DNA repair is efficient in irradiated M phase zygotes.

In somatic cells, DNA repair is attenuated during mitosis to prevent the formation of anaphase bridges and facilitate the proper segregation of sister chromatids. Irradiation-induced γH2AX foci persist for hours in M phase somatic cells. However, we observed that anaphase bridges formed in a significant fraction of mouse zygotes irradiated during mitosis. Additionally, γH2AX signals in M phase zygotes peaked 30 min after irradiation and subsequently reduced with a half-life within 1-2 h. These results suggest that the DNA repair system may operate efficiently in M phase zygotes following irradiation, leading to the frequent formation of anaphase bridges. The absence of H2AX promoted the successful segregation of sister chromatids and enhanced the development of embryos to the blastocyst stage. The DNA repair system may be differentially regulated during the M phase of the first cell cycle to ensure the immediate elimination of damaged zygotes, thereby efficiently preventing transmission of mutations to subsequent generations.

The role of telomere length modulation in delayed chromosome instability induced by ionizing radiation in human primary fibroblasts.

Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X-rays and low-energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X-rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X-ray- compared with proton-irradiated samples, suggesting a correlation between telomere length/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X-rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X-ray-treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X-ray irradiation originated from telomere attrition and consequent chromosome end-to-end fusion. This study shows that, beside an expected "early" chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability.

[Structural architectonics of apical root meristems in coherence with the quantitative assessment of its damage by radiation].

The dose dependencies of the aberrant anaphases frequency in the root meristem in 48 hours after irradiation in the range of doses of 4-10 Gy is characterized by threshold and plateau at 33% aberrant anaphase. The plateau indicates the activation of the recovery processes. Topology of cell rows in the primary meristem of the dose to 8 Gy are conserved and recovered damages. New cell rows are formed by local cell pools in the distal meristem, pericycle cells and subepidermy. It grows by intrusive character displacing the rows of damaged cells. Apparently the competition between clones of normal and aberrant cells plays the primary role in the mechanisms of recovery. Resulting to competition the promotion of aberrant cells to the extension zone is slowed down or blocked. So critical level of damage of the root apical meristem was defined about 50% of aberrant anaphase. Exceeding of this level leads to lethal consequence for meristem and it is accompanied by the inclusion of more radical process of restoration through regeneration. Regeneration leads to complete replacement of the apex tissues including the extension zone.

[Genetic effects of root extracts of Glycyrrhiza glabra L. on different test-systems].

The antimutagenic and geroprotective activities of root extracts of Glycyrrhiza glabra have been demonstrated both on plant test systems--Allium fistulosum L., Allium cepa L., Vicia faba L. and on animals--Vistar rats. The possibilities of the mobilization of Glycyrrhiza glabra root extracts as antimutagenic agents are discussed.

Spindle disturbances in human-hamster hybrid (A(L) ) cells induced by the electrical component of the mobile communication frequency range signal.

The production of spindle disturbances in a human-hamster hybrid (A(L) ) cell line by an electromagnetic field (EMF) with field strength of 90 V/m at a frequency of 900 MHz was studied in greater detail. The experimental setup presented allows investigating whether either the electrical (E) and/or the magnetic (H) field component of EMF can be associated with the effectiveness of the spindle-disturbing potential. Therefore, both field components of a transversal electromagnetic field (TEM) wave have been separated during exposure of the biological system. This procedure should give more insight on understanding the underlying mechanisms of non-thermal effects of EMF. A statistical comparison of the proportions of the fractions of ana- and telophases with spindle disturbances, obtained for five different exposure conditions with respect to unexposed controls (sham condition), showed that only cells exposed to the H-field component of the EMF were not different from the control. Therefore, the results of the present study indicate that an exposure of cells to EMF at E-field strengths of 45 and 90 V/m, as well as to the separated E component of the EMF, induces significant spindle disturbances in ana- and telophases of the cell cycle.

Terahertz radiation induces spindle disturbances in human-hamster hybrid cells.

