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.
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