of an Arg to Gln substitution at a conserved position in a consensus ATP binding motif of topo IIalpha with the at-MDR phenotype exhibited by a VM-26-resistant CEM cell line. Mutations at other nucleotide binding sites in both topo IIalpha and topo IIbeta will be introduced by in vitro mutagenesis of a topo II-expression vector in order to further define the sites and structures involved in the development of a drug-resistant topo II enzyme. A panel of at-MDR cell lines and banked DNA from clinical samples will be screened to determine if similar topo II alterations are present. The other major objective is to characterize the regulatory controls for the expression of the two topoisomerase II genes in order to further understand the specific and/or overlapping functions of the two forms of topoisomerase II. The promoter regions of topo IIalpha and beta will be isolated and characterized to identify promoter sites and cis-acting elements. Trans-acting factors that effect regulation of topoisomerase II in association with cell cycle, proliferation status or transformation state will be identified and characterized. Analysis of mutations in the at-MDR cells that are responsible for the alterations in topoisomerase II activity will yield information about the interaction of clinically important drugs with this critical enzyme, and may provide information pertinent to the mechanism of action and regulation of topoisomerase II.

拓扑异构酶II（topo II）是一种普遍存在的酶， 基本的细胞功能，如DNA复制，重组和修复 和染色体分离。 作为二聚体，拓扑异构酶II通过 双链DNA片段通过短暂的双链断裂， 第二DNA链以修饰DNA分子的拓扑结构。 Topo II是 也是核支架中的主要结构蛋白。 有两 topo II基因：一个表达170 kDa的topo II α形式，另一个表达170 kDa的topo II α形式， 编码180 kDa拓扑异构酶II β形式。 Topo II在DNA中的重要作用 复制和修复表明，topo II基因是高度和 具体规定。 几类抗肿瘤药物抑制 拓扑异构酶II通过稳定可裂解的DNA-酶 配合物 选择对单一拓扑异构酶II具有抗性的细胞- 相互作用药物通常对许多拓扑异构酶II抑制剂表现出交叉耐药性 导致改变的拓扑异构酶多药耐药表型（at- MDR）。 该项目的第一个主要目标是确定和表征 导致at-MDR表型的topo II酶突变。 一 实现这一目标的第一步将是确认直接 在一个保守的位置上的Arg到Gln取代的相关性， 具有at-MDR表型的拓扑异构酶II α的共有ATP结合基序 VM-26抗性CEM细胞系所显示的。 其他突变 拓扑异构酶II α和拓扑异构酶II β中的核苷酸结合位点将是 通过Topo II表达载体的体外诱变引入， 进一步确定发展一个 耐药拓扑异构酶II。 一组at-MDR细胞系和库存DNA 将对临床样本进行筛选，以确定是否存在类似的拓扑异构酶II 存在变化。 另一个主要目标是描述监管控制的特点， 两种拓扑异构酶II基因的表达，以进一步 理解这两种形式的具体和/或重叠的功能， 拓扑异构酶Ⅱ 拓扑异构酶II α和β的启动子区将是 分离并表征以鉴定启动子位点和顺式作用 元素 影响拓扑异构酶II调节的反式作用因子 与细胞周期、增殖状态或转化相关 国家将得到确认和定性。 分析在at-MDR细胞中的突变，所述突变负责 拓扑异构酶II活性的改变将产生关于 临床重要药物与该关键酶的相互作用，以及 可以提供与作用和调节机制有关的信息 拓扑异构酶II