The aim of this study was to investigate and quantify the production of spindle disturbances in A(L) cells, a human-hamster hybrid cell line, by 0.106 THz radiation (continuous wave). Monolayer cultures in petri dishes were exposed for 0.5 h to 0.106 THz radiation with power densities ranging from 0.043 mW/cm(2) to 4.3 mW/cm(2) or were kept under sham conditions (negative control) for the same period. As a positive control, 100 µg/ml of the insecticide trichlorfon, which is an aneuploidy-inducing agent, was used for an exposure period of 6 h. During exposure, the sample containers were kept at defined environmental conditions in a modified incubator as required by the cells. Based on a total of 6,365 analyzed mitotic cells, the results of two replicate experiments suggest that 0.106 THz radiation is a spindle-acting agent as predominately indicated by the appearance of spindle disturbances at the anaphase and telophase (especially lagging and non-disjunction of single chromosomes) of cell divisions. The findings in the present study do not necessarily imply disease or injury but may be important for evaluating possible underlying mechanisms.

Chromosome tips damaged in anaphase inhibit cytokinesis.

Genome maintenance is ensured by a variety of biochemical sensors and pathways that repair accumulated damage. During mitosis, the mechanisms that sense and resolve DNA damage remain elusive. Studies have demonstrated that damage accumulated on lagging chromosomes can activate the spindle assembly checkpoint. However, there is little known regarding damage to DNA after anaphase onset. In this study, we demonstrate that laser-induced damage to chromosome tips (presumptive telomeres) in anaphase of Potorous tridactylis cells (PtK2) inhibits cytokinesis. In contrast, equivalent irradiation of non-telomeric chromosome regions or control irradiations in either the adjacent cytoplasm or adjacent to chromosome tips near the spindle midzone during anaphase caused no change in the eventual completion of cytokinesis. Damage to only one chromosome tip caused either complete absence of furrow formation, a prolonged delay in furrow formation, or furrow regression. When multiple chromosome tips were irradiated in the same cell, the cytokinesis defects increased, suggesting a potential dose-dependent mechanism. These results suggest a mechanism in which dysfunctional telomeres inhibit mitotic exit.

Formation of the nuclear envelope permeability barrier studied by sequential photoswitching and flux analysis.

In higher eukaryotes, the nuclear envelope breaks down during mitosis. It reforms during telophase, and nuclear import is reestablished within <10 min after anaphase onset. It is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins. However, nuclear pore complex assembly is not fully completed when import capacity is regained, which raises the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. In this study, we therefore analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation. We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells show that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrate that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality.

Transformation of human mesenchymal stem cells in radiation carcinogenesis: long-term effect of ionizing radiation.

Increasing evidence on cancer stem cells suggest that stem cells are susceptive to carcinogenesis and consequently can be the origin of many cancers. We have recently established a telomerase-transduced human mesenchymal stem cell line and subsequently irradiated this in order to achieve malignant transformation. In the present study, we analyzed the long-term effect of ionizing radiation on these cells and investigate whether radiation can trigger tumor development. The cells were irradiated with a low (2.5 Gy) and a high (15 Gy) dose of gamma-rays and followed for up to 6 months after radiation. A subclone of the cells irradiated with 2.5 Gy of gamma-rays formed tumors after implantation to severe combined immunodeficiency mice. During the process of transformation, the cells showed accelerated telomere shortening, increased levels of anaphase bridges and a shift from balanced to unbalanced translocations. The tumor suppressor genes p53 and p21(CIP1) functioned normally throughout the study. Our observations indicate that radiation destabilized the telomeres and that the presence of uncapped telomeres initiated fusion-break-fusion cycles, resulting in increased chromosomal instability and tumor formation. Thus, bone marrow-derived human mesenchymal stem cells are capable of exhibiting a malignant phenotype.

Endopolyploidy in irradiated p53-deficient tumour cell lines: persistence of cell division activity in giant cells expressing Aurora-B kinase.

