Premature chromosome condensation induced by electrofusion

Premature chromosome condensation of G1, G2, and S-phase chromosomes has been achieved by the use of electrofusion in the fusion of Chinese hamster ovary (CHO) cells and HeLa cells and CHO cells with human leukocytes. Very high yields of heterokaryons, of over 80%, as well as elimination of adverse effects of chemical and viral fusion agents, facilitated induction of premature chromosome condensation of high quality.     

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.     

Chromatin condensation dynamics and implications of induced premature chromosome condensation

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing ‘prophasing’ of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.     

The kinetics of postirradiation chromatin restitution as revealed by chromosome aberrations detected by premature chromosome condensation and fluorescence in situ hybridization

In a study of X-ray-induced chromosome aberrations in human G(0) lymphocytes irradiated with 4 Gy using premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH), the time-dependent pattern of chromosome fragments and interchromosomal exchanges involving chromosome 4 was recorded after postirradiation incubation times varying from 0.5 to 46.5 h. Unattached acentric fragments and incomplete interchromosomal exchanges have high initial yields, followed by an exponential decrease, while complete interchromosomal exchanges have almost zero initial yield with a subsequent increase in their number. Plateau values of all yields are reached after about 25 h. This temporal variation of aberration yields can consistently be explained by the competition of disruptive PCC stress with the progress of postirradiation structural restitution at the sites of radiation-induced chromatin instabilities. Details of the temporal pattern of incomplete exchanges reflect the different kinetics of the alpha and beta components of the yield of aberrations. The observed large difference between late-PCC and metaphase yields of unattached acentric fragments and the almost perfect conversion from incomplete prematurely condensed chromosomes into complete metaphase exchanges are explained by a difference in the magnitude of chromosome condensation stress between PCC and mitotic conditions. Chromatin sites prone to fragmentation and incompleteness under conditions of PCC can therefore persist as genetic instabilities hidden during mitosis.     

Sister chromatid exchange and chromosome aberrations induced by curcumin and tartrazine on mammalian cells in vivo

Sister chromatid exchanges (SCEs) and chromosomal aberrations induced by curcumin (a natural dye) and tartrazine (a synthetic dye) were studied on bone marrow cells of mice and rats following acute and chronic exposure via the diet. Except for two low concentrations in the curcumin and one low concentration in the tartrazine treated series a significant increase in SCEs was observed in all the concentrations of the two dyes tested. Except for two high concentrations during the 9 months treatment no significant increase in chromosomal aberrations was observed in the curcumin treated series, whereas tartrazine showed a significant increase in chromosomal aberrations in some of the higher concentrations in all the series tested. The results indicate that tartrazine is more clastogenic than curcumin.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Analysis of chromosome aberrations in human peripheral lymphocytes induced by 5.4 keV x-rays

Irradiation of human lymphocytes by x-rays has been seen, in past studies, to produce increasing frequencies of chromosome aberrations at lower x-ray energies. However, in one earlier irradiation experiment with chromium x-rays, the relative biological effectiveness (RBE) did not appear to be larger than that of hard x-rays, especially at higher doses. A possible reason for this unexpected result may have been the irradiation and culture conditions. We have, therefore, in the present study used a technique that has been developed in our laboratory to ensure uniformity of irradiation within lymphocytes and to avoid artefacts due to the cell cycle kinetics. Monolayers of 3-h-stimulated lymphocytes were exposed to 5.4 keV x-rays. A linear-quadratic dose-response was found for dicentrics. The comparison to an earlier finding with 220 kV x-rays shows the expected result of the RBE of the 5.4 keV x-rays to be above that of 220 kV x-rays. The intercellular distribution of dicentrics did not differ significantly from a Poisson distribution. Received: 17 January 1997 / Accepted in revised form: 17 July 1997     

Complex chromosome aberrations in continuous mammalian cell lines

Complex chromosome aberrations (CCA) are described, occurring spontaneously in low frequency, in numerous mammalian cell lines. These aberrations appear similar to those reported in leukocyte cultures of some Yanamama Indians. In some cell lines the frequency of CCA is increased by the administration of cytochalasin B (CB) a drug which prevents cytoplasmic division. The frequency of CCA may also be increased by the protease inhibitor tosyl lysyl chloro methyl ketone (TLCK). TLCK may also produce binucleate cells but unlike CB does not result in high degrees of multinucleation. In one cell line, 3T12, the simultaneous administration of CB and TLCK resulted in high frequencies of CCA. Thus the induction of CCA in cell culture is reproducible. However the etiology of CCA remains unknown.     

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.     

Analysis of bleomycin- and cytosine arabinoside-induced chromosome aberrations involving chromosomes 1 and 4 by painting FISH

The genomic frequency of chromosomal aberrations obtained by chromosome painting is usually extrapolated from the observed frequency of aberrations by correcting for the DNA content of the labelled chromosomes. This extrapolation is based upon the assumption of random distribution of breakpoints from which aberrations are generated. However, the validity of this assumption has been widely questioned. While extensive investigations have been performed with ionizing radiation as chromosome breaking agent, little efforts have been done with chemical clastogens. In order to investigate interchromosomal differences in chemically-induced chromosome damage, we have used multicolour chromosome painting to analyse bleomycin-induced aberrations involving chromosomes 1 and 4, two chromosomes that differ in gene density. In addition, we have measured the effect of cytosine arabinoside upon the repair of bleomycin-induced DNA damage in chromosomes 1 and 4. Our results show that these chromosomes are equally sensitive to the clastogenic effect of bleomycin with a similar linear dose-effect relationship. However, the high gene density chromosome 1 appeared to be more sensitive to repair inhibition by Ara-C than chromosome 4. This enhanced sensitivity to repair inhibition in chromosome 1 could be mediated by preferential repair of open chromatin and actively transcribed regions.     

Effects of inhibitors of DNA,RNA and protein synthesis on frequencies and types of premature chromosome condensation from X-ray induced micronuclei

Summary Cells containing X-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interprete the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells.     

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.     

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.     

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.     

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 Bl?cher 1982) and chromosome deletions, but also from an increase in the frequency of misrepair of d.s.b. leading to exchange aberrations.     

L-Carnitine protects mammalian cells from chromosome aberrations but not from inhibition of cell proliferation induced by hydrogen peroxide

L-carnitine is a small essential molecule indispensable in fatty acid metabolism and required in several biological pathways regulating cellular homeostasis. Despite considerable progress in understanding of L-carnitine biosynthesis and metabolism, very few data are reported concerning the protective role of L-carnitine from oxidative stress-induced DNA damage that is known to be a factor in cell transformation and tumourigenesis. In order to detect the capability of L-carnitine to protect mammalian cells from oxidative stress-induced chromosomal effects, we analysed chromosome aberrations in mitotic CHO cells, which represent an appropriate cytogenetic model to study compounds that enhance cell protection against externally induced DNA damage. We chose H2O2 as an inducer of oxidative stress. Our results demonstrate for the first time a marked and reproducible reduction of H2O2-induced chromosome damage involving an L-carnitine-mediated capacity to buffer intracellular formation of reactive oxygen species (ROS). Furthermore, by studying the mitotic index and cell cycle progression, we also demonstrated that this protective effect is highly specific, since L-carnitine itself was not able to prevent the inhibition of cell growth caused by H2O2.