Ultraviolet attenuates centromere-mediated meiotic genome stability and alters gametophytic ploidy consistency in flowering plants.

Ultraviolet (UV) radiation influences development and genome stability in organisms; however, its impact on meiosis, a special cell division essential for the delivery of genetic information across generations in eukaryotes, has not yet been elucidated. In this study, by performing cytogenetic studies, we reported that UV radiation does not damage meiotic chromosome integrity but attenuates centromere-mediated chromosome stability and induces unreduced gametes in Arabidopsis thaliana. We showed that functional centromere-specific histone 3 (CENH3) is required for obligate crossover formation and plays a role in the protection of sister chromatid cohesion under UV stress. Moreover, we found that UV specifically alters the orientation and organization of spindles and phragmoplasts at meiosis II, resulting in meiotic restitution and unreduced gametes. We determined that UV-induced meiotic restitution does not rely on the UV Resistance Locus8-mediated UV perception and the Tapetal Development and Function1- and Aborted Microspores-dependent tapetum development, but possibly occurs via altered JASON function and downregulated Parallel Spindle1. This study provides evidence that UV radiation influences meiotic genome stability and gametophytic ploidy consistency in flowering plants.

External and Environmental Effects on Centrosomes.

The effects of ionizing radiation on centrosomes have been well documented and reviewed by Saladino et al. (2012) and are only briefly addressed here. These results showed that exposure of tumor cells to ionizing radiation causes centrosome overduplication and the formation of multipolar mitotic spindles, resulting in nuclear fragmentation and subsequent cell death (Sato et al. 2000). By using a variety of cell lines derived from different types of human solid tumors, it was shown that exposure to 10 Gy γ-radiation resulted in a substantial increase in cells containing an abnormally high number of aberrant centrosomes that formed multipolar spindles, resulting in imbalanced chromosome separation followed by mitotic cell death and formation of multi- or micronucleated cells.

Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification.

Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.

Altered development of Xenopus embryos in a hypogeomagnetic field.

The hypogeomagnetic field (HGMF; magnetic fields <200 nT) is one of the fundamental environmental factors of space. However, the effect of HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two-cell stage, but no later than the four-cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α-tubulin showed reorientation of the spindle of four-cell stage blastomeres. These results indicate that a brief (2-h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos.

Non-thermal effects of 2.45 GHz microwaves on spindle assembly, mitotic cells and viability of Chinese hamster V-79 cells.

The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10mW/cm(2)), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41°C, while apoptosis was induced only when the medium temperature achieved 40°C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line.

Integrin beta1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix.

Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta1-dependent phenotype, and integrin beta1 might be a potentially effective therapeutic target in combination with radiotherapy.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

Poleward force at the kinetochore in metaphase depends on the number of kinetochore microtubules.

To examine the dependence of poleward force at a kinetochore on the number of kinetochore microtubules (kMTs), we altered the normal balance in the number of microtubules at opposing homologous kinetochores in meiosis I grasshopper spermatocytes at metaphase with a focused laser microbeam. Observations were made with light and electron microscopy. Irradiations that partially damaged one homologous kinetochore caused the bivalent chromosome to shift to a new equilibrium position closer to the pole to which the unirradiated kinetochore was tethered; the greater the dose of irradiation, the farther the chromosome moved. The number of kMTs on the irradiated kinetochore decreased with severity of irradiation, while the number of kMTs on the unirradiated kinetochore remained constant and independent of chromosome-to-pole distance. Assuming a balance of forces on the chromosome at congression equilibrium, our results demonstrate that the net poleward force on a chromosome depends on the number of kMTs and the distance from the pole. In contrast, the velocity of chromosome movement showed little dependence on the number of kMTs. Possible mechanisms which explain the relationship between the poleward force at a kinetochore, the number of kinetochore microtubules, and the lengths of the kinetochore fibers at congression equilibrium include a "traction fiber model" in which poleward force producers are distributed along the length of the kinetochore fibers, or a "kinetochore motor-polar ejection model" in which force producers located at or near the kinetochore pull the chromosomes poleward along the kMTs and against an ejection force that is produced by the polar microtubule array and increases in strength toward the pole.

Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure.

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.

Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma.

Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.

