Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH)

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.     

Different effects of 1-beta-D-arabinofuranosylcytosine and aphidicolin in S-phase cells--chromosome aberrations, cell-cycle delay and cytotoxicity

Some effects of a 2-h exposure to either aphidicolin (APC) or cytosine arabinoside (ara-C) on S-phase cells of the cell line JU56 have been measured. At a concentration of 1.5 X 10(-5) M of either drug, incorporation of tritiated thymidine into log-phase cultured was reduced by 97-99%. A 2-h exposure to either drug at the same concentration induced chromosome aberrations in cells in S when they subsequently reached mitosis. However, exposure to ara-C induced small numbers of aberrations per damaged cells, and most cells were undamaged. Exposure to APC induced gross chromosomal damage (pulverized chromosomes) in damaged cells. More cells were delayed, and for longer, after exposure to APC than after exposure to ara-C. The results of clonal assays were consistent with the assumption that chromosome aberrations are the proximal cause of reproductive cell death. In the case of ara-C, the results of this and a previous study are consistent with the assumption that cell death and chromosome aberrations are correlated with incorporation of ara-C into DNA in S-phase cells, but that these biological effects manifest themselves only with doses when inhibition of semi-conservative DNA synthesis is greater than 97%.     

Induction of chromosome damage by restriction enzymes during mitosis.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline'cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo-somally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [³H]dT labelling for cells with DNA contents below the modal G_o/G₁ peak and above the modal G₂ peaks ( P <0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.     

Mechanisms of chromosomal aberration production II. Aberrations induced by 5-bromodeoxyuridine and visible light

We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G₁and G₂) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G₁ phase following incorporation induces aberrations of the chromatid type, but none of the chromosome type. All types of chromatid aberrations are induced, including isochromatid deletions and exchange types. In contrast, when cells are illuminated during the immediately following G₂ phase, large numbers of achromatic lesions and chromatic deletions are seen at the first post-illumination mitosis, but no isochromatid deletions and few exchange-type aberrations occur. When G₂-illuminated cells are examined in their second mitosis, however, chromatid aberrations of all types are again seen.

These results are interpreted within the “repair” model of chromosomal aberration production by UV light presented earlier³. The model assumes that the vertebrate chromosome is mononeme, consisting of but a single DNA double helix during the prereplication G₁ phase. The initial lesions induced by illumination of BUdR-containing DNA are believed to be single-chain breaks, and the observation that G₁ illumination produces only chromatid-type aberrations is taken as additional evidence for the mononeme chromosome. Conversion of single-chain breaks into double chain breaks through the action of a single-strand nuclease is postulated to account for the production of chromatid deletions at the first mitosis of G₂-illuminated cells. The action of this enzyme, plus a recombinational or post-replication repair mechanism, are postulated to account for the production of isochromatid deletions in G₁-illuminated cells. A rapid decline in achromatic lesion frequency with increasing time between G₂ illumination and fixation of the cells is considered evidence for rapid rejoining of most of the initial chain breaks.     

Effect of Nutrient Availability on Progenitor Cells in Zebrafish (Danio Rerio)

In zebrafish brains, populations of continuously proliferating cells are present during an entire life span. Under normal conditions, stem cells give rise to rapidly proliferating progenitors that quickly exit the cell cycle and differentiate. Hence fish are favorable models to study what regulates postembryonic neurogenesis. The aim of this study was to determine if optic tectum (OT) cell proliferation is halted during nutritional deprivation (ND) and whether or not it can be restored with refeeding. We examined the effect of ND on the proliferation of Neuroepithelial/Ependymal Progenitor cell (NeEPC) and transitory‐amplifying progenitors (TAPs). Following ND, no PCNA immunostaining was found in OT of starved fish, while positive cell populations of PCNA positive progenitors are found at its periphery in control fish. This indicated that active proliferation stopped. To label retaining progenitor cells, BrdU was applied and a chase‐period was accompanied by ND. Positive NeEPCs were detected in the external tectum marginal zone of starved fish suggesting that these progenitors are relatively immune to ND. Moreover in the internal tectum marginal zone labeled retaining cells were observed leaving the possibility that some arrested TAPs were present to readily restart proliferation when nutrition was returned. Our results suggest that neurogenesis was maintained during ND and that a normal proliferative situation was recovered after refeeding. We point to the mTOR pathway as a necessary pathway in progenitors to regulate their mitosis activity. Thus, this study highlights mechanisms involved in neural stem and progenitor cell homeostatic maintenance in an adverse situation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 26–38, 2017     

Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered "new," "derived," or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Cell survival and radiation induced chromosome aberrations

Human peripheral lymphocytes were irradiated in whole blood with 0.5-4.0 Gy of 220 kVp X-rays and the frequency of chromosome aberrations was determined in 1st or 2nd division metaphases discriminated by fluorescence plus giemsa staining. Using the empirical distributions of aberrations among cells, cell survival and transmission of aberrations were investigated. Considering both daughter cells, we found that 20% of fragments and 55% of dicentrics or ring chromosomes are lost during the 1st cell division; i.e. cell survival rate from 1st to 2nd generation is mainly influenced by anaphase bridging of these two-hit aberrations. Cell survival to 2nd mitosis was calculated considering this situation and compared with the survival derived from the fraction of M 1 cells without unstable aberrations. The resulting shouldered survival curves showed significantly different slopes, indicating that cell reproductive death is overestimated in the latter approach.     

Hydroxyurea retards the progression of G2 cells

Continuous treatment of monolayer cultures of Chinese hamster ovary (CHO) cells with 1.0 mM HU reduced the rate at which G2 cells entered mitosis. Although all cells in G2+M at the start of treatment eventually entered mitosis, early G2 cells, which were exposed to HU the longest, entered mitosis 45 min later than untreated G2 cells. This perturbation of G2 progression should be considered when employing HU as a synchronizing agent in mammalian cell populations.     

DNA polymerase zeta is required for proliferation of normal mammalian cells

Unique among translesion synthesis (TLS) DNA polymerases, pol ζ is essential during embryogenesis. To determine whether pol ζ is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O(2) to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3-4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30-50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol ζ is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells. Loss of pol ζ in slowly proliferating mouse cells in vivo may allow accumulation of chromosomal aberrations that could lead to tumorigenesis. Pol ζ is unique amongst TLS polymerases for its essential role in cell proliferation.     

Echinococcus granulosus: regulation of leukocyte growth by living protoscoleces from horses, sheep, and cattle

To determine whether living hydatid tissue can, like hydatid fluid, regulate leukocyte growth, T-cell, B-cell, and macrophage lines were cocultured with protoscoleces of Echinococcus granulosus and their growth was compared with that of control cultures by thymidine uptake estimates and chemiluminescent assays of cell number. Protoscoleces supported mitosis of IL-1-deprived D10 T cells, but did not increase D10 count. The action of protoscoleces was affected by the species and organ of their origin and the length of time in culture. Unusually marked mitotic reaction, unaffected by parasite age and origin, was recorded in the B-cell line, BSM, also without commensurate count increase, indicating that induced mitosis resulted in cell loss. It is concluded that protoscoleces can induce mitosis in B and T cells of particular lineages and that this is a potential means of producing the pathological proliferation and depletion of B- and T-cell areas which characterize local reaction to hydatids.     

The relation between tumor cell proliferation and vascularization in differentiated and undifferentiated colon carcinomas in the rat

Tumor cell proliferation and tumor vascularization were investigated in differentiated and undifferentiated colon carcinomas of the rat. The following results were found: 1, in both tumor types, vascularization is developed equally well; 2, both in the differentiated and in the undifferentiated carcinomas, the number of proliferating tumor cells decreases with increasing distance from the capillaries; at a distance of about 80 microns, there is no longer any noteworthy tumor cell proliferation; 3, the differentiated and undifferentiated carcinomas differ both in the level of the mitosis index and 3H-thymidine labeling index as well as in their local proliferation pattern despite equal vascularization; 4, these differences must be based on factors which are independent of vascularization; they are attributed to proliferation properties which are inherent in the differently differentiated tumor cell populations. It is concluded from this that the actual proliferation behavior of a carcinoma is primarily dependent on the proliferation properties of the tumor cell population.     

Dissimilar characteristics of umbilical cord mesenchymal stem cells from donors of different ages

