Browsing by Author "Silkworth, William T."

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    Changes in Gene Expression and Cellular Architecture in an Ovarian Cancer Progression Model
    Creekmore, Amy L.; Silkworth, William T.; Cimini, Daniela; Jensen, Roderick V.; Roberts, Paul C.; Schmelz, Eva M. (PLOS, 2011-03-03)
    Background Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment. Methodology/Principal Findings Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII. Conclusions/Significance Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.
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    Chromosome Bridges Maintain Kinetochore-Microtubule Attachment throughout Mitosis and Rarely Break during Anaphase
    Pampalona, Judit; Roscioli, Emanuele; Silkworth, William T.; Bowden, Brent; Genesca, Anna; Tusell, Laura; Cimini, Daniela (PLOS, 2016-01-19)
    Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers) bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.
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    Multipolar Spindle Pole Coalescence Is a Major Source of Kinetochore Mis-Attachment and Chromosome Mis-Segregation in Cancer Cells
    Silkworth, William T.; Nardi, Isaac K.; Scholl, Lindsey M.; Cimini, Daniela (PLOS, 2009-08-10)
    Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed γ-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells.
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    Transient defects of mitotic spindle geometry and chromosome segregation errors
    Silkworth, William T.; Cimini, Daniela (2012-08-11)
    Assembly of a bipolar mitotic spindle is essential to ensure accurate chromosome segregation and prevent aneuploidy, and severe mitotic spindle defects are typically associated with cell death. Recent studies have shown that mitotic spindles with initial geometric defects can undergo specific rearrangements so the cell can complete mitosis with a bipolar spindle and undergo bipolar chromosome segregation, thus preventing the risk of cell death associated with abnormal spindle structure. Although this may appear as an advantageous strategy, transient defects in spindle geometry may be even more threatening to a cell population or organism than permanent spindle defects. Indeed, transient spindle geometry defects cause high rates of chromosome mis-segregation and aneuploidy. In this review, we summarize our current knowledge on two specific types of transient spindle geometry defects (transient multipolarity and incomplete spindle pole separation) and describe how these mechanisms cause chromosome mis-segregation and aneuploidy. Finally, we discuss how these transient spindle defects may specifically contribute to the chromosomal instability observed in cancer cells.