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Abstract
Grant Number: 1R21NS053666-01 Project Title: GFP-Based Assays to Probe Transcriptional Controls(RMI)
PI Information: Name Title KATZ, RICHARD A. richard.katz@fccc.edu Abstract: DESCRIPTION (provided by applicant): Two (2) green fluorescence protein (GFP)-based assays will be developed to probe mechanisms of transcriptional and post-transcriptional control. The first assay will utilize cells that contain a GFP gene that is silenced by epigenetic mechanisms. Treatment of these cells with small molecules that are known histone deacetylase (HDAC) inhibitors (HDIs), results in robust reactivation of GFP. This finding indicates that the silencing mechanisms likely include (but are not limited to) histone deacetylation. HDIs have been recognized as anti-cancer drugs and high throughput screens (HTSs) of chemical libraries using this GFP cell-based assay should identify new HDIs, as well as compounds that target other proteins that are required for maintenance of silencing. Preliminary results include several proof of-principle experiments that address specificity, sensitivity and background of the assay. The proposed experiments will test the adaptability of the assay to a multi-well format. Pilot screens with known compounds (HDIs) and chemical libraries will be carried out. GFP-silent cells will also be challenged with siRNAs that will knockdown the expression of HDACs and this treatment is also predicted to reactivate the silent GFP gene. Additional experiments will also be carried out to assess the feasibility of a genome-wide siRNA HTS to identify other genes involved in the maintenance of epigenetic silencing. A second GFP based assay will be used to probe the pathway of posttranscriptional control that coordinates DNA replication and histone biosynthesis, through histone mRNA stability. In this assay, a fusion transcript will be generated encoding GFP and the 3'-untranslated region of histone mRNA. It is predicted that inhibition of DNA synthesis will result in specific degradation of the GFP fusion transcript. Chemicals or siRNAs that interrupt this regulatory pathway are predicted to stabilize the fusion transcript and allow continued GFP expression. Feasibility studies will be carried out to validate this assay. As both assays are GFP-based, optimized conditions for GFP detection in a HTS format will be applicable to both systems. Both of these assays address fundamental aspects of cell regulation (epigenetic silencing, cell cycle control) that have been implicated in cancer development. The assays have the potential to reveal new lead compounds, as well as new gene targets for therapeutics.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
amidohydrolase, drug screening /evaluation, enzyme inhibitor, genetic regulation, genetic transcription, green fluorescent protein, high throughput technology, technology /technique development
DNA replication, cell cycle, chemical stability, gene induction /repression, messenger RNA, protein biosynthesis, small interfering RNA, small molecule
biotechnology, cell line, clinical research, human tissue
Institution: INSTITUTE FOR CANCER RESEARCH 333 Cottman Avenue PHILADELPHIA, PA 191112434 Fiscal Year: 2005 Department: Project Start: 30-SEP-2005 Project End: 31-AUG-2007 ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE IRG: ZNS1