p53 dependent centrosome clustering prevents multipolar mitosis in tetraploid cells

Backgroundp53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy.Conclusionsp53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.     

Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.     

The fate of lipopolysaccharide in cultured rat Kupffer cells

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.     

Kinetochores and microtubules in multipolar mitosis and chromosome orientation.

The ultrastructure of the kinetochore and the orientation of kinetochore microtubules were studied in multipolar divisions of cultured rat-kangaroo cells (Pt-K1). The metaphase kinetochore exhibited a lamellar structure and most of the chromosomes expressed a bipolar microtubule orientation. A multipolar kinetochore orientation was observed in some chromosomes. Equal or unequal portions of a chromatid's kinetochore and a corresponding number of kinetochore microtubules may be oriented to two different poles. Curved or bent continuous microtubules were observed in the vicinity of chromosomes showing multipolar orientation. The findings are in accordance with Östergren's theory of ‘auto-orientation’ [8]. It is speculated that orientation of a chromatid kinetochore to more than one pole might be a possible regular event during the process of chromosome orientation prior to full metaphase in bipolar mitosis.     

The fate of mosaic aneuploid embryos during mouse development

More than any other species, humans have difficulty reproducing. As recent studies show that human infertility is ever increasing, much efforts are needed towards the understanding of our low fecundity. While aneuploidy is the leading cause of spontaneous pregnancy loss in humans, we still know surprisingly little about the developmental consequences of chromosomal abnormalities. We have here used a mouse model that spontaneously incites chromosomal primary aneuploidy in female haploid oocytes and find that after fertilization, these primary aneuploid cells become cytological unstable, generating diverse karyotypic mosaic embryos. The mosaic aneuploid embryos can develop and implant into the female uterine tissue and initiate the gastrulation process (E6.5) but quickly degrade and succumb by E8.0. We find that loss of embryo viability due to chromosomal mosaicism is caused by the activation of a spatially and temporally controlled p53-independent apoptotic mechanism and does not result from a failure to progress through mitosis. We conclude that an initial state of primary aneuploidy within an embryo results in a rapid evolution of mosaicism and early embryonic death. This gestational loss due to aneuploid mosaicism could account for the large proportion of human pregnancy loss prior to clinical recognition.     

Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6β-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.     

Pattern of DNA segregation in multipolar anatelophases of different ploidy in euploid and aneuploid mammalian cells cultivated in vitro

A microdensitometrical analysis of multipolar anatolophases of cuploid and aneuploid mammalian cells in vitro has been made in order to study the quantitative distribution of DNA to the poles.In the tripolar anatelophases of aneuploid Chinese hamster cells (obtained using colchicine) DNA is distributed to poles in an almost random way. In the spontaneous tripolar anatelophases of euploid cells of Rhesus, DNA is distributed to the poles in whole number multiples of 1c. The following distributions were observed in tetraploid cells: 14 cases of 3 : 3 : 2 and 3 cases of 4 : 2 : 2. In triploid cells, 6 cases of 2 : 2 : 2 and 1 case of 3 : 2 : 1.Thus we have deduced that each ploidy degree has a preferential distribution of sets and in contrast to the original hypothesis, random segregation of the sets is not the common occurrence. Hypotheses have been made to explain the origin of multipolar mitosis and the preferential distribution of chromatid sets to the poles. The results would confirm the existence of haploid sets of chromatids and would explain the appearance of triploid, pentaploid and hexaploid cells in tissues and cell cultures.     

Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

Background  

Aurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barrett's adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.     

Myofibril organisation and mitosis in cultured cardiac muscle cells.

Isolated cardiac muscle cells grown in vitro have been studied with respect to their ability to contract spontaneously and maintain myofibrillar organisation during division. These cells do not round up to undergo mitosis; division is achieved by the cell pinching itself in two in a selected area. This adaptation minimises disturbance to cell attachment sites and to myofibrils running between them. We correlated this with the persistence of beating during division and the maintenance of myofibrils with intact Z bands, even in close proximity to the nucleus, through division in many cells. Cessation of beating and disorganisation of myofibrils are therefore not prerequisites for division of cardiac muscle cells, as reported previously.     

