Project 1

癌症涉及诱导细胞中不受控制的DNA复制和有丝分裂，以及确保细胞逃避细胞死亡或衰老并在特定的组织微环境中存活的过程。Project 1在研究人类基因组的遗传机制和控制方面处于领先地位，已经确定了许多参与复制叉DNA合成的关键蛋白质和其他参与DNA复制起始的蛋白质。在拟议的研究中。项目1将继续关注人类细胞中DNA复制的起始是如何被控制的，以及这个过程在肿瘤细胞中是如何出错的。特异性目标1将关注如何在染色体中标记DNA复制的起源，以便在有丝分裂结束或Gl期形成复制前复合体，从而使DNA复制在细胞分裂周期的S期开始。由于起源识别复合体，特别是其最大的亚基Orel在有丝分裂前期就被装载到染色体上，并且Orel是结合最稳定的染色质蛋白，因此Orel在人类基因组中的结合位置将被确定。此外，还将确定在M期和G1期与ORC动态相互作用的蛋白。细胞周期调节因子Cyclin E-CDK2和Cyclin A-CDK2，前者在乳腺癌中经常过度活跃，通过与Orel和Cdc6的直接相互作用来控制，这些相互作用如何影响中心体中DNA复制的起始和中心粒复制将被研究。最近有证据表明，ORC亚基在着丝点中起着关键作用，这些着丝点结合微管纺锤体，在染色体分离之前形成染色体。在Specific Aim 2中，将研究ORC的Orc2和Orc3亚基与纺锤体组装检查点激酶BubRI之间的相互作用，以及ORC亚基在中期到后期转变期间维持纺锤体稳定附着的作用。在Specific Aim 3中，将研究DEAD-box RNA解旋酶DDX5对DNA复制的控制及其与转录因子E2F1的相互作用。DDX5在25%的人类乳腺癌细胞的基因组中被扩增，正是这些细胞对DDX5蛋白水平的抑制表现出选择性敏感性。在细胞周期的Gl期，DDX5如何影响e2f1驱动的DNA复制基因的表达将被研究。项目1还将研究DDX5拷贝数扩增的肿瘤细胞与正常、非肿瘤细胞和许多其他癌细胞相比，如何对其持续表达上瘾。最后，研究DDX5耗竭联合曲妥珠单抗（赫赛汀）治疗对肿瘤细胞增殖抑制的叠加效应。

Cancer involves the induction of uncontrolled DNA replication and mitosis in cells, as well as processes that ensure cells evade cell death or senescence and survive in specific tissue micro-environments. Project 1 has been a leader in studying the mechanisms and control of inheritance of the human genome and has identified many of the key proteins that are involved in DNA synthesis at the replication fork and other proteins that are involved in the initiation of DNA replication. In the proposed studies. Project 1 will continue to focus on how the initiation of DNA replication is controlled in human cells and how this process goes awry n tumor cells. Specific Aim 1 will focus on how the origins of DNA replication are marked in chromosomes so that they can form pre-replicative complexes during exit from mitosis or during Gl phase, thereby enabling the initiation of DNA replication in S phase of the cell division cycle. Since the Origin Recognition Complex, particularly its largest subunit Orel is loaded onto chromosomes beginning in prophase of mitosis and Orel is the most stably bound chromatin protein, the locations within the human genome for Orel binding will be determined. In addition, proteins that dynamically interact with ORC during M and G1 phases will be determined. The cell cycle regulators Cyclin E-CDK2 and Cyclin A-CDK2, the former often over-active in breast cancer, are controlled by direct interactions with Orel and Cdc6 and how these interactions influence the initiation of DNA replication and centriole duplication in centrosomes will be investigated. Recent evidence has emerged that ORC subunits play a critical role at kinetochores that bind microtubule spindles for congression of chromosomes prior to their segregation. In Specific Aim 2, interactions between the Orc2 and Orc3 subunits of ORC and the Spindle Assembly Checkpoint kinase BubRI will be investigated, as will the role of the ORC subunits in maintenance of stable spindle attachment during the metaphase to anaphase transition. In Specific Aim 3, the control of DNA replication by the DEAD-box RNA helicase DDX5 and its interaction with the transcription factor E2F1 will be studied. DDX5 is amplified in the genome of cells in 25% of human breast cancers and it is these cells that display selective sensitivity to inhibition of DDX5 protein levels. How DDX5 influences E2F1-driven expression of DNA replication genes in the Gl phase of the cell cycle will be investigated. Project 1 will also investigate how tumor cells with amplified copy number of DDX5 become addicted to its continued expression, in contrast to normal, non-tumor cells and many other cancer cells. Finally, the additive effects on inhibition of tumors cell proliferation with the combination of DDX5 depletion and Trastuzumab (Herceptin) treatment will be examined.