Recent findings including computerised live imaging suggest that polyploidy cells transiently emerging after severe genotoxic stress (and named 'endopolyploid cells') may have a role in tumour regrowth after anti-cancer treatment. Until now, mostly the factors enabling metaphase were studied in them. Here we investigate the mitotic activities and the role of Aurora-B, in view of potential depolyploidisation of these cells, because Aurora-B kinase is responsible for coordination and completion of mitosis. We observed that endopolyploid giant cells are formed via different means in irradiated p53 tumours, by: (1) division/fusion of daughter cells creating early multi-nucleated cells; (2) asynchronous division/fusion of sub-nuclei of these multi-nucleated cells; (3) a series of polyploidising mitoses reverting replicative interphase from aborted metaphase and forming giant cells with a single nucleus; (4) micronucleation of arrested metaphases enclosing genome fragments; or (5) incomplete division in the multi-polar mitoses forming late multi-nucleated giant cells. We also observed that these activities can release para-diploid cells, although infrequently. While apoptosis typically occurs after a substantial delay in these cells, we also found that approximately 2% of the endopolyploid cells evade apoptosis and senescence arrest and continue some form of mitotic activity. We describe here that catalytically active Aurora-B kinase is expressed in the nuclei of many endopolyploid cells in interphase, as well as being present at the centromeres, mitotic spindle and cleavage furrow during their attempted mitotes. The totally micronucleated giant cells (containing sub-genomic fragments in multiple micronuclei) represented only the minor fraction which failed to undergo mitosis, and Aurora-B was absent from it. These observations suggest that most endopolyploid tumour cells are not reproductively inert and that Aurora-B may contribute to the establishment of resistant tumours post-irradiation.

Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1).

Checkpoints monitor the state of DNA and can delay or arrest the cell cycle at multiple points including G1-S transition, progress through S phase and G2-M transition. Regulation of progress through mitosis, specifically at the metaphase-anaphase transition, occurs after exposure to ionizing radiation (IR) in Drosophila and budding yeast, but has not been conclusively demonstrated in mammals. Here we report that regulation of metaphase-anaphase transition in Drosophila depends on the magnitude of radiation dose and time in the cell cycle at which radiation is applied, which may explain the apparent differences among experimental systems and offer an explanation as to why this regulation has not been seen in mammalian cells. We further document that mutants in Drosophila Chk1 (Grapes) that are capable of delaying the progress through mitosis in response to IR are incapable of delaying progress through mitosis when DNA synthesis is blocked by mutations in an essential replication factor encoded by double park (Drosophila Cdt1). We conclude that DNA damage and replication checkpoints operating in the same cell cycle at the same developmental stage in Drosophila can exhibit differential requirements for the Chk1 homolog. The converse situation exists in fission yeast where loss of Chk1 is more detrimental to the DNA damage checkpoint than to the DNA replication checkpoint. It remains to be seen which of these two different uses of Chk1 homologs are conserved in mammals. Finally, our results demonstrate that Drosophila provides a unique opportunity to study the regulation of the entry into, and progress through, mitosis by DNA structure checkpoints in metazoa.

Backward chromosome movement in crane-fly spermatocytes after UV microbeam irradiation of the interzone and a kinetochore.

Single anaphase chromosomes (in crane-fly spermatocytes) moved backwards after double irradiations with an ultraviolet light (UV) microbeam, first of the interzone and then of a kinetochore: the chromosome irradiated at the kinetochore moved backwards rapidly, across the equator and into the other half-spindle. High irradiation doses at the kinetochore were required to induce backward movement. Single irradiations of kinetochores or interzones were ineffective in inducing backward movements.

Sticky anaphase aberrations after G2-phase arrest of gamma-irradiated human skin fibroblasts: TP53 independence of formation and TP53 dependence of consequences.