Chromatid distribution at mitosis in cultured Wallabia bicolor cells

An analysis of labelled centromere regions of chromosomes in metaphase cells of the Swamp Wallaby (Wallabia bicolor) demonstrates conclusively that chromatids do not co-segregate in sets which contain DNA template strands of identical age. Also, there is no tendency for chromatids of homologous chromosome pairs to distribute non-randomly. The data are consistent with the assumption of random distribution of chromatids at mitosis.     

Identification of cytokinins associated with mitosis in synchronously cultured tobacco cells

The amount of endogenous cytokinins in the butanol-soluble fractionincreased about 5 times during the mitosis of synchronouslydividing cultured tobacco cells (Nicotiana tabacum cv. Xanthi).The free cytokinins were identified as trans-zeatin and zeatinribonucleoside by paper and thin layer chromatography and byhigh-performance liquid chromatography combined with mass spectrometryand bioassay. The water-soluble fraction contained zeatin ribonucleotide,isopentenyladenosine monophosphate, phosphoglucosylzeatin andphosphoglucosylzeatin ribonucleoside. Changes in the cytokininsin the water-soluble and transfer RNA fractions during the cellcycle did not support the hypothesis that they might be thesource of the butanol-soluble cytokinins which increased duringmitosis. These seem to be synthesized de novo during mitosis. (Received February 1, 1980; )     

Assembly of nucleolus-derived foci in various cultured mammalian cells during mitosis

During mitosis, rebuilding of the nucleolus is a step-wise process that, above all, includes an assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells. In this study, we performed a comparative analysis of NDF formation in mitotic cells of various mammalian cell cultures, such as green monkey CV1 cells, human HeLa cells, mouse 3T3 cells, and pig PK cells, both in control and following inhibition of rRNA synthesis by actinomycin D or by an adenosine analogue, DRB. The results obtained show that in all examined cell types NDF are formed shortly after or simultaneously with the onset of chromosome segregation to the poles of the mitotic spindle. However, an efficiency of NDF assembly, i.e. the number of NDF per anaphase or telophase cell, and the portion of anaphase and telophase cells containing NDF vary in different cell cultures, being most prominent in CVI and HeLa cells. In these cells, the vast majority of NDF accumulate several proteins of the mature nucleolus, such as B23/nucleophopmin, C23/nucleotin, fibrillarin, and, to a lesser extent, Nop52. The rRNA harbored by NDF is synthesized several hours prior mitosis, and plays an essential role the maintenance of NDF structural integrity. Starting from early stages of the assembly onwards, the NDF are predominantly located in the area occupied by aster microtubules of the mitotic spindle.     

The side population in human lung cancer cell line NCI-H460 is enriched in stem-like cancer cells

Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate. Recent studies indicate that tumors contain a subset of stem-like cancer cells that possess certain stem cell properties. Herein, we used Hoechst 33342 dye efflux assay and flow cytometry to isolate and characterize the side population (SP) cells from human lung cancer cell line NCI-H460 (H460). We show that the H460 SP cells harbor stem-like cells as they can readily form anchorage-independent floating spheres, possess great proliferative potential, and exhibit enhanced tumorigenicity. Importantly, the H460 SP cells were able to self-renew both in vitro and in vivo. Finally, we show that the H460 SP cells preferentially express ABCG2 as well as SMO, a critical mediator of the Hedgehog (HH) signaling, which seems to play an important role in H460 lung cancer cells as its blockage using Cyclopamine greatly inhibits cell-cycle progression. Collectively, our results lend further support to the existence of lung cancer stem cells and also implicate HH signaling in regulating large-cell lung cancer (stem) cells.     

