CELLULAR UPTAKE AND INTERACTION OF IRON AND GALLIUM

铁和镓的细胞吸收和相互作用

• 批准号: 3446871
• 负责人: CHRISTOPHER R CHITAMBAR
• 金额: $ 5.54万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3446871
• 关键词: affinity chromatography; antireceptor antibody; autoradiography; biopsy; cell growth regulation; chelating agents; ferritin; fibroblasts; gallium; growth inhibitors; hereditary hemochromatosis; high performance liquid chromatography; human subject; immunochemistry; ion transport; iron; iron metabolism; metal metabolism; phosphorylation; radioimmunoassay; radiotracer; receptor mediated endocytosis; tissue /cell culture; transferrin

The long-term objectives and specific aims of the projects in this research proposal are 1) to obtain a better understanding of cellular iron requirements during cellular proliferation and to define the interaction of gallium with cellular iron metabolism since gallium appears to inhibit cell growth by interfering with cellular iron utilization in an unknown manner and has been used in clinical cancer trials as a cytotoxic agent. An understanding of this interaction may provide new information regarding cellular requirements for iron during proliferation and information as to how these requirements may be exploited to control tumor cell growth. 2) To establish fibroblast cultures from patients with Hemochromatosis (a disease characterized by iron-overload resulting in multi-organ damage) and study transferrin receptor regulation and factors influencing iron uptake in these cells. The studies may elucidate new information regarding this disease. Studies of the effect of gallium on intracellular distribution of 59Fe will include gel filtration of solubilized cytoplasmic contents and immunoprecipitation of the peak radioactive fractions for transferrin, transferrin receptor and ferritin using specific antisera. Subcellular fractionation will also be performed using sucrose gradient centrifugation. Cellular isoferritins will be studied during gallium-controlled cell proliferation using isoelectric focusing. Studies of 67gallium uptake and intracellular distribution will be performed using similar methodology. Gallium-binding proteins will be identified by gel electrophoresis. Studies of cellular receptosome unloading of iron in gallium treated cells will employ studies of receptosomal pH changes using FITC-labeled transferrin-metal probes and analysis in a spectrofluorometer. Studies of effects of different transferrin-metals on receptor cycling/recycling will include 59Fe/125I-transferrin in pulse-chase experiments. Studies of transferrin receptor synthesis and phosphorylation will employ 35S-methionine and 32P labeling of the receptor with isolation by affinity chromatography. 32P labeled phosphorylated transferrin receptor will be identified on gels using autoradiography. The different transferrin-metals, iron chelators and antitransferrin receptor antibodies will be used to study transferrin receptor regulation and iron uptake in fibroblasts from patients with Hemochromatosis. Transferrin receptors will be assayed using 125I-transferrin binding, radioimmunoassay, and measurement of immunofluorescent cell surface transferrin receptor with a fluorocytometer.

该研究中项目的长期目标和具体目标 建议是1）更好地了解细胞铁 细胞增殖期间的要求，并定义 甘露与细胞铁代谢，因为甘露似乎会抑制细胞 通过以未知方式干扰细胞铁的生长 并已在临床癌症试验中用作细胞毒性剂。 一个 对这种互动的理解可能会提供有关有关的新信息 在增殖过程中铁的细胞要求和信息 如何利用这些需求来控制肿瘤细胞的生长。 2） 从血色素症患者中建立成纤维细胞培养物（A 铁超载的特征是导致多器官损害）和 研究转铁蛋白受体调节和影响铁吸收的因素 在这些细胞中。 研究可能阐明有关此的新信息 疾病。 甘露对59FE细胞内分布的影响的研究将 包括溶解性细胞质含量的凝胶过滤和 转铁蛋白的峰值放射性分数的免疫沉淀， 使用特定抗血清的转铁蛋白受体和铁蛋白。 亚细胞 分馏也将使用蔗糖梯度进行 离心。 细胞等铁蛋白将在 使用等电聚焦的镀膜控制细胞增殖。 研究 将使用67gallium摄取和细胞内分布进行 类似的方法。 凝胶将鉴定凝胶蛋白 电泳。 铁中铁卸载的研究 甘露处理的细胞将采用使用受体pH变化的研究 FITC标记的转铁蛋白 - 金属探针和分析 光谱荧光计。 研究不同的转铁蛋白金属对 受体循环/回收将包括59fe/125i-Transferrin 脉搏追踪实验。 转铁蛋白受体合成和 磷酸化将采用35s-甲硫代氨酸和32p标记的受体标记 通过亲和色谱分离。 32p被标记的磷酸化 转铁蛋白受体将在凝胶上使用放射自显影仪进行鉴定。 这 不同的转铁蛋白金属，铁螯合剂和抗转铁蛋白受体 抗体将用于研究转铁蛋白受体调节和铁 血色素症患者的成纤维细胞摄取。 转铁蛋白 将使用125-Transferrin结合，放射免疫测定法测定受体， 并测量与免疫荧光细胞表面转移蛋白受体的测量 荧光细胞计。