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.

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.

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.

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.

[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.

[The effect of centrosome UV microbeam irradiation on cell behavior. II. The radiation sequelae in the anaphase: the completion of division and the fate of the interphase cell]. / Vliianie UF-mikrooblucheniia tsentrosomy na povedenie kletok. II. Posledstviia oblucheniia v anafaze: zavershenie deleniia i sud'ba interfaznoi kletki.

Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.

Does actin produce the force that moves a chromosome to the pole during anaphase?

Chromosomes move towards spindle poles because of force produced by chromosomal spindle fibres. I argue that actin is involved in producing this force. Actin is present in chromosomal spindle fibres, with consistent polarity. Physiological experiments using ultraviolet microbeam irradiations suggest that the force is due to an actin and myosin (or myosin-equivalent) system. Other physiological experiments (using inhibitors in "leaky" cells or antibodies injected into cells) that on the face of it would seem to rule out actin and myosin on closer scrutiny do not really do so at all. I argue that in vivo the "on" ends of chromosomal spindle fibre microtubules are at the kinetochores; I discuss the apparent contradiction between this conclusion and those from experiments on microtubules in vitro. From what we know of treadmilling in microtubules in vitro, the poleward movements of irradiation-induced areas of reduced birefringence (arb) can not be explained as treadmilling of microtubules: additional assumptions need to be made for arb movements toward the pole to be due to treadmilling. If arb movement does indeed represent treadmilling along chromosomal spindle fibre microtubules, treadmilling continues throughout anaphase. Thus I suggest that chromosomal spindle fibres shorten in anaphase not because polymerization is stopped at the kinetochore (the on end), as previously assumed, but rather because there is increased depolymerization at the pole (the "off" end).

Differential rates of loss of chromosomes aberrations in rat thyroids after X rays or neutrons.

Rat thyroid glands were exposed in vivo to 5.5-Gy X rays or 2.75-Gy neutrons (14.7 MeV) and cell proliferation was stimulated by goitrogen treatment at various intervals up to 48 weeks postirradiation. The amount of chromosome damage in stimulated follicular cells declined much more slowly after neutron than X irradiation, suggesting differential repair. This observation may be relevant to the question of residual cellular damage and oncogenesis after X rays and neutrons.

9-beta-D-arabinofuranosyladenine increases the frequency of X-ray induced chromosome abnormalities in mammalian cells.

Treatment of X-irradiated stationary Ehrlich ascites tumour cells with the DNA synthesis inhibitor beta-ara A (120 mumol/l, 30 min before and for 7 hours after irradiation) is shown to lead to a large increase in the incidence of anaphase chromosome abnormalities (anaphase bridges and fragments) at the first mitosis following irradiation. This increase is similar to the increase in cell killing observed for this cell line when treated with beta-ara A under the same conditions (Iliakis 1980). The results suggest that the increased frequency of chromosome abnormalities caused by beta-ara A may result not only from the inhibition of DNA double strand break repair, leading to additional unrepaired d.s.b. (Bryant and Blöcher 1982) and chromosome deletions, but also from an increase in the frequency of misrepair of d.s.b. leading to exchange aberrations.

The relationship between cell killing, chromosome aberrations, spindle defects and mitotic delay in mouse lymphoma cells of differential sensitivity to X-rays.

The ultrasensitivity of a subline of L5178Y mouse lymphoma cells to X-rays was thought to result from chromosome structural aberrations which are much more frequent in these cells than in radiation-resistant cells derived from them (Scott, Fox and Fox 1974). However, Ehmann, Nagasawa, Peterson and Lett (1974) in time-lapse photography studies of the sensitive line, concluded that the induction of multipolar mitoses by X-rays might be a more important mechanism of cell killing than chromosome aberrations. We have now shown that at survival levels above about 20 per cent, chromosome structural aberrations which lead to bridges and fragments at anaphase are about four times more frequent than spindle defects. We have confirmed the higher frequency of structural aberrations and spindle defects, and the greater mitotic delay in the X-ray-sensitive than in the X-ray-resistant cell line and have proposed a model which causally relates these end-points to cell killing and DNA repair.