Chromosome damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin

The clastogenic effect of furocoumarins psoralen and angelicin in the presence of near-UV (320-380 nm) differs greatly, as do their modes of interaction with DNA. Psoralen, which requires only one-fifth as much light energy to produce the same lethal effect as angelicin at equimolar concentrations, is able to cross-link DNA whereas angelicin cannot. The frequency of micronuclei which arise from chromosomal fragments shows the same differential effect as lethality. Indeed aberrations account for much or all of the lethality observed. Metaphase analysis at comparable aberration frequencies revealed that angelicin and psoralen both induce chromatid deletions and a wide spectrum of chromatid exchanges. These data show that both cross-links and monoadducts to the DNA can result in chromosomal aberrations. The relative contributions of cross-links and monoadducts to chromosomal aberrations still remain to be determined. It is noteworthy that extensive chromosomal damage is induced in mammalian cells by the combination of psoralen and near-UV, a treatment which is currently widely used in the therapy of psoriasis.     

Sensitivity of bone marrow cells damaged by antitumor drugs to cytotoxic action of effectors of a normal spleen

Different cytogenetic effects in bone marrow cells induced by antitumor drugs with different mechanisms of action was studied. The treatment of adriablastine causes the appearance of chromatid deletion, vinblastine-polyploid cells, cyclophosphamide-chromosomal and chromatid aberration in mice. It was shown that bone marrow cells with cytogenetic damage have altered susceptibility to normal spleen cells-effectors cytotoxic action.     

Post-treatments with sodium arsenite during G2 enhance the frequency of chromosomal aberrations induced by S-dependent clastogens

Treatment with sodium arsenite during the G2 phase potentiated the chromatid breaks and chromatid exchanges induced by ultraviolet light or 4-nitroquinoline 1-oxide but not those induced by methyl methanesulfonate, ethyl methanesulfonate, mitomycin C or cisplatin in Chinese hamster ovary cells. A comparison was made between the effects of treatment during G2 with sodium arsenite, cytosine-β- -arabinofuranoside, aphidicolin, hydroxyurea, caffeine, 3-aminobenzamide and novobiocin on the frequency of chromosomal aberrations induced by the above-mentioned S-dependent clastogens. It was found that the effects varied considerably, both quantitatively and qlalitatively. However, potentiation was more often observed in the chromosomal aberrations induced by ultraviolet light and 4-nitroquinoline 1-oxide than by other S-dependent clastogens, and the frequency of chromatid exchanges was potentiated only in cells pretreated with ultraviolet light or 4-nitroquinoline 1-oxide. Furthermore, for all of the S-dependent clastogens studied, treatment with cytosine-β- -arabinofuranoside during the G2 phase potentiated the frequency of chromatid breaks but not the frequency of chromatid exchanges.     

Comparative investigations on cytogenetic effects of X-irradiation on the germinal epithelium of male mice and Chinese hamsters

Summary In one short-term-experiment and one long-term-experiment spermatogonia of mice and Chinese hamsters were compared for their sensitivity of X-ray induced chromosome aberrations.Short-term-experiment: Six hours after varying doses of X-rays the spermatogonia of both species were analysed and the number of induced chromatid breaks determined. At the dose range from 25–125 R the number of induced chromatid breaks per cell per roentgen is 0.01 in mice. In Chinese hamsters this value is 0.0072.The frequencies of chromatid breaks were studied in both species after a single dose of 100 R until 48 h p.i. The frequency in mice decreased more slowly than in hamster spermatogonia. After 12 h p.i. the ratio breaks in mice cells: breaks in hamster cells was 3.5:1, after 24 h this ratio was 5.2:1 after 48 h both frequencies were on the same level.Long-term-experiment: Analysis of spermatogonia and primary spermatocytes has been done 5 weeks after irradiation of the mice and 2, and 4 months after irradiation of the Chinese hamsters. The number of observed reciprocal translocations turned out to be higher in spermatogonial mitoses than in diakinesis-metaphases I in each animal.The conclusion is drawn for mice that a selection against abnormal cells is taking place already during pre-meiosis. In hamster pre-meiosis, the results are only indicative for a similar effect.These investigations were sponsored by the Deutsche Forschungsgemeinschaft within the SFB 35 (Klinishe Genetik).     

First glance at the plant APC/C,a highly conserved ubiquitin-protein ligase

The anaphase-promoting complex or cyclosome (APC/C) is a cell-cycle-regulated ubiquitin-protein ligase that has been extensively studied in both fungal and animal cells. Many APC/C protein targets have been identified, and their sequential degradation during the cell cycle is essential for chromatid separation and mitotic exit. APC/C-dependent ubiquitylation of proteins not involved in cell-cycle progression has also been documented in animal cells. By contrast, the plant APC/C's structure and functions remained unexplored until recently. Here, we discuss recent developments in this field and explore the Arabidopsis genome sequence to identify plant APC/C components. Details of the APC/C ubiquitylation pathway in Arabidopsis are also available on a website that will be regularly updated.     

