Res Proj 2: Molecular Imaging Strategies to Study Cdc25A Regulation in vivo

我们的广泛目标包括描绘细胞分裂周期是如何调节的，确定癌细胞是如何 破坏细胞周期调控途径，并最终将这些信息用于诊断和治疗 人类癌症我们建议将我们的努力集中在Cdc 25 A蛋白磷酸酶上，因为这种酶是一种 哺乳动物细胞分裂周期的关键调节因子，在广泛的人类肿瘤中过量产生。 将产生稳定的细胞系和小鼠集落，使得能够在细胞中研究Cdc 25 A调节 和活体老鼠的实验中。特别地，诱导表达融合蛋白的细胞系 人Cdc 25 A和萤火虫荧光素酶之间的蛋白质（Cdc 25 A-FLuc）将以高通量使用 筛选以鉴定调节蛋白质稳定性的丝氨酸/苏氨酸蛋白磷酸酶全酶。 Cdc 25 A体内。原则上，本研究中鉴定的蛋白质可以为成像提供新的靶点 癌症治疗中的药物和治疗干预。此外，将产生敲入小鼠，其 表达内源性Cdc 25 A与甲虫红色荧光素酶的融合蛋白（Cdc 25 A-CBRLuc）， Cdc 25 A基因座以这种方式，可以非侵入性地监测内源Cdc 25 A蛋白水平 并在稳态条件下在活小鼠中重复进行，并响应于DM治疗剂， 针对细胞周期检查点的药物。此外，分子成像策略将应用于小鼠 乳腺癌模型，以验证合理的抗癌治疗方案的靶特异性， 联合治疗本研究的试剂和结果对今后的临床应用具有一定的指导意义 将联合收割机DNA损伤剂与新的Chk 1抑制剂或新的检查点消除剂组合的试验。的 预期拟议的研究将增强我们对细胞周期控制基本原理的理解， 哺乳动物，并可能影响未来的癌症治疗策略。

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.