MECHANISM OF ACTION OF INSULIN-LIKE GROWTH FACTORS

胰岛素样生长因子的作用机制

• 批准号: 3774284
• 负责人: S P NISSLEY
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 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/3774284
• 关键词: chromosome deletion; growth factor receptors; high performance liquid chromatography; hormone binding protein; human tissue; insulinlike growth factor; neoplastic cell; radioimmunoassay; receptor expression; tissue /cell culture; western blottings

A rapid, sensitive, dot-blot assay for insulin-like growth factor-II (IGF-II) based on chemiluminescence methodology has been further evaluated for possible interference by IGF binding proteins (IGFBPs). Six different IGFBPs were tested in the dot blot assay at concentrations up to 60-fold the concentration of IGF-II. The IGF-II signal was not diminished. By contrast, in a conventional radioimmunoassay using the same monoclonal antibody to IGF-II, the binding proteins produced significant interference, causing 23% to 87% competition of binding of 125I-IGF-II to the antibody. Thus the IGF-II dot blot assay is unique in allowing direct measurement of IGF-II in conditioned medium from cells in culture without interference by IGFBPs. Addition of IGF-I to MG-63 human osteosarcoma cells resulted in rapid activation of extracellular signal regulated kinase 2 (ERK2) as assessed by ion exchange HPLC and ERK kinase antibodies. ERK1 appeared to be already activated in serum-starved MG-63 cells and not to be further activated following addition of IGF-I. Thus ERK2 activation may be part of the signaling pathway for the IGF-I receptor. Further evaluation of fibroblasts from two patients with deletion of the distal long arm of chromosome 15 showed evidence for decreased expression of IGF-I receptor mRNA in one of the patients compared to age-matched controls. Evaluation of receptor function in a bioassay which measured the incorporation of [3H]thymidine into DNA in the presence of a full range of IGF-I concentrations showed no difference in the ED50 for IGF-I between the patient and control fibroblasts. We conclude from these findings and our earlier results that although there is evidence for decreased IGF-I receptor expression in fibroblasts from patients with chromosome 15q deletion syndrome, receptor function as assessed by cellular response to IGF-I is not impaired.

胰岛素样生长因子-II的快速、灵敏、斑点杂交法 基于化学发光方法学的IGF-II的研究已经被进一步证实。 评估IGF结合蛋白（IGFBPs）的可能干扰。 在斑点印迹试验中测试了六种不同的IGFBPs，浓度为 高达IGF-II浓度的60倍。 IGF-II信号不是 减少了 相比之下，在常规的放射免疫测定中， IGF-II的相同单克隆抗体，产生的结合蛋白 显着干扰，导致23%至87%的结合竞争 125 I-IGF-II的抗体。 因此，IGF-II斑点印迹法是独特的 在允许直接测量IGF-II的条件培养基， 细胞培养物中没有IGFBPs的干扰。 将IGF-I加入MG-63人骨肉瘤细胞中， 细胞外信号调节激酶2（ERK 2）的活化， 通过离子交换HPLC和ERK激酶抗体。 ERK 1似乎是 已经在血清饥饿的MG-63细胞中活化，并且不需要进一步的 在添加IGF-I后激活。 因此，ERK 2激活可能是 IGF-I受体的信号通路。 进一步评价来自两名缺失了 15号染色体的远端长臂显示出减少的证据， IGF-I受体mRNA的表达在一个病人相比， 年龄匹配的对照组。 生物测定中受体功能的评价 它测量了[3 H]胸苷掺入DNA的情况， IGF-I浓度的全范围的存在没有显示出差异 在患者和对照成纤维细胞之间IGF-I的ED 50中。 我们 从这些发现和我们早期的结果中得出结论， 这是IGF-I受体在成纤维细胞中表达降低的证据， 染色体15 q缺失综合征患者，受体功能如 通过对IGF-I的细胞应答评估的细胞毒性未受损。