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.

Centrosome separation can be completed either before or after nuclear envelope breakdown (NEB). A combination of experimental and computational approaches shows that incomplete centrosome separation at NEB decreases the accuracy of chromosome segregation and thus represents a severe threat to genome stability.

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10 -20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.assembly speed and accuracy ͉ merotelic attachments ͉ microtubules ͉ search and capture T he mitotic spindle is a complex molecular machine segregating chromosomes (1, 2). Molecular inventory and general principles of the spindle dynamics are becoming clear (3), but quantitative understanding of spindle mechanics in general and its self-assembly in particular is lacking. The first hypothesis of how the spindle assembles, elegantly called ''search and capture'' (Fig. 1A), was put forward in ref. 4 after the discovery of the dynamic instability phenomenon: Microtubules (MTs) grow and shorten rapidly and repeatedly from the centrosomes in random directions ''searching'' for the kinetochores (KTs), specialized chromosome structures that function as an interface between the chromosomes and the mitotic spindle. Whenever a growing MT plus end runs into a KT, this MT is stabilized; the assembly is complete when all KTs are thus captured transforming two MT asters into a typical bipolar spindle. Capture of a single astral MT by a KT has been visualized directly in newt lung cell cultures (5).How can hundreds of MTs turning over in tens of seconds capture tens of chromosomes within 10-20 min (6) is one of the fundamental questions of mitosis. Mathematical modeling has been instrumental in attempts to answer this question, because it is very hard to experimentally resolve individual MTs, follow their formation, and perturb their dynamics (7). First applications of modeling were the analyses (8, 9) suggesting that the dynamic instability parameters have to be optimized to ensure fast assembly, so that a MT switches from growth to shortening when it is as long as the distance between the centrosome and the chromosome. This analysis was extended (10) to simulate hundreds of MTs searching for tens of KTs in realistic geometry. The simulation...

BackgroundOvarian 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 FindingsMouse 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/SignificanceOur 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.

No abstract