CELLULAR UPTAKE AND INTERACTION OF IRON AND GALLIUM

铁和镓的细胞吸收和相互作用

基本信息

批准号：
3182487
负责人：
CHRISTOPHER R CHITAMBAR
金额：
$ 15.59万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-01-01 至 1994-05-31
项目状态：
已结题

项目摘要

Iron is an essential requirement for cellular proliferation. The metal gallium appears to inhibit cell growth by interfering with iron uptake and with iron-dependent steps in DNA synthesis. The long-term objectives of this proposal are: a) to study the interaction of gallium with iron metabolism and with the proteins of iron transport (transferrin and its receptor) and storage (ferritin); b) define factors involved in the uptake, intracellular localization and release of gallium from cells; c) isolate and characterize intracellular gallium-binding proteins; d) further study the mechanism of inhibition of DNA synthesis by gallium (which, in preliminary studies, appears to be due to its action on the iron-containing M2 subunit of ribonucleotide reductase). Although gallium is being used in clinical trials as an anti- neoplastic agent, little lead to a more judicious use of gallium in the treatment of specific malignancies, and may provide new information regarding iron requirements during cellular proliferation. Human leukemic K562 and HL60 cells adapted to long-term growth in serum-free media (with or without transferrin) will be used to study gallium uptake by transferrin-dependent versus transferrin- independent pathways. Studies will use gallium-67, dual-labeled 125I-transferrin-67Ga, and a fluorescent method for the detection of intracellular gallium. Agents which affect receptor internalization, receptosomal acidification, energy-dependent processes, and transferrin recycling will be used to define the role these steps play in gallium uptake. Cellular gallium-binding proteins will be isolated by gel filtration, ion-exchange chromatography and gel electrophoresis. Transferrin, transferrin receptor and ferritin will be immunoprecipitated by specific antibodies. Electron spin resonance spectroscopy and high performance liquid chromatography will be used to study the effect of gallium on ribonucleotide reductase (M2 tyrosyl radical) and nucleotide pools respectively; a specific assay will be used for DNA polymerase. Cell cycle analysis will be done by flow cytometry. Transferrin receptors shed from cells will be isolated by affinity chromatography, identified by gel electrophoresis, 125I-anti-receptor monoclonal antibody and autoradiography. 35S- methionine and 125I will be used to label and study the synthesis and release of the transferrin receptor from cells. It will be determined if the free transferrin receptor influences gallium cytotoxicity.

铁是细胞增殖的重要要求。这金属着甘油似乎通过干扰细胞的生长铁吸收并在DNA合成中具有铁依赖性步骤。这该提议的长期目标是：a）研究凝固膜与铁代谢和蛋白质的相互作用铁运输（转铁蛋白及其受体）和存储（铁蛋白）; b）定义涉及摄取的因素从细胞中定位和释放； c）分离和表征细胞内凝固型蛋白； d）进一步研究凝胶抑制DNA合成的机制（在初步研究中，这似乎是由于其对含铁核苷酸还原酶的含铁M2亚基）。尽管在临床试验中使用甘露作为抗肿瘤剂，很少导致更明智地使用；在治疗特定的恶性肿瘤中，可能会提供新的有关蜂窝中铁要求的信息增殖。人类白血病K562和HL60细胞适应了长期生长无血清培养基（有或没有转铁蛋白）将用于依赖转铁蛋白依赖性与转铁蛋白的研究易载剂独立途径。研究将使用镀与双标签 125-Transferrin-67GA和一种检测的荧光方法细胞内危险。影响受体的药物内在化，受体酸化，能量依赖性过程和转铁蛋白回收将用于定义这些步骤在凝胶吸收中发挥作用。细胞结合蛋白质将通过凝胶过滤，离子交换来隔离色谱和凝胶电泳。转铁蛋白，转铁蛋白受体和铁蛋白将通过特异性免疫沉淀抗体。电子自旋共振光谱和高性能液相色谱法将用于研究效果在核糖核苷酸还原酶（M2酪酶自由基）和核苷酸池分别；特定的测定将用于 DNA聚合酶。细胞周期分析将通过流量进行细胞仪。从细胞中脱离的转铁蛋白受体将分离通过凝胶电泳鉴定的亲和色谱法， 125-ANTI受体单克隆抗体和放射自显影。 35s- 蛋氨酸和125i将用于标记和研究合成并从细胞中释放转铁蛋白受体。这将是确定游离蛋白受体是否影响胆管是否会细胞毒性。