STUDIES OF MITOSIS IN NORMAL AND NEOPLASTIC CELLS

肿瘤细胞的一个主要特征是频率较高。 非整倍体和不稳定的核型。尽管这一事件的发生率 正常细胞的有丝分裂错误很低，癌细胞经常改变 他们的染色体数目。尽管很难确定 非整倍体是否与基因表达有因果关系 恶性表型或后来的后果，很明显 致癌物质和抗肿瘤药物的数量增加了 非整倍体的频率。这项研究旨在了解更多信息 关于正常染色体运动的分子基础和 并阐明非整倍体发生的可能原因。 正常细胞和肿瘤细胞。这项研究是专门针对 关于有丝分裂器的结构和生理学 特别强调动粒，一种三层状的板状结构 位于中期染色体着丝粒的结构 纺锤形微管连接到其上并通过其 有丝分裂力量起到移动染色体的作用。我们将利用几个 新的技术发展使调查成为可能 体内外动粒的结构和功能。少校 该提案的目的是：(1)分离完整的、有功能的 中期染色体中的动点并对其进行研究 它们的结构、分子组成和运动性； 鉴定和鉴定动粒蛋白并测定 它们在着丝点内的特定定位以及 与着丝粒DNA、微管蛋白和微管的相互作用； 比较“活跃的”有丝分裂着丝点的蛋白质 “不活跃”的间期核前运动；(4) 芒果染色体“复合”着丝点特征的研究 作为真核细胞动粒进化的模型和(5) 阐明着丝点和纺锤体极在 染色体异常运动和非整倍体。方法论 本研究中使用的方法包括细胞培养、标准生化 以及分析技术，杂交瘤技术， 免疫荧光和免疫电子显微镜使用 单抗和多克隆抗体以及人类自身抗体， 显微注射、原位杂交和基因芯片技术。细胞 将使用的品系包括HeLa，中国仓鼠卵巢(CHO)， BalbC/3T3、SV3T3、印度和中国仓鼠以及PtK1大鼠 袋鼠细胞。这些研究对于理解 染色体分离和非整倍体的分子基础。

A major characteristic of tumor cells is their elevated frequency of aneuploidy and unstable karyotypes. Although the incidence of mitotic error in normal cells is low, cancer cells frequently alter their chromosome number. Although it is difficult to ascertain whether aneuploidy is causally related to the expression of the malignant phenotype or a later consequence, it is clear that a number of carcinogens and antitumor drugs increase the frequency of aneuploidy. This research is designed to learn more about the molecular basis of normal chromosome movement and distribution and to elucidate the probable causes of aneuploidy in normal and neoplastic cells. The research is focused specifically on the structure and physiology of the mitotic apparatus with special emphasis on the kinetochore, a trilaminar plate-like structure located at the centromere of metaphase chromosomes to which spindle microtubules are attached and through which mitotic forces act to move chromosomes. We will exploit several new technical developments which make it possible to investigate kinetochore structure and function in vitro and in vivo. The major aims of the proposal are: (1) to isolate intact, functional kinetochores from metaphase chromosomes and to investigate their structure, molecular composition and motility; (2) to identify and characterize kinetochore proteins and to determine their specific localization, within the kinetochore as well as interaction with centromeric DNA, tubulin and microtubules; (3) to compare the proteins of "active" mitotic kinetochores with "inactive" prekinetochores of interphase nuclei; (4) to characterize "compound" kinetochores of muntjac chromosomes as models for kinetochore evolution in eukaryotic cells and (5) to elucidate the role of the kinetochore and spindle poles in abnormal chromosome movements and aneuploidy. Methodology to be used in this study includes cell culture, standard biochemical and analytical techniques, hybridoma technology, immunofluorescence and immunoelectron microscopy using monoclonal and polyclonal antibodies and human autoantibodies, microinjections, in situ hybridization and cDNA technology. Cell lines to be used include HeLa, Chinese hamster ovary (CHO), BalbC/3T3, SV3T3, Indian and Chinese muntjac, and PtK1 rat kangaroo cells. These studies are essential for understanding the molecular basis of chromosome segregation and aneuploidy.