We have studied the impact of TP53 status on the extent and nature of chromosome damage seen in human skin fibroblasts after gamma irradiation beyond the G1-phase checkpoint but prior to the G2-phase checkpoint. Mitotic cells were examined in the absence and presence of treatment with nocodazole and the yield of aberrations was scored as a function of time postirradiation. The results revealed substantially greater damage in the absence of nocodazole, indicating that damage was being masked in its presence. While metaphase aberrations were seen exclusively in the presence of nocodazole, anaphase aberrations were seen principally in its absence. Furthermore, these were mostly of an unseparated, or "sticky", type that showed separation of the chromatids in the centromeric region, indicating normal degradation of cohesin, with retention of adhesion further out on the chromatid arms. Using postirradiation BrdU labeling and the absence of nocodazole, we were able to identify mitotic figures up to the third postirradiation mitosis. Analysis of the data revealed that in cells wild-type for TP53 the aberrant anaphases were lost after the first postirradiation mitosis, although they were still found in gradually decreasing amounts into the second and third postirradiation mitoses in E6-expressing cells. The data indicate that the formation of these sticky anaphases is independent of TP53 status, an observation that is consistent with the TP53 independence of transient G2-phase arrest. However, the consequences of the formation of these lesions appear to be very different. In the case of cells wild-type for TP53 this is chronic G1-phase arrest, while in E6 cells it is anaphase catastrophe.

[Chromosomal radiosensitivity as associated with chromatin diminution in cyclops (Crustacea, Copepoda)]. / Radiochuvstvitel'nost' khromosom v sviazi s diminutsiei khromatina u tsiklopov (Crustacea, Copepoda).

Chromosomal radiosensitivity inferred from the yield of chromosome aberrations (CAs) was for the first time studied in Cyclops (Crustacea, Copepoda) before and after chromatin diminution (CD). A comparison was made for C. kolensis, in which CD denudes somatic embryo cells of the greatest (94%) DNA amount known for multicellular organisms, and C. insignis, which lacks CD. The two species have similar genome sizes, 4.6 and 4.3 pg. respectively. Radiosensitivity of C. kolensis chromosomes proved to be extremely high during prediminution cleavage divisions. This was attributed to membrane damage in granules that contain enzymes (topoisomerases) normally involved in cleavage and ligation of chromosomal DNA during CD.

[The dose dependence of cytogenetic damages and the adaptive response of mammalian cells under the action of ionizing radiation at low doses]. / Dozovaia zavisimost' tsitogeneticheskikh povrezhdenii i adaptivnyi otvet kletok mlekopitaiushchikh pri deistvii ioniziruiushchego izlucheniia v malykh dozakh.

The dose-effect dependence of cytogenetic damage after single dose irradiation in the dose range of 0.1-2 Gy and the adaptive response after double-dose irradiation were studied on Chinese hamster and human melanoma cells in culture. The non-linear dose dependencies were found for the induction of chromosome aberrations with decrease in cell radiosensitivity in the definite dose range. This decrease started at 10 and 20 cGy for melanoma and Chinese hamster cells respectively. The maximal adaptive response was induced at 1 cGy for melanoma cells and at 20 cGy for Chinese hamster cells. It can be supposed that the same inducible repair processes are responsible for non-linearity of dose-effect curves and induction of the adaptive response. These processes are similar in mechanisms and different in quantitative proportion for different cell types.

A cytoplasmic dynein required for mitotic aster formation in vivo.

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

[Characterization of the adaptive response to the action of gamma-rays, induced by low doses of 14C in Chinese hamster fibroblasts]. / Kharakteristika adaptivnogo otveta k deistviiu gamma-luchei, indutsirovannogo malymi dozami 14C v fibroblastakh kitaiskogo khomiachka.

It has been studied the correlation of the mitotic activity of the chromosome aberrations and apoptosis, in the V-79 cells pre-exposure to an adapting dose of ionizing radiation from 14C-thymidine prior to an acute challenge dose of gamma-rays. In spite of that the incubation of the cells with isotope increased of the yield of the chromosome aberrations, but the cells became more resistant to following gamma-irradiation. Increasing the adaptive dose of the 14C on degree didn't influence on the present of the adaptive response. However, using concentrations of the 14C damaged metaphase/anaphase transition and cells blocked in this check-point by apoptotic death. The results suggest, that the cellular selection has been involved in 14C-induced adaptive response, estimated by level of asymmetric chromosome aberrations in V-79 cells.

Effect of 1 Gy X-rays in G1 phase on activation of cell cycle checkpoints in human lymphocytes: role played by chromosomal aberrations.

