Regulation of Chromosome Segregation in Human Cells

描述(申请人提供)：染色体分离对所有生物体的健康和生存至关重要。越来越多的证据表明，绝大多数人类癌症表现出很高的染色体错分率。这种现象被称为染色体不稳定(CIN)，被认为是通过促进恶性潜能增加的非整倍体克隆的出现来推动肿瘤的发生。因此，必须更好地了解调节人类细胞染色体分离过程的分子和机制，并保持其高保真。 我们已经开发了在人类体细胞中“敲除”基因的新方法，并使用这些方法来表征一种名为securin的小有丝分裂调节蛋白的作用。这种蛋白质与一种名为分离酶的大型半胱氨酸蛋白酶形成紧密的复合体，该酶消化将姐妹染色单体连接在一起的物理链接。根据我们的初步研究，我们假设Securin分离酶复合体在人类有丝分裂进程和高保真染色体分离中发挥核心作用。对这个复合体的精确调控被认为是将姐妹染色单体分离与上游检查点联系起来，并将其限制在细胞周期的适当阶段，即后期。为了验证这些假设，我们将调查特定的翻译后修饰如何有助于分离酶-Securin复合体的功能。在目标1中，我们使用遗传学方法来阐明分离酶自动切割在体内的作用，以确定其与有丝分裂进程和染色体动力学的相关性。在目标2中，我们利用我们早期研究中开发的hSecurin‘基因敲除’细胞，测试了一种已提出的抑制早熟姐妹染色单体分离的Securin非依赖性机制。在目标3中，我们评估了对姐妹染色单体分离以外的过程进行分离酶依赖性调节的潜在机制。总之，这些研究将极大地促进我们对人类有丝分裂染色体分离的理解。这些信息对于开发更有效的癌症治疗方法至关重要，这些治疗方法可以攻击大多数癌细胞中发现的CIN表型。

DESCRIPTION (provided by applicant): Chromosome segregation is crucial to the health and survival of all organisms. Accumulating evidence indicates that the vast majority of human cancers display high rates of chromosome missegregation. This phenomenon, termed chromosomal instability (CIN), has been proposed to drive tumorigenesis by facilitating the emergence of aneuploid clones with increased malignant potential. Thus, it is imperative to obtain a better understanding of the molecules and mechanisms that regulate the process of chromosome segregation in human cells and sustain its high fidelity. We have developed novel methods for 'knocking out' genes in human somatic cells, and we have used these methods to characterize the role of a small mitosis-regulating protein termed securin. This protein forms a tight complex with a large cysteine protease termed separase, which digests the physical links that hold sister chromatids together. Based on our preliminary studies, we hypothesize that the securin-separase complex plays a central role in mitotic progression and high-fidelity chromosome segregation in humans. Precise regulation of this complex is proposed to link sister chromatid separation to upstream checkpoints and restrict it to the the appropriate phase of the cell cycle, i.e., anaphase. To test these hypotheses, we will investigate how specific post-translational modifications contribute to the function of the separase-securin complex. In Aim 1, we use a genetic approach to elucidate the in vivo role of separase auto-cleavage, to determine its relevance to mitotic progression and chromosome dynamics. In Aim 2, we test a proposed securin-independent mechanism for inhibiting premature sister chromatid separation, taking advantage of the hSecurin- 'knockout' cells developed in our earlier studies. In Aim 3, we evaluate potential mechanisms for separase-dependent regulation of processes other than sister chromatid separation. Together, these studies will significantly advance our understanding of mitotic chromosome segregation in humans. Such information is essential to developing more effective cancer therapies that attack the CIN phenotype found in most cancer cells.