Notch signaling is an evolutionary conserved mechanism used by metazoans to direct cell fatedecisions, proliferation and apoptosis at all stages of development, including self-renewing adult tissues.Aberrant Notch signaling is implicated in cancer, especially in the emerging field of cancer stem cells.Mammalian cells contain four Notch receptors and five Delta and Jagged cognate ligands; the overall goal ofthis proposal is to elucidate the significance of context on individual Notch receptor subtype-mediated signalsusing non-invasive molecular imaging strategies. We propose to develop enabling technologies that willfacilitate high throughput screening for agents and gene products that are capable of modulating the Notchsignaling pathway in cancer, inherited diseases and facilitate tissue engineering. A comprehensivemechanistic understanding of how to manipulate individual receptors in a context-dependent manner stilleludes investigators. We propose to develop a real time imaging system that will go beyond the currentlyavailable reporters in providing real time, quantitative accounting of the activation status of individual Notchreceptor subtypes. The reporter system we are developing is based on the luciferase complementationimaging technology developed in the ICMIC Molecular Reporter Core at Washington University School ofMedicine and enables visualization of the interactions between a specific Notch intracellular domain (NICD)and the common nuclear cofactor RBPjk. The system is versatile; it can be adaptedfor studying the pathwayin different cell types and can be easily modified to monitor interaction with other cancer-relevant partnerssuch as components of the NF-kB pathway. While the imaging technology being developed in thisapplication is not directly applicable to imaging in patients as it requires protein engineering, thesemethodologies will help develop and evaluate novel therapies for Notch-related diseases.
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