Quantitative Measurements and Modeling of the Mitotic Checkpoint

描述（由申请人提供）：细胞分裂是生长、发育和更新的基本特征。每次细胞分裂时，都必须生成一份精确的基因组拷贝，并且每条复制的染色体均匀地分配给后代细胞。如果染色体分布不均，这个细胞就被称为非整倍体——染色体数量不正确的状态。非整倍体是癌细胞的病理标志，是多种出生缺陷的基础。防止非整倍体的细胞机制被称为有丝分裂检查点，它的作用是阻止染色体的分布，直到它们附着在有丝分裂纺锤体上。与导致遗传性癌症的基因（如Rb、BRCA1、p53等）不同，很少发现有丝分裂检查点基因在疾病中发生突变。然而，癌症和出生缺陷的非整倍体的压倒性证据强烈暗示有丝分裂检查点机制的缺陷。最近的观察现在集中在一个模型上，即基因，特别是在着丝酶介导的Mad2信号通路中，不一定直接突变，但基因的表达水平和因此降低的有丝分裂检查点的活性可能导致非整倍体。在这里，我们建议定量测量活细胞中着丝点信号传导的动力学，并将这些测量结果置于检查点信号传导的预测计算机模型中。这些测量是通过现代分子生物学技术与尖端的显微镜和基于光谱的生物物理仪器的结合进行的。通过确定由未附着的着丝点产生的信号阵列，以防止染色体过早分离，并确定这些信号的产生速度，我们将能够开发出该过程的计算机模型。该模型将允许我们测试信号通路中这些基因的定量扰动。这将允许产生关于有丝分裂检查点的基本机制的新假设和实验，并开始了解该过程中的细微缺陷如何可能成为疾病的基础。

DESCRIPTION (provided by applicant): Cell division is an essential feature of growth, development and renewal. Each time a cell divides an exact copy of the genome must be made and each replicated chromosome equally distributed to the progeny cells. If chromosomes are unequally distributed, the cell is said to be aneuploid - the state of having the incorrect number of chromosomes. Aneuploidy is a pathological hallmark of cancerous cells and underlies a variety of birth defects. The cellular machinery to prevent aneuploidy is called the mitotic checkpoint and acts to prevent distribution of the chromosomes until they are attached to the mitotic spindle. Unlike genes that cause hereditable cancers (e.g. Rb, BRCA1, p53 etc.), very few mitotic checkpoint genes have been found mutated in disease. However, the overwhelming evidence of aneuploidy in cancer and birth defects strongly implicates a deficiency in the mitotic checkpoint machinery. Recent observations are now converging on a model whereby genes, particularly in the kinetochore-mediated Mad2 signaling pathway, are not necessarily mutated directly, but that the expression levels of genes and thereby reduced activity of the mitotic checkpoint can result in aneuploidy. Here we propose to quantitatively measure the kinetics of kinetochore signaling in living cells and house these measurements in a predictive in silico model of checkpoint signaling. These measurements are made through the marriage of modern molecular biological techniques with cutting- edge microscopy and spectroscopy-based biophysical instrumentation. By determining the array of signals produced by unattached kinetochores to prevent untimely chromosome segregation and the rates at which these are made we will be able to develop an in silico model of the process. The model will allow us to test the quantitative perturbations of these genes in the signaling pathway. This will permit the generation of new hypotheses and experiments regarding the basic mechanisms of the mitotic checkpoint and begin to understand how subtle defects in this process may underlie disease. Quantitative measurements and in silico modeling will provide a solid foundation upon which to study the checkpoint response of varied cell lines and human tumors. Our sensitivity analyses can identify those proteins in this pathway that would be most sensitive to therapeutic intervention. Ultimately, such models may provide predictive capacity to choose chemotherapeutic/anti-mitotic treatment regimen based on the proteomic signature of a human tumor.