TUMORIGENESIS IN MICE LACKING OSTEOPONTIN

骨桥蛋白(OPN)是一种分泌型磷蛋白，可与两者结合 细胞和矿化表面。有相当多的 间接和直接证据表明OPN参与了 肿瘤发生的过程。我们已经开发出了一种小鼠 含有OPN基因的靶向干扰，并且无法 表达骨桥蛋白。这些小鼠是有生存能力和繁殖力的。这个 这项提案中描述的研究目标是使用这些小鼠 定义OPN在分子和生理水平上的作用 在肿瘤发生中起着重要作用。我们将获得小鼠胚胎成纤维细胞 可以和不能表达OPN的小鼠细胞株，并转化为 通过将不同的癌基因导入这些细胞。的能力 不同状态的细胞在软琼脂中生长并形成肿瘤 将在裸鼠身上进行评估。其他人已经表明，改变了 表达OPN反义RNA的细胞能力受损 在软琼脂中生长并形成肿瘤，所以我们预计会看到 OPN基因敲除小鼠的细胞表型相似。这一缺陷 在这些细胞中将通过转染OPN表达而得到纠正 构造。开始定义OPN的分子机制 行动后，OPN缺陷细胞将被导入构建体 OPN突变体的表达及其对人骨桥蛋白表达的影响 肿瘤发生的不同方面将与它们的 几种体外试验中的活性。定义OPN在以下方面的角色 在肿瘤发生的生理水平上，我们将培育OPN 具有转基因小鼠的缺陷小鼠，其中肿瘤迅速生长和 频率很高。肿瘤形成的动力学和频率 在表达转基因但不能表达的动物中 将对OPN进行评估，并与具有OPN的动物进行比较 快递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 OPN的水平与某些肿瘤相似，可分泌 蛋白质进入液体，如尿液或牛奶。因此，目前还不清楚 肿瘤产生的骨桥蛋白在哪里，为了回答这个问题，我们将 开发新的、高亲和力的小鼠骨桥蛋白抗体，并验证其 使用OPN-/-小鼠的特异性。我们将使用这些抗体来 建立了一种检测不同液体中小鼠骨桥蛋白的酶标法，并对其进行了检测 对肿瘤和小鼠组织进行免疫组织化学。

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.