Binomial mitotic segregation of MYCN-carrying double minutes in neuroblastoma illustrates the role of randomness in oncogene amplification.

BACKGROUND: Amplification of the oncogene MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development. PRINCIPAL FINDINGS: We show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers. CONCLUSION: Binomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.

High-resolution molecular cytogenetic analysis of Wilms tumors highlights diagnostic difficulties among small round cell kidney tumors.

Many solid tumors exhibit characteristic gene fusions, which are reflected by balanced translocations at the cytogenetic level. These changes might be useful diagnostic and prognostic tools. In Wilms tumor (WT, nephroblastoma) no fusions genes or recurrent balanced translocations have been described thus far. To screen for cryptic balanced translocations, we have analyzed 17 renal neoplasms, histopathologically classified as WT, by a combination of G-banding, multicolor FISH, and subtelomeric FISH. This approach revealed several submicroscopic chromosomal aberrations and three different seemingly balanced translocations, resulting in a heterozygous deletion of HACE1, an EWSR1/ERG fusion, and an EWSR1/FLI1 fusion, respectively. As EWSR1 rearrangements are known to be a characteristic of Ewing tumors (ET), our findings illustrate the diagnostic problems regarding small cell kidney tumors and strongly argue for the need of adjuvant diagnostic techniques in this group of neoplasms. In summary, our genomic screening approach proved efficient in finding structural chromosomal aberrations. The fact that no recurrent translocations were found in the WTs of this study argues against the presence of a frequent pathognomonic translocation in this disease entity.

When the genome plays dice: circumvention of the spindle assembly checkpoint and near-random chromosome segregation in multipolar cancer cell mitoses.

BACKGROUND: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally. PRINCIPAL FINDINGS: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells. CONCLUSION: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.

Defective chromosome segregation and telomere dysfunction in aggressive Wilms' tumors.

PURPOSE: In many childhood neoplasms, prognostic subgroups have been defined based on specific chromosome changes. In Wilms' tumor (WT), such subclassification has been hampered by the diverse and relatively unspecific pattern of chromosomal imbalances present in these tumors. Unspecific patterns of cytogenetic imbalances in tumors are often caused by mitotic segregation errors due to short dysfunctional telomeres. As an alternative to cytogenetic classification, we therefore have evaluated whether the rate of telomere-dependent chromosomal instability could influence the clinical course in WT patients. EXPERIMENTAL DESIGN: Telomere function and mitotic segregation errors were assessed in 12 cultured tumors and in tumor tissue sections from 41 WT patients. RESULTS: Abnormal telomere shortening was found in cultured cells and in tissue sections from highly aggressive tumors. In vitro, dysfunctional telomeres were associated to specific cell division abnormalities, including anaphase bridges and multipolar mitoses. Assessment of mitotic figures in tissue sections revealed that anaphase bridges and multipolar mitoses were predominantly, but not exclusively, present in high-risk tumors and were predictors of poor event-free and overall survival. CONCLUSIONS: Telomere-dependent mitotic instability is present in a subgroup of WT, predominantly consisting of high-risk tumors.

Distinct mitotic segregation errors mediate chromosomal instability in aggressive urothelial cancers.

PURPOSE: Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth. EXPERIMENTAL DESIGN: We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation. RESULTS: Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival. CONCLUSION: Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.

Euploidy in somatic cells from R6/2 transgenic Huntington's disease mice.

BACKGROUND: Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the HD gene. The huntingtin protein expressed from HD has an unknown function but is suggested to interact with proteins involved in the cell division machinery. The R6/2 transgenic mouse is the most widely used model to study HD. In R6/2 fibroblast cultures, a reduced mitotic index and high frequencies of multiple centrosomes and aneuploid cells have recently been reported. Aneuploidy is normally a feature closely connected to neoplastic disease. To further explore this unexpected aspect of HD, we studied cultures derived from 6- and 12-week-old R6/2 fibroblasts, skeletal muscle cells, and liver cells. RESULTS: Cytogenetic analyses revealed a high frequency of polyploid cells in cultures from both R6/2 and wild-type mice with the greatest proportions of polyploid cells in cultures derived from skeletal muscle cells of both genotypes. The presence of polyploid cells in skeletal muscle in vivo was confirmed by fluorescence in situ hybridisation with centromeric probes. Enlarged and supernumerary centrosomes were found in cultures from both R6/2 and wild-type mice. However, no aneuploid cells could be found in any of the tissues. CONCLUSION: We conclude that polyploid cells are found in fibroblast and skeletal muscle cultures derived from both R6/2 and wild-type littermate mice and that aneuploidy is unlikely to be a hallmark of HD.

Structural and numerical chromosome changes in colon cancer develop through telomere-mediated anaphase bridges, not through mitotic multipolarity.

Telomere dysfunction has been associated with chromosomal instability in colorectal carcinoma, but the consequences of telomere-dependent instability for chromosome integrity and clonal evolution have been little explored. We show here that abnormally short telomeres lead to a wide spectrum of mitotic disturbances in colorectal cancer cell lines, including anaphase bridging, whole-chromosome lagging, and mitotic multipolarity. These abnormalities were found in both the presence and absence of microsatellite instability. The mean telomere length varied extensively between cells from the same tumor, allowing the establishment of tumor cell subpopulations with highly different frequencies of mitotic disturbances. Anaphase bridging typically resulted in either inter-centromeric chromatin fragmentation or centromere detachment, leading to pericentromeric chromosome rearrangements and loss of whole chromosomes, respectively. There was a strong correlation between anaphase bridges and multipolar mitoses, and the induction of dicentric chromosomes by gamma irradiation and telomerase inhibition led to an elevated frequency of multipolar mitotic spindles, suggesting that multipolarity could result from polyploidization triggered by anaphase bridging. Chromatid segregation in multipolar mitoses was close to random, resulting in frequent nullisomies and nonviable daughter cells. In contrast, there was a high clonogenic survival among cells having gone through anaphase bridging in bipolar mitoses. Bridging of telomere-deficient chromosomes could thus be a major mutational mechanism in colorectal cancer, whereas mitotic multipolarity appears to be a secondary phenomenon that rarely, if ever, contributes to clonal evolution.