The labelled mitosis curve for a population consisting of fast and slowly cycling cells

In studies of tumour growth, and particularly of tumour treatment with phase-specific chemotherapeutic agents, the fraction labelled mitosis technique is frequently used to estimate the kinetic properties of the cell population making up the tumour. We show here that the FLM technique is in principle very insensitive to the behaviour of slowly cycling cells, even if these constitute a large proportion of the total cell population. Furthermore, since the rate of DNA synthesis is frequently lower in slowly growing cells than in those growing rapidly, there is a higher probability of labelling error associated with the former cells. In view of these theoretical and experimental considerations, it is suggested that considerable caution be used when applying the FLM technique to heterogeneous cell populations such as those of solid tumours.     

Induction of multiple microtubule-organizing centers, multipolar spindles and multipolar division in cultured V79 cells exposed to diethylstilbestrol, estradiol-17beta and bisphenol A

Inducibility of multipolar spindles and multipolar division by diethylstilbestrol (DES) and estradiol-17beta (E2) was investigated in terms of the mechanism of induction of aneuploidy by the estrogens. DES, E2 and bisphenol A (Bp-A), a structural analogue of DES, caused mitotic arrest and aberrant spindles, such as tripolar and multipolar spindles, in a concentration-dependent manner. Gamma-tubulin, a well-characterized component of microtubule-organizing centers (MTOCs), was co-localized with the aberrant spindles induced by estrogens and Bp-A. The number of gamma-tubulin signals in the mitotic cells coincided with that of the aberrant spindles and rose with an increasing concentration of the chemicals. The incidence and location of gamma-tubulin in interphase cells were not influenced by the chemicals. These results suggest that multiple MT nucleating sites were induced by the estrogens and Bp-A during the transition from interphase to the mitotic phase. DES, E2 and Bp-A induced multipolar division in a concentration-dependent process associated with the induction of aneuploidy.     

Induction of centrosome injury, multipolar spindles and multipolar division in cultured V79 cells exposed to dimethylarsinic acid: role for microtubules in centrosome dynamics

Role for microtubules in the induction of multiple microtubule organizing centers (MTOCs) and multipolar spindles by dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, was investigated with respect to the effects of microtubule disruption and reorganization. DMAA induced multiple signals of gamma-tubulin, a well-characterized component of MTOCs in the centrosome, in a manner specific to mitotic cells. The multiple signals of gamma-tubulin were co-localized with multipolar spindles caused by DMAA. Disruption of microtubules by nocodazole (NOZ) suppressed the appearance of centrosome injury caused by DMAA while disorganization of actin microfilaments by cytochalasin D did not. Post-treatment incubation of cells in which multiple signals of gamma-tubulin caused by DMAA had been coalesced to one or two dots by NOZ caused the reappearance of mitotic cells with multiple signals of gamma-tubulin, in conjunction with reorganization of the microtubules. These results suggest a role for microtubules in the dynamic behavior of the mitotic centrosome. DMAA induced aberrant cytokinesis, such as tripolar and quadripolar division, in a concentration-dependent manner. These results, together with the findings of earlier studies, suggest that the centrosome is the primary target for the induction of multipolar spindles by DMAA and the resultant induction of multinucleation and multipolar division.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

The aggregation and inheritance of damaged proteins determines cell fate during mitosis

Recent evidence suggests that proliferating cells polarize damaged proteins during mitosis to protect one cell from aging, and that the structural conformation of damaged proteins mediates their toxicity. We report that the growth, resistance to stress, and differentiation characteristics of a cancer cell line (PC12) with an inducible Huntingtin (Htt) fused to enhanced green fluorescent protein (GFP) are dependent on the conformation of Htt. Cell progeny containing inclusion bodies have a longer cell cycle and increased resistance to stress than those with diffuse Htt. Using live imaging, we demonstrate that asymmetric division resulting from a cell containing a single inclusion body produces sister cells with different fates. The cell that receives the inclusion body has decreased proliferation and increased differentiation compared with its sister cell without Htt. This is the first report that reveals a functional consequence of the asymmetric division of damaged proteins in mammalian cells, and we suggest that this is a result of inclusion body-induced proteasome impairment.