The effect of X-irradiation (1 Gy) during G1 on the transition from G1 to S and on the length of G2 over cell cycles subsequent to irradiation was studied in human lymphocytes from six different donors. After irradiation a delay was observed in the onset of S phase, as was an extension of the G2 phase lasting throughout the three to four subsequent cell divisions. The extension of G2 and of the cell cycle as a whole is partly related to the presence of chromosome aberrations in the cell. This is demonstrated by: (i) the presence of a larger number of chromosome aberrations in M1 cells corresponding to sampling times longer after that of irradiation; (ii) the presence of a larger number of chromosome aberrations in cells with a longer G2. The most significant chromosome aberrations in this respect are isochromatid fragments. Lastly, we observed that irradiation during G1 activates another checkpoint governing the way mitosis proceeds. This takes the form of an extension of metaphase; in this case also, in some cells, activation of a possible checkpoint during preanaphase seems to be related to the presence of aberrations.

Coordinated movements between autosomal half-bivalents in crane-fly spermatocytes: evidence that 'stop' signals are sent between partner half-bivalents.

During anaphase-I in crane-fly spermatocytes, sister half-bivalents separate and move to opposite poles. When we irradiate a kinetochore spindle fibre with an ultraviolet microbeam, the associated half-bivalent temporarily stops moving and so does the partner half-bivalent with which it was paired during metaphase. To test whether a 'signal' is transmitted between partner half-bivalents we irradiated the spindle twice, once in the interzone (the region between separating partner half-bivalents) and once in a kinetochore fibre. For both irradiations we used light of wavelength 290 microns and a dose that, after irradiating a spindle fibre only, altered movement in 63% of irradiations (12/19); in 11 of the 12 cells both partner half-bivalents stopped moving after the irradiation. In control experiments we irradiated the interzone only: these irradiations generally did not stop chromosomal poleward motion but sometimes (14/29) caused poleward movement to each pole to be abruptly reduced to about half the velocity prior to irradiation. In double irradiation experiments we varied the order of the irradiations. In some double irradiation experiments we irradiated the interzonal region first and the spindle fibre second; in 75% (9/12) of the cells the half-bivalent associated with the irradiated fibre stopped moving while the partner half-bivalent moved normally, i.e. in 9/12 cells the interzonal irradiations uncoupled the movements of the partner half-bivalents. In other double irradiation experiments we irradiated the spindle fibre first and the interzone second: in 80% (4/5) of the cells the half-bivalents not associated with the irradiated spindle fibre resumed movement immediately after the irradiation while the other half-bivalent remained stopped. Interzonal irradiations therefore uncouple the poleward movements of sister half-bivalents and the uncoupling does not depend on the order of the irradiation. Our experiments suggest therefore that the irradiation of a spindle fibre causes negative ('stop') signals to be transmitted across the interzone and that irradiation of the interzone blocks the transmission of the stop signal.

[The effect of ultraviolet microbeam irradiation of the centrosome on cellular behavior. IV. Synthetic activity, cell spreading and growth with an inactivated centrosome]. / Vliianie ul'trafioletovogo mikrooblucheniia tsentrosomy na povedenie kletok. IV. Sinteticheskaia aktivnost', rasplastyvanie i rost kletok s inaktivirovannoi tsentrosomoi.

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam at anaphase. After irradiation, cell division completed with a minor delay and two daughter cells were spreading synchronously. Later on, cells with irradiated centrosomes slightly shrunk, while their sister cells enlarged normally. Sister cells entered S-phase, some of them undergoing mitosis. In the cells with irradiated centrosomes the formation of nucleoli was disturbed and numerous primary nucleoli remained for 50 h (the maximum time of observation). RNA synthesis in the cells with irradiated centrosomes was twice less than in the sister cells, with ribosomal RNA synthesis being suppressed predominantly. Cells with irradiated centrosomes did not enter S-phase for as long as 24 h. The same irradiation of a portion of cytoplasm outside the spindle performed during anaphase did not change the pattern behaviour in daughter cell. In is concluded that the centrosome regulates progression throughout the cell cycle, and that centrosome irradiation induces specific and irreversible damage of interphase cells.