Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform.
Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform.
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发表时间：
1999-07
期刊：
影响因子：
11.2
通讯作者：
I. Wessel;L. Jensen;P. B. Jensen;J. Falck;A. Rose;M. Roerth;J. Nitiss;M. Sehested
中科院分区：
文献类型：
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作者：
I. Wessel;L. Jensen;P. B. Jensen;J. Falck;A. Rose;M. Roerth;J. Nitiss;M. Sehested
ICRF-187等双二氧代哌嗪药物是DNA拓扑异构酶II的催化抑制剂，对该酶至少有两种作用：即，将其锁定在闭合钳形式并抑制其ATP酶活性。这与拓扑异构酶 II 毒物如依托泊苷和安吖啶 (m-AMSA) 形成鲜明对比，后者通过在 DNA 门链断裂且蛋白质共价连接到 DNA 的阶段稳定酶-DNA-药物复合物发挥作用。选择对 ICRF-187 (NYH/187) 具有抗性的人小细胞肺癌 NYH 细胞显示拓扑异构酶 IIα 水平增加了 25%，而 β 异构体的表达没有变化。对来自 NYH/187 细胞的整个拓扑异构酶 IIα cDNA 进行测序，结果显示核苷酸 485 处存在纯合 G-->A 点突变，导致 Walker A 共有 ATP 结合位点（α 同种型中的残基 161-165）发生 R162Q 转换，这是该位点描述的第一个药物选择突变。与 ICRF-187 孵育后的蛋白质印迹显示，与野生型 (wt) 细胞相比，NYH/187 细胞中的 α 同工型没有耗尽，而在两个亚系中观察到了相同的 β 同工型耗尽。碱性洗脱测定表明，在 NYH/187 细胞中，依托泊苷诱导的 DNA 单链断裂缺乏抑制作用，而这种抑制作用在 NYH 细胞中很明显。将人拓扑异构酶 IIalpha 定点诱变引入具有温度条件酵母 TOP2 突变体的酿酒酵母菌株中，证明 R162Q 赋予双二氧代哌嗪 ICRF-187 和 -193 抗性，但不赋予依托泊苷或 m-AMSA 抗性。与野生型相比，依托泊苷和 m-AMSA 使用纯化的 R162Q 酶诱导更多的 DNA 切割。与野生型相比，R162Q 酶的催化能力降低了 20-25%，并且与野生型相比，在 <0.25 mM ATP 时几乎没有活性。与 wt 相比，R162Q 酶在 1 mM ATP 下对动质体 DNA 的去连接对 ICRF-187 没有抗性，但在低 ATP 浓度下，它对 ICRF-187 的敏感性明显低于 wt。这表明，这是酶催化循环中平衡向开钳状态的转变，这是由于导致双二氧代哌嗪抗性的突变酶与 ATP 的结合减少所致。
Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.