Increasing attention is being placed on the use of adult stem cells due to their potential therapeutic applications for unmet medical needs. Among all adult stem cell types, umbilical cord mesenchymal stem cells (UCMSCs) can be collected easily and does not harm newborns and the mothers. The excellent pluripotency and proliferation potential of UCMSCs has been previously demonstrated by many groups. However, little is known about the different characteristics of these cells due to diversity of donors (e.g., the mothers’ age). Regarding this, we examined the effect of mothers’ age-related variation on the basic properties (proliferation, colony forming and differentiation potential) of UCMSCs in vitro. The data evidenced that elder mother results in reduction of both proliferative and colony forming capacity of UCMSCs, and impacts proliferative potential more significantly. Data also evidenced that a decline in osteogenic potential, but an increase in adipogenic potential of UCMSCs from the elder donors. Additionally, no phenotypic differences were observed by flow cytometry analysis using a panel of 6 main surface antigen markers. We observed no differences in karyotyping and both UCMSCs populations exhibited diploid cells without chromosomal aberrations. The results of our study are the basis for banking UCMSCs, which may be labeled with donors’ information for different experiments, and the mother age-related differences in characteristics of UCMSCs should be taken into account when these cells are considered for further practical applications.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

The Plk1 inhibitor BI 2536 temporarily arrests primary cardiac fibroblasts in mitosis and generates aneuploidy in vitro

BI 2536 is a new anti-mitotic drug that targets polo-like kinase 1 (Plk1) and is currently under clinical development for cancer therapy. The effect of this drug on cancer cells has been extensively investigated, but information about the effects on primary dividing cells and differentiated non-dividing cells is scarce. We have investigated the effects of this drug on primary neonatal rat cardiac fibroblasts and on differentiated cardiomyocytes and explored the possibility to use this drug to enrich differentiated cell populations in vitro. BI 2536 had a profound effect on cardiac fibroblast proliferation in vitro and arrested these cells in mitosis with an IC50 of about 43 nM. Similar results were observed with primary human cells (HUVEC, IC50 = 30 nM), whereas the cancer cell line HeLa was more sensitive (IC50 of 9 nM). Further analysis revealed that prolonged mitotic arrest resulted in cell death for about 40% of cardiac fibroblasts. The remaining cells showed an interphase morphology with mostly multi- and micro-nucleated nuclei. This indicates that a significant number of primary fibroblasts are able to escape BI 2536 induced mitotic arrest and apparently become aneuploid. No effects were observed on cardiomyocytes and hypertrophic response (growth) upon endothelin-1 and phenylephrine stimulation was normal in the presence of BI 2536. This indicates that BI 2536 has no adverse effects on terminally differentiated cells and still allows proliferation independent growth induction in these cells. In conclusion, cardiomyocytes could be enriched using BI 2536, but the formation of aneuploidy in proliferating cells most likely limits this in vitro application and does not allow its use in putative cell based therapies.     

Structural centrosome aberrations promote non‐cell‐autonomous invasiveness

Centrosomes are the main microtubule‐organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein‐like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP‐induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP‐induced centrosome aberrations trigger a re‐organization of the cytoskeleton, which stabilizes microtubules and weakens E‐cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non‐cell‐autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.     

Intercellular Correlations: Relation between DNA Synthesis and Cell Division in Early Stages of in Vitro Bud Neoformation

As well as showing the existence, during the first stages of in vitro bud neoformation, of cell populations in a tissue composed of a single cell layer (stem epidermis of Torenia fournieri Lind), some new physiological characteristics of mitosis are defined. Most of the cells which divide during organogenesis synthesize their DNA between 20 and 48 hours of culture. On an epidermal strip (10 × 2.5 millimeters composed of about 5,500 cells) 20% of the original cells enter the S-phase. The first division takes place at the 20-hour stage after the entry into the S-phase of a cell population of about 25 cells. Almost none of the cells of this population divide. The greatest percentage of divisions occurs in cells which synthesize DNA near the 48-hour stage. The relation [Formula: see text] has a value of about 25 at the beginning of cell division (20 hours) and falls to a value of about 1.4 for cells which synthesize DNA near the 48-hour stage. A hypothesis of the existence of a mitotic stimulant in the epidermis is put forward; this stimulant, at first weak, increases progressively.     

Chromosome aberrations, sister-chromatid exchanges and cell-cycle kinetics in human peripheral blood lymphocytes exposed to organoselenium in vitro

The ability of 2 synthetic organoselenium compounds, a dimer of p-methoxybenzeneselenol (DPMBS) and benzylselenocyanate (BSC), to induce sister-chromatid exchanges (SCE) and chromosome aberrations (CA) as well as to alter the progression of the cell through mitosis has been investigated in cultured human lymphocytes. Cultures treated with the highest concentration (2.27 x 10(-5) M) of the 2 compounds exhibited about a 3-fold increase in the level of SCE and about 2-3-fold increase in the incidence of CA. In addition, the 2 selenium compounds led to an inhibition of cell proliferation as was evidenced by the depression of the proliferation rate index (PRI).