AG-NOR PROTEINS USED FOR CANCER DIAGNOSIS

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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.