Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34^cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis.     

Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.     

Various chemical agents can induce premature chromosome condensation in Vicia faba

A number of chemical agents known to influence the key cell cycle regulatory factors were used to assess the requirements of hydroxyurea-treated root meristem cells of Vicia faba for premature condensation of chromosomes (PCC). These included caffeine and 2-aminopurine (inhibitors of ATM/ATR sensor kinases activated by DNA damage or stalled replication forks), inhibitors of protein kinases (staurosporine and wortmannin), inhibitors of protein phosphatases (sodium vanadate and calyculin A), and other compounds like 1,2-dioctyl-sn-glycerol, an activator of protein kinase C, 5-azacytidine, an inhibitor of DNA methyltransferase, dithiothreitol and N-etylmaleimide, capable to up- and down-regulate the activity of Cdc25 phosphatase. Cytological parameters used to evaluate quantitative aspects of PCC allowed us to discriminate various phenotypes of cells and, consistent with the extent of chromosomal fragmentation, to classify them as S- or G2-PCC. Two significant aspects relevant to the induction of premature mitosis in plants seem to emerge: one concerns the inverse relationship between the incidence of mitotic and PCC events, the other refers to the extent with which a variety of chemical agents may activate mechanisms that override the S-M replication checkpoint. 1,2-dioctyl-sn-glycerol, an activator of protein kinase C in animal cells proved extremely effective in stimulation of PCC, in spite of evident lack of molecular targets in plants.     

Effect of low-level, 60-Hz electromagnetic fields on human lymphoid cells: I. Mitotic rate and chromosome breakage in human peripheral lymphocytes

Dividing human peripheral lymphocytes from 10 normal adults (5 males and 5 females) were exposed in vitro to low level 60-Hz electromagnetic fields for 69 hours. The current density of the electrical field was 30 microA/cm2, while the magnetic field was either 1 or 2 gauss. The cytological endpoints measured were mitotic rate and chromosome breakage. No statistically significant differences, indicative of a field effect, were observed between treated and control cells whether exposed to an electric field, a magnetic field, or to various combinations of the two.     

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.     

Premature expression of cyclin B sensitizes human HT1080 cells to caffeine-induced premature mitosis

Eukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S-phase arrested hamster cells undergo mitosis-specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine-induced PCC and the level of cyclin B-associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S-phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S-phase arrested HT1080 cells. The transfected cell population displayed a 5-fold increase in the frequency of caffeine-induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope-tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically-induced uncoupling of mitosis from the completion of DNA replication. © 1995 Wiley-Liss, Inc.     

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01–0.5 mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

Elevated sister chromatid exchange frequencies in dividing human peripheral blood lymphocytes exposed to 50 Hz magnetic fields

The in vitro cytomolecular technique, sister chromatid exchange (SCE), was applied to test the clastogenic potentiality of extremely low frequency (ELF) electromagnetic fields (EMFs) on human peripheral blood lymphocytes (HPBLs). SCE frequencies were scored in dividing peripheral blood lymphocytes (PBLs) from six healthy male blood donors in two rounds of experiments, R1 and R2, to determine reproducibility. Lymphocyte cultures in the eight experiments conducted in each round were exposed to 50 Hz sinusoidal (continuous or pulsed) or square (continuous or pulsed) MFs at field strengths of 1 microT or 1 mT for 72 h. A significant increase in the number of SCEs/cell in the grouped experimental conditions compared to the controls was observed in both rounds. The highest SCE frequency in R1 was 10.03 for a square continuous field, and 10.39 for a square continuous field was the second highest frequency in R2. DNA crosslinking at the replication fork is proposed as a model which could explain the mechanistic link between ELF EMF exposure and increased SCE frequency.     

The cytogenetic effect of bleomycin on human peripheral lymphocytes in vitro and in vivo.

The cytogenetic effect of bleomycin (BLM) in human lymphocytes was studied after exposure to different doses during the G0 and G2 phases. BLM produced a marked specific effect on the cell cycle. The main aberration types after exposure in tg0 were dicentrics and deletions; and after exposure in G2, open chromatid breaks. A linear dose--response was calculated for all these aberration types as well as for the number of aberrant cells. In the G2 experiments, partially and totally pulverized cells also increased linearly with dose. The intercellular distributions of the most frequent aberration types after exposure in G0 and G2--the dicentrics and chromatid breaks, respectively--showed over-dispersion. These results show that the cytogenetic effect of BLM may be compared with that of densely ionizing irradiation. Preliminary results of chromosome analysis of three cancer patients in the course of BLM therapy showed effects similar to those in the G0 experiments.     

