Multipolar Mitotic Spindles Research Articles

Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Calcium, Calmodulin, and CaMKII Requirement for Initiation of Centrosome Duplication in Xenopus Egg Extracts

Aberrant centrosome duplication is observed in many tumor cells and may contribute to genomic instability through the formation of multipolar mitotic spindles. Cyclin-dependent kinase 2 (Cdk2) is required for multiple rounds of centrosome duplication in Xenopus egg extracts but not for the initial round of replication. Egg extracts undergo periodic oscillations in the level of free calcium. We show here that chelation of calcium in egg extracts or specific inactivation of calcium/calmodulin-dependent protein kinase II (CaMKII) blocks even initial centrosome duplication, whereas inactivation of Cdk2 does not. Duplication can be restored to inhibited extracts by addition of CaMKII and calmodulin. These results indicate that calcium, calmodulin, and CaMKII are required for an essential step in initiation of centrosome duplication. Our data suggest that calcium oscillations in the cell cycle may be linked to centrosome duplication.

HSP70 overexpression increases resistance of V79 cells to cytotoxicity of airborne pollutants, but does not protect the mitotic spindle against damage caused by airborne toxins

Exposure of Chinese hamster V79 cells to extracts of airborne pollutants induced formation of multipolar or incomplete mitotic spindles. To find out whether overexpression of the HSP70 chaperone protein could protect spindles against airborne toxins we constructed V79 cells stably transfected with an expression vector containing rat heat-inducible hsp70.1 gene under the control of a constitutive CMV promoter. When cells were incubated with extracts of airborne pollutants (5–20 μg/ml) no protective effect of the HSP70 protein against mitotic spindle damage was observed. Moreover, at 20 μg/ml of extracts of airborne toxins the frequency of mitotic malformations was even higher in HSP70-overexpressing cells than in control ones. Extracts of airborne pollutants of 50 μg/ml blocked the formation of mitotic figures both in control and HSP70-overexpressing cells and led to destruction of cell nuclei. However, the HSP70-overproducing cells exhibited higher survival rates when exposed to heat shock and airborne toxins than the control ones, as determined by MTT assay. This suggests that HSP70 overexpression—a frequent feature of cancer cells—should be considered as a factor facilitating survival of cells with damaged mitotic spindles and aberrantly segregated chromosomes.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that γ-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to γ-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Stepwise Progression of Centrosome Defects Associated with Local Tumor Growth and Metastatic Process of Human Pancreatic Carcinoma Cells Transplanted Orthotopically into Nude Mice

Recent evidence indicates that loss of centrosome integrity may be a major cause of genetic instability underlying various human cancers. The aim of this study was to define the role of centrosome defects during the in vivo tumor progression of pancreatic carcinoma using an orthotopic implantation model. Injection of Suit-2 human pancreatic cancer cells into the pancreata of nude mice reproduced the pattern of local tumor growth and distant metastasis observed in humans. Pancreatic xenografts, peritoneal disseminations, and hepatic metastases were harvested, and tumor cells were examined for centrosomes by immunofluorescence microscopy. Centrosome abnormalities, characterized by increased numbers of centrosomes, were detected in only a small fraction of parental Suit-2 cells in culture, whereas the frequency was markedly increased in cells isolated from the pancreatic xenografts. Abnormal centrosome numbers were found at higher frequencies in metastatic foci than in pancreatic xenografts. A significant positive correlation existed between the fraction of cells with multiple centrosomes and that with multipolar mitotic spindles, suggesting a functional involvement of aberrant centrosomes in spindle disorganization and chromosome missegregation. In addition, the increased frequency of abnormal centrosomes was associated with an enhanced degree of chromosomal instability. These findings suggest a novel model of pancreatic tumor progression whereby a stepwise increase in the magnitude of centrosomal abnormalities confers an increased chance for aberrant mitotic events, thus accelerating genetic instability and causing the tumor to progress to a more advanced stage.

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Multiple Centrosome Formation Induced by the Expression of Vpr Gene of Human Immunodeficiency Virus

We previously established a cell line called MIT-23 in which expression of theVprgene of human immunodeficiency virus 1 (HIV-1) can be controlled by the addition of tetracycline. Vpr expression induces multiple nuclear formation and increased ploidy in MIT-23 cells. We herein report that multipolar mitotic spindles were formed upon induction of Vpr. Further analysis of centrosomes with anti-γ-tubulin immunostaining revealed that a significant population of cells 1 week after expression of Vpr gene product had an increased number of centrosomes in the cells with abnormal nuclei. Taking into account that the centrosome plays an important role in genome integrity, the abnormal number of centrosomes in cells expressing Vpr may be directly related to aneuploidy or the formation of micronuclei in MIT-23 cells, suggesting that Vpr has an oncogenic role in HIV infected cells.

Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Heat shock alters centrosome organization leading to mitotic dysfunction and cell death

To identify the cellular target(s) responsible for thermal killing in the G1 phase of the cell cycle, synchronous cultures of Chinese hamster ovary cells (CHO) were heat shocked and studied for one cell cycle by time-lapse videomicroscopy and immunocytochemistry. At the first mitosis post-heating, the fraction of cells giving rise to multinucleated progeny approximately equaled the nonclonogenic fraction. In addition, the cells yielding multinucleated progeny were delayed in prophase-metaphase relative to the cells yielding two uninucleated progeny (clonogenic cells). To study the basis for the delay in prophase-metaphase and subsequent formation of multinucleated cells, cells in mitosis were examined by immunofluorescence for spindle abnormalities. Multipolar mitotic spindles and chromosome misalignment were induced by heat. All multiple spindle poles induced by heat stained for pericentriolar material (PCM), the microtubule nucleating material of centrosomes. Heated cells in mitosis also contained additional foci of PCM which were not associated with the spindle. Cells made thermotolerant by a nonlethal heat shock were resistant to both thermal killing and the induction of multiple foci of PCM. Quantitative analysis revealed a good correlation between the fraction of cells with multipolar spindles, the fraction with more than two foci of PCM, and the nonclonogenic fraction. These data indicate that heat-induced alterations to the PCM of centrosomes resulted in multipolar mitotic spindles, delay in prophase-metaphase, and formation of multinucleated cells which were nonclonogenic. These results identify the centrosome as a G1 target for cell killing.

A carbamate herbicide causes microtubule and microfilament disruption and nuclear fragmentation in fibroblasts

Isopropyl( N-(3-chlorophenyl)carbamate (CIPC) is a widely used herbicide thought to inhibit mitosis by selectively perturbing either the microtubules or microtubule organizing centers of susceptible plant species. Its effects on microtubule and microfilament organization in mouse 3T3 fibroblasts are analyzed here by immunofluorescence techniques using anti-tubulin and anti-actin antibodies. We report that relatively high levels of CIPC (but not higher than the usual levels employed in plant studies) inhibit fibroblast growth, cause the disappearance of cytoplasmic microtubules and also lead to disorientation of microfilament bundles. These responses develop within 24 h of exposure of 3T3 cells to CIPC and persist as long as the herbicide is present in the culture medium. More importantly, cells returned to drug-free medium following 5–7 days exposure to CIPC resume rapid division but show several remarkable morphological responses. In particular they develop multipolar spindles during mitosis and show an unequal partitioning of nuclei between daughter cells. Consequently a high proportion of interphase cells contains multiple and multilobed nuclei. Such fibroblast cultures exposed to CIPC and then returned to drug-free medium may provide chromosome-deficient cells of value for genetic mapping and recombinant studies. On the other hand if similar responses to CIPC exposure and withdrawal occur in vivo then the herbicide may pose a genetic risk to man that is not revealed by usual testing procedures. The mechanism(s) by which CIPC produces these responses remains unclear. Fibroblasts cultured in the continued presence of the known microtubule-specific drugs colchicine and nocodazole (10 −6 M) show microtubule disassembly within 30 min followed by cell rounding and detachment. By contrast CIPC-treated cells lose microtubule integrity more slowly and remain highly elongated and firmly glass adherent even with prolonged treatment. These dissimilar morphological responses, as well as the failure of CIPC to affect microtubule assembly in vitro, indicate that tubulin is not the exclusive target of CIPC in animal cells. The distribution of assembled microtubules into the arms of multipolar mitotic spindles is clearly observed following withdrawal of CIPC. The abnormality could reflect an irreversible effect of CIPC on microtubule organizing centers. However, the redistribution of cytoplasmic microfilaments into dense, discontinuous submembranous bundles in the presence of CIPC and the failure of microfilaments to resume an entirely normal distribution following herbicide withdrawal suggests that microfilaments are additional targets of herbicide action.

The effect of spindle inhibitors applied before meiosis on meiotic chromosome pairing.

ABSTRACT Injection of 0·5 % colchicine into immature tillers of genotypes of Triticum aestivum, T. aestivum × Aegilops mutica and T. aestivum x Secale cereale hybrids induces asynapsis at first meiotic metaphase irrespective of the homologous or homoeologous nature of the potential pairing chromosomes. The induction of asynapsis occurs at a time during and immediately following the last premeiotic mitosis of pollen mother cells. No disruption of synapsis and chiasma formation occurs in anthers having pollen mother cells originally at leptotene or immediately prior to leptotene when cultured in White’s medium plus colchicine. Tetraploid and octaploid pollen mother cells resulting from the disruption of premeiotic spindles by colchicine show pairing of chromosomes only in bivalents, in genotypes normally having a degree of multivalent pairing configurations. The induction of multipolar mitotic spindles with 0·01 % colchicine results in the development of pollen mother cell mosaics with different numbers of chromosomes. Such cells show high levels of chromosome pairing, including multivalents, in some genotypes that normally have very little chromosome pairing. The injection of 0·5 % chloral hydrate during the last premeiotic mitosis of the archesporium causes no disturbances of meiotic pairing. The results are discussed with reference to the hypothesis that the control mechanism of meiotic chromosome pairing involves centromeric microtubules of the spindle (not affected by chloral hydrate) that are responsible for the positional adjustment, during the last mitotic anaphase, of potential pairing partners.