AG-NOR PROTEINS USED FOR CANCER DIAGNOSIS

这是香农奖，为这项研究提供部分支持 项目低于指定研究所的资助范围，但 都处于最优秀的边缘。香农奖旨在提供 支持测试该方法的可行性；开发进一步的测试 改进研究方法；对可用的数据进行二次分析 数据集；或执行可演示PI的离散项目 研究能力或为已经功成名就的 申请。下面的摘要摘自原始文件 由首席调查员提交。 描述：Ag-NOR蛋白是银染蛋白，位于 真核细胞的核仁组织区(NOR)。这些 蛋白质被用于癌症诊断，以区分恶性和恶性 良性肿瘤：恶性病变含有更多的Ag-NOR蛋白 良性病变。Ag-NOR的鉴定与表征 蛋白质具有生物学意义和临床意义。他们将会是 有助于理解两个重要的细胞 过程：在细胞的NOR处开始形成核仁。 核糖体RNA的分裂和转录。调查人员还将 为改进癌症诊断提供有用的信息。仅限 已知的核仁素/C23和核磷蛋白/B23属于这一组 蛋白质。然而，它们在银染方面的特定活性 尚未与其他不明身份人员的具体活动进行比较 Ag-NOR蛋白。这项拟议项目的长期目标是 鉴定其他Ag-NOR蛋白，鉴定它们的特性，并确定 它们被染成银色的机制。要实现这些目标 目的：用银染色法筛选人的c DNA文库 它识别硝酸纤维滤膜上的银染蛋白质和 通过使用P32标记的DNA探针的杂交技术 核磷蛋白/B23的第二个酸性区域被证明是负责的 为核磷蛋白/B23银染性能。分离的cDNA克隆 与银染蛋白对应的蛋白质将被测序并 在细菌中表达。在HeLa细胞中的定位将通过 用针对这些蛋白质产生的抗体进行免疫染色。已知 具有酸性结构域的核仁蛋白，如UBF1、HMG1和HMG2将 测试他们是否有能力染上银色。对这一问题的解释 Ag-NOR染色的机制将基于他们之前的发现 天冬氨酸残基是银染的原因 核磷脂/B23。将引入突变以找出是否相同 机制适用于其他Ag-NOR蛋白，并确定 银染所需的最小天冬氨酸残留量。 与Ag-NOR蛋白结合的核仁蛋白也将被识别出来。 癌细胞的高增殖活性可能与其 高水平的核糖体RNA表达。因为Ag-NOR蛋白是 参与核糖体RNA合成，这项研究的结果可能 详细解释Ag-NOR蛋白与 恶毒。这个项目也可能会导致更好的发展 癌症诊断工具，如使用针对Ag-NOR蛋白的抗体， 在设计药物时，可能会通过干扰 Ag-NOR蛋白与其他核仁蛋白的相互作用。

This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION: Ag-NOR proteins are silver-stainable proteins located in the nucleolar organizer region (NOR) of a eukaryotic cell. These proteins are used for cancer diagnosis to distinguish malignant from benign tumors: malignant lesions contain more Ag-NOR proteins than benign lesions. Identification and characterization of the Ag-NOR proteins have both biological and clinical significance. They will be useful in understanding the mechanisms of two important cellular processes: nucleolar formation which begins at the NOR during cell division, and transcription of ribosomal RNA. The investigators will also provide useful information for improved cancer diagnosis. Only nucleolin/C23 and nucleophosmin/B23 are known to belong to this group of proteins. However, their specific activities in terms of silver staining are yet to be compared to the specific activities of other unidentified Ag-NOR proteins. The long-term objectives of this proposed project are to identify other Ag-NOR proteins, to characterize them and to determine the mechanism through which they are silver stained. To accomplish these goals, a human cDNA library will be screened by Ag-NOR staining protocol which identifies silver stainable proteins on nitrocellulose filters and by hybridization technique using a P32-labeled DNA probe which codes for the second acidic domain of nucleophosmin/B23 shown to be responsible for the nucleophosmin/B23 silver staining property. Isolated cDNA clones corresponding to the silver staining proteins will be sequenced and expressed in bacteria. Localization in HeLa cells will be done by immunostaining using antibodies raised against these proteins. Known nucleolar proteins with acidic domains such as UBF1, HMG1, and HMG2 will be tested for their ability to be silver stained. The elucidation of the mechanism of Ag-NOR staining will be based on their previous finding that aspartate residues are responsible for silver staining of nucleophosmin/B23. Mutations will be introduced to find if the same mechanism holds true for other Ag-NOR poteins and to determine the minimum number of aspartic acid residues required for silver staining. Nucleolar proteins that bind to Ag-NOR proteins will also be identified. The high proliferative activity of cancer cells may be related to their high level of ribosomal RNA expression. Since Ag-NOR proteins are involved in ribosomal RNA synthesis, the results form this study may explain in detail the relationship between Ag-NOR proteins and malignancy. This project may also lead to the development of better cancer diagnostic tools like using antibodies against Ag-NOR proteins, and in designing drugs that may inhibit cancer cell growth by disrupting the interaction of Ag-NOR proteins wiht other nucleolar proteins.