REDIRECTION OF REDOX PROTEINS TO THE NUCLEUS OF EUKARYOTIC CELLS

氧化还原蛋白重定向至真核细胞的细胞核

• 批准号: 3752414
• 负责人: A RUSSO
• 金额: --
• 依托单位: DIVISION OF CANCER TREATMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3752414
• 关键词: catalase; cell nucleus; cytotoxicity; detoxification; eukaryote; genetic manipulation; intracellular transport; polymerase chain reaction; protein transport; site directed mutagenesis; superoxide dismutase; tissue /cell culture; transfection /expression vector

Active oxygen species are implicated in ionizing radiation's and some antineoplastic drugs' cytotoxic mechanism of action. We already have shown that cells engineered to overexpress glutathione peroxidase (a protein known to detoxify hydrogen peroxide and alkylperoxides) are no more resistant to adriamycin or radiation induced cytotoxicity than are control cells. The lack of effect can be explained by the fact that glutathione peroxidase, although overexpressed, is localized normally in the cytoplasmic compartment and the primary target of the two damaging agents is DNA within the nuclear compartment. Therefore, glutathione peroxidase gene has been re-engineered by site directed mutagenesis to localize to the nucleus. Cooper/zinc dependent superoxide dismutase (detoxifies superoxide anion radical) and catalase (detoxifies hydrogen peroxide) genes have also been engineered to traffic to the nucleus. In the case of catalase, the peroxisome directing signal had to be removed from the gene and replaced by a nuclear targeting signal. Eukaryotic cells are being transformed with vectors containing the engineered constructs to test if active oxygen species are: l) responsible for cytotoxicity and 2) can be detoxified within the nuclear compartment. The reengineered proteins were chosen to provide information of which active oxygen species is promoting cell death. The projects require mRNA isolation, RT/PCR to isolate the respective gene of interest, SDM for redirection of the expressed proteins, tissue culture and transfection vector and regulatable promoter containing plasmid construction. This project has the potential to provide greater understanding of the mechanism of action of antineoplastic drugs and radiation.

活性氧与电离辐射有关，并且一些 抗肿瘤药物的细胞毒作用机制。 我们已经有 研究表明，经过改造的细胞可以过度表达谷胱甘肽过氧化物酶（一种 已知可以解毒过氧化氢和烷基过氧化物的蛋白质）是没有的 对阿霉素或辐射诱导的细胞毒性的抵抗力比 控制细胞。 缺乏效果可以用以下事实来解释： 谷胱甘肽过氧化物酶虽然过度表达，但通常位于 细胞质区室和两个损害的主要目标 试剂是核室内的DNA。 因此，谷胱甘肽 过氧化物酶基因已通过定点突变重新设计 定位于细胞核。 库珀/锌依赖性超氧化物歧化酶 （解毒超氧阴离子自由基）和过氧化氢酶（解毒氢） 过氧化物）基因也被设计用于运输到细胞核。 在 对于过氧化氢酶，必须去除过氧化物酶体定向信号 来自基因并被核靶向信号取代。 真核生物 细胞正在用含有工程改造的载体进行转化 构建来测试活性氧是否： l) 负责 细胞毒性和2)可以在核室内解毒。 这 选择重新设计的蛋白质来提供哪些活性的信息 氧物质正在促进细胞死亡。 该项目需要 mRNA 分离、RT/PCR 来分离相应的基因 感兴趣的，SDM 用于重定向表达的蛋白质，组织培养 以及含有质粒的转染载体和可调控启动子 建造。 该项目有潜力提供更多 了解抗肿瘤药物的作用机制 辐射。