Taxane-mediated radiosensitization derives from chromosomal missegregation on tripolar mitotic spindles orchestrated by AURKA and TPX2.

Taxane-based radiochemotherapy is a central treatment option for various cancer entities in locally advanced stages. The therapeutic synergism of this combined modality approach due to taxane-mediated radiosensitization of cancer cells is well-known. However, the underlying molecular mechanisms remain largely elusive, and mechanism-derived predictive markers of taxane-based radiochemotherapy are currently not available. Here, we show that clinically relevant doses of Paclitaxel, the prototype taxane, stimulate a tripolar mode of mitosis leading to chromosomal missegregation and aneuploidization rather than interfering with cell cycle progression. This distinct mitotic phenotype was interlinked with Paclitaxel-mediated radiosensitization via overexpression of mitotic Aurora kinase A (AURKA) and its cofactor TPX2 whose knockdown rescued the bipolar mode of cell division and largely attenuated the radiosensitizing effects of Paclitaxel. In the cancer genome atlas (TCGA) lung adenocarcinoma cohort, high expression levels of AURKA and TPX2 were associated with specifically improved overall survival upon taxane-based radiochemotherapy, but not in case of non-taxane-based radiochemotherapy, chemo- or radiotherapy only. Thus, our data provide insights into Paclitaxel-mediated radiosensitization on a mechanistic and molecular level and identify AURKA and TPX2 as the first potential mechanism-based, predictive markers of taxane-based radiochemotherapy.

Centrosome Clustering Is a Tumor-selective Target for the Improvement of Radiotherapy in Breast Cancer Cells.

BACKGROUND/AIM: Owing to the frequent observation of centrosome amplification in human cancers, cancer cells have a unique mechanism to suppress detrimental multipolar division by clustering multiple centrosomes into two functional spindle poles, known as centrosome clustering. This study investigated whether inhibition of centrosome clustering enhances the radiation sensitivity of breast cancer cells. MATERIALS AND METHODS: In this study, inhibition of centrosome clustering was examined by using various centrosome-declustering agents and KIFC1 siRNA in three breast cancer cell lines and two normal fibroblast cell lines. The combination effect of radiation and centrosome declustering was evaluated by cell viability, clonogenic, immunofluorescence assay. RESULTS: This study showed that targeting centrosome clustering enhanced the efficacy of radiotherapy of breast cancer cells with less damage to normal cells. Ionizing radiation induced centrosome amplification in breast cancer cells, but not in normal fibroblast cells. Notably, we showed that centrosome declustering efficiently radiosensitized the centrosome-amplified breast cancer cells through induction of multipolar spindles but did not affect the viability of normal fibroblasts in response to irradiation. Furthermore, KIFC1 mediated the radiosensitivity of the centrosome-amplified breast cancer cells. CONCLUSION: Our data provided the first evidence that centrosome clustering is a tumor-selective target for the improvement of radiotherapy in breast cancer cells.

Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B.

INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.

27 T ultra-high static magnetic field changes orientation and morphology of mitotic spindles in human cells.

Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology.

A possible role for centrosome overduplication in radiation-induced cell death.

Radiotherapy plays a key role in the treatment of many tumors; however, the precise mechanisms responsible for radiation-induced cell death remain uncertain. We have reported previously that ionizing radiation induces centrosome overduplication in human tumor cells. The present study was designed to elucidate a possible link between centrosome dysregulation and radiation-induced cell death. Exposure to 10 Gy gamma-radiation resulted in a substantial increase in cells containing an abnormally high number of centrosomes in a variety of cell lines derived from different types of human solid tumors. These aberrant centrosomes contribute to the assembly of multipolar spindles, thereby causing an unbalanced division of chromosomes and mitotic cell death characterized by the appearance of multi- or micronucleated cells. An extensive analysis of a panel of 10 tumor cell lines revealed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with these nuclear abnormalities after irradiation. When the centrosome overduplication was blocked by enforced expression of p21Waf1/Cip1, the radiation-induced lethality was drastically rescued. Taken together, these results indicate that centrosome overduplication may be a critical event leading to mitotic failure and subsequent cell death following exposure to ionizing radiation.

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint.

Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule-kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.

Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus.

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.