Alterations in protein kinase activity following exposure of cultured human lymphocytes to modulated microwave fields

Cultures of human tonsil lymphocytes were exposed in a Crawford cell to a 450-MHz field (peak envelope intensity 1.0 mW/cm2), sinusoidally amplitude modulated (depth 80%) at frequencies between 3 and 100 Hz for periods up to 60 min. The Crawford cell was housed in a temperature-controlled chamber (35 degrees C) and control cultures were placed in the same chamber. Activity of cAMP-dependent protein kinase relative to controls remained unaltered by fields modulated at 16 or 60 Hz with exposures of 15, 30, and 60 min. By contrast, total non-cAMP-dependent kinase activity fell to less than 50% of unexposed control levels after 15 and 30 min exposures, but, despite continuing field exposure, returned to control or preexposure levels by 45 and 60 min. A smaller reduction (20-25%) also occurred with 60-Hz modulation and was also restricted to exposure durations of 15 and 30 min. CW 450-MHz fields were without effect. Reduced enzyme activity occurred with 16-, 40-, and 60-Hz modulation frequencies, but not with 3-, 6-, 80-, or 100-Hz modulation. The specific identity of this kinase is unknown. This rapid but transient reduction in lymphocyte protein kinase activity restricted to modulation frequencies between 16 and 60 Hz and to less than 30 min exposure is consistent with "windowing" with respect to modulation frequency and exposure duration.     

Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/μm), and were then stimulated to obtain dividing cells. PBLs were treated with 100 nM calyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30 Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy⁻¹. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.     

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.     

Changes in the proliferation of human lymphocytes induced by several cytostatics and revealed by the premature chromosome condensation technique

Premature chromosome condensation was induced by cell fusion in stimulated human lymphocytes treated with different cytostatics. Changes in the proportion of the cell-cycle stages were investigated after 72 h of culture. Although it has been reported that some agents which induce severe DNA damage accumulate cells in G2, our results have shown some differences in the modes of action of the different tested chemicals. These variations could be due to several factors like mechanisms of action of the drugs, sensitivity of lymphocyte subpopulations to the cytostatics, inter- and intra-individual variability in the response of donors.     

Loss of chromosome 13 in cultured human vascular endothelial cells

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.     

Cell division,chromosomal aberration,and micronuclei formation in human peripheral blood lymphocytes

Age related cytotoxicity of stannic chloride was evaluated in human lymphocytes considering mitotic index (MI), damaged cell (DC), chromosome aberration (CA), and micronuclei formation (MNC) as endpoints. Significant elevation of DCs, CAs, MNCs, and reduction of MI were observed in all classified age groups compared to each control set. The mean frequencies of abnormalities show a statistically significant increase with subject's age. Linearity of the effect of age was common for both untreated and treated lymphocytes of both sexes.     

Formation of multilayer rosettes in phytohemagglutinin-stimulated lymphocytes: correlation with chromatin condensation conformation in premature chromosome condensation

We found that the formation of multilayer rosettes by transformed human blood lymphocytes after phytohemagglutinin (PHA) stimulation is correlated with conformational changes of the chromatin as seen by premature chromosome condensation (PCC). The frequency distribution of grades of PCC and multilayer rosette formation suggests that changes in chromatin are a prerequisite for rosette formation. Rosette formation was most pronounced for 24-h and 48-h cultures. Chromatin decondensation and rosette formation showed identical patterns. The possibility that multilayer rosette formation is directly dependent on conformational changes of chromatin is discussed.     

Genotoxic evaluation of the antimalarial drug,fansidar, in cultured human lymphocytes

Fansidar (pyrimethamine-sulfadoxine) has been used extensively worldwide for the treatment of chloroquine resistant Plasmodium falciparum malaria, toxoplasmosis and Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Because of the wide usage of pyrimethamine-sulfadoxine in developing countries and the lake of information from open literature and reports from manufacturers about the genotoxicity of such antimalarial drug, the present work was suggested. The possible genetic toxicity of fansidar has been evaluated in human peripheral blood lymphocyte cultures. The frequencies of sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests covering a wide range of induced genetic damage as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up by using blood samples from two healthy donors and the treatment was done using different fansidar concentrations ranging from 1:20 to 10:200 g/ml. From our results, it appears that this drug is able to induce moderate genotoxic effects, as revealed by the increases found in SCE and MN frequencies in cultures from the two donors at the two highest concentrations tested (5:100 and 10:200 g/ml). In addition, cyotoxic/cytostatic effects of fansidar were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI). Our findings suggest that the use of this drug should be restricted to situations where other antimalarial drugs cannot be used. The drug should never be given to pregnant women.     

Nuclear and territorial topography of chromosome telomeres in human lymphocytes

Nuclear and territorial positioning of p- and q-telomeres and centromeres of chromosomes 3, 8, 9, 13, and 19 were studied by repeated fluorescence in situ hybridization, high-resolution cytometry, and three-dimensional image analysis in human blood lymphocytes before and after stimulation. Telomeres were found on the opposite side of the territories as compared with the centromeres for all chromosome territories investigated. Mutual distances between telomeres of submetacentric chromosomes were very short, usually shorter than centromere-to-telomere distances, which means that the chromosome territory is nonrandomly folded. Telomeres are, on average, much nearer to the center of the cell nucleus than centromeres; q-telomeres were found, on average, more centrally localized as compared with p-telomeres. Consequently, we directly showed that chromosome territories in the cell nucleus are (1) polar and (2) partially oriented in cell nuclei. The distributions of genetic elements relative to chromosome territories (territorial distributions) can be either narrower or broader than their nuclear distributions, which reflects the degree of adhesion of an element to the territory or to the nucleus. We found no tethering of heterologous telomeres of chromosomes 8, 9, and 19. In contrast, both pairs of homologous telomeres of chromosome 19 (but not in other chromosomes) are tethered (associated) very frequently.     

Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95-105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103-1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.