TUMORIGENESIS IN MICE LACKING OSTEOPONTIN

缺乏骨桥蛋白的小鼠的肿瘤发生

• 批准号: 2405121
• 负责人: Susan R Rittling
• 金额: $ 20.66万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2405121
• 关键词: antitumor antibody; carcinogenesis; cell line; enzyme linked immunosorbent assay; gene expression; genetically modified animals; immunocytochemistry; laboratory mouse; molecular oncology; neoplasm /cancer genetics; neoplastic cell; oncogenes; osteopontin; phenotype; tissue /cell culture; transfection

Osteopontin (OPN) is a secreted phosphoprotein which binds both to cells and to mineralized surfaces. There is considerable circumstantial and direct evidence indicating that OPN participates in the process of tumorigenesis. We have developed mice which contain a targeted disruption of the OPN gene, and are unable to express osteopontin protein. These mice are viable and fertile. The goal of the research described in this proposal is to use these mice to define the role at the molecular and physiological level that OPN plays in tumorigenesis. We will derive mouse embryo fibroblast lines from mice which can and cannot express OPN, and transform there cells by transfection with different oncogenes. The ability of the cells of differing ON status to grow in soft agar and form tumors in nude mice will be assessed. Others have shown that transformed cells expressing antisense RNA to OPN are impaired in their ability to grow in soft agar and to form tumors, so we expect to see a similar phenotype in cells from the OPN knockout mice. The defect in these cells will be corrected by transfection with OPN expression constructs. To begin to define the molecular mechanism of OPN's action, the OPN deficient cells will be transfected with constructs expressing mutant forms of OPN, and the effects of these mutants on different aspects of tumorigenesis will be correlated with their activity in several in vitro assays. To define the role of OPN in tumorigenesis at the physiological level, we will breed the OPN deficient mice with transgenic mice in which tumors arise rapidly and with high frequency. The kinetics and frequency of tumor formation in animals which express the transgenes but are unable to express OPN will be assessed, and compared with that in animals that do express OPN. Finally, we will address the question of the localization of OPN in tumors. Most normal tissues which express OPN at similar levels to that found in some tumors secrete the protein into a fluid, such as urine or milk. Thus, it is not clear where the OPN made by tumors is, To answer this question, we will develop new, high affinity antibodies to mouse OPN, and verify their specificity using the OPN -/- mice. We will use these antibodies to develop an ELISA to measure mouse OPN in different fluids, and to perform immunohistochemistry on tumors and mouse tissues.

骨桥蛋白（OPN）是一种分泌的磷蛋白，其结合骨桥蛋白和骨桥蛋白。 细胞和矿化表面。 有相当大 间接和直接证据表明，OPN参与 肿瘤发生的过程 我们培育出了 含有OPN基因的靶向破坏，并且不能 表达骨桥蛋白。 这些小鼠是可存活和可繁殖的。 的 这项研究的目的是利用这些小鼠， 明确OPN在分子和生理水平上的作用， 在肿瘤发生中起作用。 我们将小鼠胚胎成纤维细胞 来自能够和不能表达OPN的小鼠的细胞系， 这些细胞通过转染不同的癌基因。 的能力 不同ON状态的细胞在软琼脂中生长并形成肿瘤 将在裸鼠中进行评估。 其他研究表明， 表达OPN反义RNA的细胞在其能力上受损， 在软琼脂中生长并形成肿瘤，所以我们希望看到 在来自OPN敲除小鼠的细胞中具有相似的表型。 缺陷 在这些细胞中，将通过转染OPN表达来校正 结构。 开始定义OPN的分子机制 作用时，将用构建体转染OPN缺陷细胞， 表达突变形式的骨桥蛋白，以及这些突变体对 肿瘤发生的不同方面将与其 活性在几个体外测定。 明确OPN在以下方面的作用： 在生理水平上的肿瘤发生，我们将培育OPN 缺陷小鼠与转基因小鼠，其中肿瘤迅速出现， 高频率。 肿瘤形成的动力学和频率 在表达转基因但不能表达 将对OPN进行评估，并与接受OPN治疗的动物进行比较。 表达OPN。 最后，我们将讨论 骨桥蛋白在肿瘤中的定位。 大多数正常组织表达 与某些肿瘤中发现的水平相似的OPN分泌 将蛋白质转化为液体，如尿液或牛奶。 因此， 为了回答这个问题，我们将 开发新的，高亲和力的小鼠OPN抗体，并验证其 使用OPN -/-小鼠的特异性。 我们将用这些抗体 开发ELISA来测量不同液体中的小鼠OPN， 对肿瘤和小鼠组织进行免疫组织化学。