Induction of endoreduplication in double minutes of the human neuroblastoma cells and the replication pattern.

Induction of endoreduplication (ERD) using Hoechst 33258 as well as colcemid was carried out in cultured neuroblastoma (NB) line cells. In these endoreduplicated cells, the majority of double minutes (DMs) appeared to take a diplochromosome like configuration to form a cluster consisting of four minute elements, assuming a complex DM. Sister chromatid differential staining (SCD) using 5-bromo-2'-deoxyuridine (BrdUrd) revealed the non-random distribution of the stained chromatids among four chromatids composing each diplochromosome, suggesting the occurrence of so-called "outside replication" of DNA strands during the process of ERD. The same pattern of differential staining was also found in the quadruple minutes of each endoreduplicated DM. Since DMs are acentric, the present results suggest that centromeres do not play any essential role in the formation of diplochromosomes observed in the conventional cytologic preparations and that centromeres are probably not responsible for the phenomenon of the "outside replication" of DNA strands.     

MTBP plays a crucial role in mitotic progression and chromosome segregation

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.     

Interspecific hybridization induced amplification of Mdm2 on double minutes in a Mus hybrid

We report a case of interspecific hybridization induced amplification of Chromosome 10 on double minutes (dm) in the karyotype of a hybrid Mus embryo. Stable, non-mosaic dm were previously found in tissues of a 16.5-day Mus Musculus x Mus Caroli hybrid (Graves, 1984). Dm in tissues of the hybrid was of interest to us because of previous reports of genomic instability in interspecific hybrids (O'Neill et al., 1998) and thus we decided to characterize the dm in the hybrid karyotypes. Whole chromosome painting of the hybrid cell lines showed amplification of Chromosome 10 sequences. Southern analysis with a probe for the candidate gene Mdm2 showed amplification of the paternal allele of this oncogene. Overexpression of Mdm2 was confirmed by a western analysis that also showed an associated inactivation of the tumor suppressor, Trp53. Evidence indicates that the event leading to the instability observed was an early adaptive response to stress on the genome, i.e. interspecific hybridization.     

DNA amplification associated with double minutes originating from chromosome 19 in mouse hepatocellular carcinoma

DNA amplification is associated with genomic instability, the main characteristic of cancer cells, and it frequently involves protooncogenes. Double minute chromosomes (DM) and homogeneously stained regions (HSR) are cytological manifestations of DNA amplification. Gain of chromosome 19 is a recurrent alteration in mouse hepatocellular carcinoma (HCC). In one tumor cell line established from HCC developed in myc transgenic mice, DM derived from chromosome 19 were identified by spectral karyotyping and confirmed by fluorescence in situ hybridization (FISH). A probe generated by PCR from microdissected DM was localized by FISH on normal and HCC-derived cell lines on DM and chromosome 19 at two sites separated by several medium size G-bands. This organization of DM containing amplified sequences from separate loci of the same chromosome, indicates a complex mechanism of DNA amplification, possibly involving more than one gene. DM or HSR were not previously identified in mouse HCC and adult human HCC. The recognition of these loci could lead to the cloning of new genes or identification of known genes important in development or progression of HCC.     

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.Key words: BubR1, sumoylation, kinetochores, centromeric cohesion, spindle checkpoint, Sgo1     

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.     

PICH promotes mitotic chromosome segregation: Identification of a novel role in rDNA disjunction

PICH is an SNF2-family DNA translocase that appears to play a role specifically in mitosis. Characterization of PICH in human cells led to the initial discovery of “ultra-fine DNA bridges” (UFBs) that connect the 2 segregating DNA masses in the anaphase of mitosis. These bridge structures, which arise from specific regions of the genome, are a normal feature of anaphase but had escaped detection previously because they do not stain with commonly used DNA dyes. Nevertheless, UFBs are important for genome maintenance because defects in UFB resolution can lead to cytokinesis failure. We reported recently that PICH stimulates the unlinking (decatenation) of entangled DNA by Topoisomerase IIα (Topo IIα), and is important for the resolution of UFBs. We also demonstrated that PICH and Topo IIα co-localize at the rDNA (rDNA). In this Extra View article, we discuss the mitotic roles of PICH and explore further the role of PICH in the timely segregation of the rDNA locus.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

Studies of the genetic mechanism of radiation-induced mitotic segregation in yeast

Summary Sectoring was induced with x-rays or ultraviolet in a diploid yeast strain heterozygous for seven genes located on one chromosome arm. The frequencies of sectoring of different genes were approximately linearly related to their distance from the centromere. If two or more adjacent genes sectored, the event could be explained by mitotic crossing over. Sectoring of single genes, however, was mostly nonreciprocal and resembled a conversion-type event. Approximately 80% of the sectored colonies could be explained single mitotic crossovers in one of the intergenic regions.     

Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: Evidence for gene amplification

We have isolated a metaphase chromosome fraction highly enriched in double minutes (dm) from a mouse adrenocortical tumor cell line (Y1-DM). We have cloned DNA from this dm-enriched fraction in the λ vector Charon 4A, and have characterized two randomly chosen recombinant bacteriophage clones from this dm DNA library. When ³²P-labeled DNA from each recombinant was hybridized to Southern blots of restriction endonuclease-digested DNA from different mouse cell lines, large differences were seen in the intensity of the resulting autoradiographic images, depending on the source of the genomic DNA. A very strong signal was obtained with DNA from the Y1-DM cells and with DNA from a related Y1 subline that lacks dm but contains a marker chromosome bearing a large homogeneously staining region (HSR). Hybridization to DNA from parental inbred mice and from two unrelated mouse cell lines produced a significantly weaker signal than that obtained with DNA from the Y1 cells, but the DNA fragments from these sources were of similar size. Based on results from filter hybridization analysis, we estimate that sequences homologous to the cloned fragments are approximately 100- to 200-fold more abundant in the genome of the Y1-DM cells than in the parental mouse cells. The data are consistent with the hypothesis that dm and HSRs in these cells contain amplified genes.     

Function and regulation of dynein in mitotic chromosome segregation

Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing. How is a single motor able to fulfill this large array of functions and how are these activities temporally and spatially regulated? The answer lies in the unique composition of the dynein motor and in the interactions it makes with multiple regulatory proteins that define the time and place where dynein becomes active. Here, we will focus on the different mitotic processes that dynein is involved in, and how its regulatory proteins act to support dynein. Although dynein is highly conserved amongst eukaryotes (with the exception of plants), there is significant variability in the cellular processes that depend on dynein in different species. In this review, we concentrate on the functions of cytoplasmic dynein in mammals but will also refer to data obtained in other model organisms that have contributed to our understanding of dynein function in higher eukaryotes.     

A novel role for the mitotic spindle during DNA segregation in yeast: promoting 2 microm plasmid-cohesin association

The 2 microm circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal "selfish" DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2 microm circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.     

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.     

The spatial segregation of pericentric cohesin and condensin in the mitotic spindle

In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring.     

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.     

EBP2 plays a key role in Epstein-Barr virus mitotic segregation and is regulated by aurora family kinases

Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.     

Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint

Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.     

Ultrastructural aspects of rapid plasma membrane growth in mitotic neuroblastoma cells

In murine C1300 neuroblastoma cells, clone Neuro 2A, the major fraction of the necessary increase in cell surface area during the cell cycle occurs within a short period around mitosis. During this period cell cycle-related modulations in a number of structural, dynamic and transport properties are most prominent. In this study we have examined the mechanism of rapid plasma membrane growth during mitosis, and the resulting changes in the ultrastructural features of the plasma membrane, by scanning and freeze-fracture electron microscopy as well as by electron microscopy of ultrathin sections. Our observations show that plasma membrane growth occurs by the fusion with and the incorporation into the plasma membrane of cytoplasmic multilamellar, lipidic membrane vesicles. Such vesicles are not observed at other times in the cell cycle. As a consequence, IMP-free domains appear transiently in the mitotic and early post-mitotic plasma membrane. Comparison of replicas prepared from glutaraldehyde-fixed cells and unfixed, ultrarapidly frozen cells showed that aldehyde fixation artefactually induces a bleb-like appearance of these domains. The IMP-free domains disappear in the G1-phase as a result of the mobilization and lateral redistribution of membrane components. It is argued that mitotic membrane growth by preferential incorporation of membrane lipids not only serves to accomodate for the necessary increase in cell surface area, but also provides a mechanism for plasma membrane-mediated regulation of the cell cycle.