TOPISOMERASE II--GENE REGULATION AND DRUG RESISTANCE

拓扑异构酶II(Topo II)是一种普遍存在的酶，是许多 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通过一个 通过瞬间双链断裂的双链DNA片段 第二条DNA链来改变DNA分子的拓扑结构。Topo II是 也是核支架中的主要结构蛋白。有两个 TOPO II基因：一个表达170 kDa的TOPO II阿尔法型，另一个表达 编码180 kDa的Topo IIbeta形式。Topo II在DNA中的关键作用 复制和修复将表明Topo II基因高度和 特别受到监管。几类抗肿瘤药物的抑制作用 拓扑异构酶II对可切割DNA酶的稳定作用 复合体。选择对单一TOPO II具有抗性的细胞- 相互作用的药物通常对许多Topo II抑制剂表现出交叉耐药性 导致拓扑异构酶多药耐药表型改变(at- MDR)。 该项目的第一个主要目标是确定和描述 导致at-mdr表型的topo II酶突变。一个 实现这一目标的第一步将是确认直接 精氨酸与谷氨酰胺在细胞内保守位置上取代的相关性 Topo IIpha与at-mdr表型的一致ATP结合基序 由一株耐VM-26的CEM细胞株展示。其他地方的突变 Topo IIpha和Topo IIbeta中的核苷酸结合位点都将是 TOPO II-表达载体的体外有序诱变 为了进一步定义参与开发 耐药的Topo II酶。一组at-mdr细胞系和储存的DNA 将对临床样本进行筛查，以确定是否存在类似的TOPO II 变化是存在的。 另一个主要目标是描述以下监管控制的特征 两种拓扑异构酶II基因的表达 了解以下两种形式的特定和/或重叠功能 拓扑异构酶II。Topo IIα和β的启动子区域将是 分离和鉴定启动子位点和顺式作用 元素。影响拓扑异构酶II调控的反式作用因素 与细胞周期、增殖状态或转化有关 国家将被识别和表征。 分析导致At-MDR细胞的突变 拓扑异构酶II活性的变化将提供关于 临床重要药物与这种关键酶的相互作用，以及 可提供与行动和监管机制有关的信息 拓扑异构酶II。

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.