Our broad goals include delineating how the cell division cycle is regulated, determining how cancer cells derail cell cycle regulatory pathways, and ultimately using this information for diagnosing and treating human cancers. We propose to focus our efforts on the Cdc25A protein phosphatase as this enzyme is a key regulator of the cell division cycle in mammals and is overproduced in a wide range of human tumors. Stable cell lines and mouse colonies will be generated that enable Cdc25A regulation to be studied in cells and living mice using molecular imaging technologies. In particular, cell lines that inducibly express a fusion protein between human Cdc25A and firefly luciferase (Cdc25A-FLuc) will be used in a high throughput screen to identify the serine/threonine protein phosphatase holoenzymes that regulate the stability of Cdc25A in vivo. In principal the proteins identified in this study could provide novel targets for imaging agents and therapeutic intervention in cancer treatment. In addition, knock-in mice will be generated that express a fusion protein between endogenous Cdc25A and click beetle red luciferase (Cdc25A-CBRLuc) from the Cdc25A locus. In this way endogenous Cdc25A protein levels can be monitored non-invasively and repetitively in living mice under steady state conditions and in response to DMAdamaging agents and drugs targeting cell cycle checkpoints. In addition, molecular imaging strategies will be applied to mouse models of breast cancer to validate target specificity of rational anti-cancer therapeutic regimens in combination therapies. The reagents and results obtained in these studies will be useful in future clinical trials that combine DNA damaging agents with novel Chk1 inhibitors or novel checkpoint abrogators. The proposed studies are expected to enhance our understanding of basic principles of cell cycle control in mammals and may impact future therapeutic strategies for cancer treatment.
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