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High-Content FRET-FLIM Screening in Inhibitors of Oncogenic Transcription by C-Myc in Breast Cancer

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Final rept. 1 Jun 2007-31 May 2009

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To develop a novel high content screen to identify inhibitors that block MycTRRAP interaction, in the first year of this project we constructed several fluorescent fusion protein constructs of Myc and TRRAP, and evaluated their ability to bind and engage in fluorescence energy transfer FRET in vivo. All year-one tasks were completed in the first year of the grant. During the second year we discovered that expression of Myc-CFP was toxic to some cells and in other cells the Myc-CFP did not show the expected blue fluorescence. Due to the short half-life of Myc most of the CFP-Myc proteins were turning over before the CFP matured and became fluorescent. To overcome this unexpected issue, we introduced a mutation T58A to increase the stability of the CFP-Myc fusion protein and we evaluated additional cell lines, as well as new expression constructs. Conditions were identified in which sufficient CFP-Myc folded correctly and became fluorescent. To define MycTRRAP co-regulated target genes we established a Myc-dependent transformation system in the MCF10A cells and conducted Myc-specific ChIP-on-chip in these cells with and without ectopic Myc expression. Expression profiling of these cells was also conducted and data analysis revealed the subset of genes whose promoter was bound and regulated by Myc. During validation of the compound screen we discovered an engineering problem with the FRET-FLIM screening device that prevented the analysis of large numbers of samples. To rectify this we designed and built a pulse stretcher for the FLIM-Opera screening instrument.

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  • Medicine and Medical Research

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