Cellular Targets for Gallium Compounds in Lymphoma

淋巴瘤中镓化合物的细胞靶点

• 批准号: 7027683
• 负责人: CHRISTOPHER R CHITAMBAR
• 金额: $ 26.3万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7027683
• 关键词: antineoplastics; apoptosis; chemosensitizing agent; clinical research; cytotoxicity; electron spin resonance spectroscopy; fluorescent dye /probe; free radical oxygen; gallium; gene expression; gene mutation; gene targeting; high performance liquid chromatography; immunologic assay /test; intracellular transport; iron; lymphoma; mitochondria; neoplasm /cancer therapy; northern blottings; pharmacokinetics; ribonucleotide reductase; transferrin receptor; western blottings

DESCRIPTION (provided by applicant): The mortality from lymphoma is high; thus, there is a great need to not only develop new therapeutic agents for this disease but to also further develop agents that have already shown promise in prior investigations. In early clinical trials, gallium nitrate (NSC15200) demonstrated significant clinical activity in lymphoma but its use in lymphoma was never rigorously pursued and information regarding its mechanisms of action has remained largely incomplete. There is now a renewed clinical interest in using gallium for lymphoma and clinical trials are in progress. The long-term objectives of this project are to optimally develop gallium compounds for the treatment of lymphoma by understanding its mechanisms of action. Our preliminary data suggest that the hemochromatosis (HFE) gene influences gallium uptake and cytotoxicity in lymphoblastoid cells and that gallium targets the mitochondria. HFE mutations, C282Y and H63D, occur with high frequency in the population. Specific Aim 1 will further investigate the role of HFE [wild type (wt) and mutant] as modulators of transferrin receptor-targeted gallium uptake and cytotoxicity in lymphoma cells. This Aim will investigate why lymphoblastic cells with the HFE C282Y mutation are more sensitive to gallium than cells with wt HFE. Studies will investigate the effects of HFE on gallium transport, gallium-induced apoptosis, gallium's action on iron homeostasis and ribonucleotide reductase. Specific Aim 2 will examine the effects of gallium on the generation of reactive oxygen species (ROS), mitochondrial function, and apoptosis induction. The effect of HFE mutations on gallium's antineoplastic action will be investigated with immortalized cell lines developed from individuals with wt HFE and HFE mutations. Lymphoma cells transfected with an inducible HFE gene (wt or mutant) will also be utilized. Ga-67 and Fe-59-transferrin will be used for uptake studies. mRNA and protein levels of transferrin receptor, ferritin, and HFE will be measured by Northern blotting, ligand binding, immunoblotting and immunoassays. Iron regulatory protein-RNA binding and ribonucleotide reductase R2 subunit will be studied by bandshift assay and ESR spectroscopy, respectively. ROS in cells will be detected using fluorescent probes and HPLC. Mitochondria-targeted antioxidants will be employed to investigate the source of gallium-induced ROS production. Mitochondrial electron transport enzymes will be assayed by measuring oxygen consumption and ATP production in cells and isolated mitochondria using substrates and inhibitors. Assays will measure aconitase and glucose consumption and caspase activity. Our studies will provide new information regarding: a) the impact of HFE mutations on the response of lymphoma to gallium, and, b) the mechanism of action of gallium at the mitochondrial level.

描述（由申请人提供）：淋巴瘤的死亡率很高；因此，不仅需要为该疾病开发新的治疗剂，而且还需要进一步发展那些已经在先前研究中显示出希望的药物。在早期的临床试验中，硝酸盐（NSC15200）在淋巴瘤中表现出显着的临床活性，但从未严格追求其在淋巴瘤中的使用，有关其作用机理的信息在很大程度上仍然不完整。现在，在使用胆管进行淋巴瘤方面具有新的临床兴趣，并且正在进行临床试验。该项目的长期目标是通过了解其作用机制来最佳地开发包胶化合物来治疗淋巴瘤。我们的初步数据表明，血色素症（HFE）基因影响淋巴母细胞细胞中的凝胶摄取和细胞毒性，并且胆靶靶向线粒体。 HFE突变，C282Y和H63D，人口频率高。具体的目标1将进一步研究HFE [野生型（WT）和突变体]作为转运蛋白受体靶向的凝胶摄取和细胞毒性在淋巴瘤细胞中的作用。这个目标将调查为什么具有HFE C282Y突变的淋巴细胞细胞比具有WT HFE的细胞更敏感。研究将研究HFE对凝胶运输的影响，炮诱导的凋亡，炮对铁稳态和核糖核苷酸还原酶的作用。具体目标2将检查凝胶对活性氧（ROS），线粒体功能和凋亡诱导产生的影响。通过从WT HFE和HFE突变的个体发展出永生的细胞系，将研究HFE突变对胆管抗肿瘤作用的影响。还将利用用诱导HFE基因（WT或突变体）转染的淋巴瘤细胞。 GA-67和FE-59-Transferrin将用于摄取研究。转铁蛋白受体，铁蛋白和HFE的mRNA和蛋白质水平将通过北印迹，配体结合，免疫印迹和免疫测定法测量。铁调节蛋白-RNA结合和核糖核苷酸还原酶R2亚基将分别通过带班试验和ESR光谱研究。使用荧光探针和HPLC检测细胞中的ROS。将采用靶向线粒体的抗氧化剂来研究镀膜诱导的ROS产生的来源。线粒体电子转运酶将通过测量细胞中的氧气消耗和ATP产生以及使用底物和抑制剂分离的线粒体来测定。测定将测量丙型酶和葡萄糖消耗和caspase活性。我们的研究将提供有关以下方面的新信息：a）HFE突变对淋巴瘤对胆管反应的影响，b）凝固膜在线粒体水平上的作用机理。