Induction of chromatid breaks by carbon K-shell ultrasoft X rays

Chromatid breaks have previously been shown to be induced in G2-phase cells after exposure to ionizing radiation (X and gamma rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for 60Co gamma rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.     

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.     

Failure of centromere separation leads to formation of diplochromosomes in next mitosis in okadaic acid treated HeLa cells.

High concentrations of okadaic acid, sufficient to inhibit phosphatase 1 and 2A activities, induces formation of diplochromosomes in HeLa cells. It has been shown that this is due to a failure of sister chromatid separation in earlier mitosis in the presence of okadaic acid in the medium and not due to bypassing of mitosis (endoreduplication). Moreover, it has been demonstrated that the sister chromatid adherence does not depend on any under-replicated chromatin segment shared by the sister chromatids which might happen in okadaic acid induced premature mitosis, but due to the failure of the centromeres to separate at metaphase - anaphase transition. The role of phophatase 1 in sister chromatid separation has been discussed     

A selection system for unequal crossing-over: Plasmid construction and initial studies in Escherichia coli

Unequal crossing-over is involved in genetic duplication and deletion in such diverse genetic systems as Drosophila, bacteria, and animal viruses. It is proposed to be involved in the form of unequal sister chromatid exchange in gene amplification in cultured animal cells and during carcinogenesis. Studies of the process of unequal crossing-over have been hampered by the lack of genetic systems allowing specific selection for cells that have undergone such unequal crossing-over. We report here on the construction of plasmids designed to provide specific selection of unequal crossing-over. One such plasmid was studied in Escherichia coli. We show that kanamycin resistance is generated, as predicted, by the expected unequal crossover event.     

Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety''s collaborative study on plant systems for the detection of environmental mutagens

A review has been made of the four compounds (maleic hydrazide, methyl nitrosourea, sodium azide, azidoglycerol) tested in the International Program on Chemical Safety's collaborative study systems. Maleic hydrazide (MH) is a weak cytotoxic/mutagenic chemical in mammalian tissues and is classified as a class 4 chemical. In contrast, with few exceptions such as Arabidopsis, MH is a potent mutagen/clastogen in plant systems. The difference in its response between plant and animal tissue is likely due to differences in the way MH is metabolized. MH appears to be noncarcinogenic and has been given a negative NCI/NTP carcinogen rating.

Methyl nitrosourea (MNU) is a toxic, mutagenic, radiomimetic, carcinogenic, and teratogenic chemical. It has been shown to be a mutagen in bacteria, fungi, Drosophila, higher plants, and animal cells both in vitro and in vivo. MNU is a clastogen in both animal and human cell cultures, plant root tips and cell cultures inducing both chromosomes and chromatid aberrations as well as sister-chromatid exchanges. Carcinogenicity has been confirmed in numerous studies and involves the nervous system, intestine, kidney, stomach, bladder and uterus, in the rat, mouse, and hamster. MNU produces stage-specific teratogenic effects and also interferes with embryonic development. The experimental evidence that strongly indicates the mutagenic effects of MNU underlines the possible hazard of this compound to human beings. The experimental evidence for the stringent handling of this compound is clear.

Sodium azide (NaN₃) is cytotoxic in several animal and plant systems and functions by inhibiting protein synthesis and replicative DNA synthesis at low dosages. It is mutagenic in bacteria, higher plants and human cells and has been used as a positive control in some systems. In general, tests for clastogenicity have been negative or weakly positive. No evidence of carcinogenicity has been reported in a 2-year study seeking carcinogenic activity in male and female rats. Its advantages in comparison to other efficient mutagens are claimed to be a high production of gene mutations accompanied by a low frequency of chromosomal rearrangements and safer handling because of its nonclastogenic and noncarcinogenic action on humans.     

Chromosomenaberrationen,hervorgerufen durch Scopolaminum hydrobromatum

Final concentration of 1% Scopolaminum hydrobromatum induced chromatid aberrations, but not reunion figures in HeLa cells. Duration of treatment was 5 hs, begin of treatment 22 resp. 46 hs before harvesting. In contrast to these findings no aberration was induced by Scop. hydr. in human leukocytes and BSC-K cells. It is concluded that mere findings in HeLa cells do not prove mutageneity of a chemical compound in man.     

Distribution of spontaneous and 8-methoxypsoralen-induced sister chromatid exchanges along the length of the 1st chromosome of Chinese hamster cells

Distributions of spontaneous, induced by monoadducts and induced by crosslinks sister chromatid exchanges (SCE) along the first chromosome of Chinese hamster cells are nonrandom. All experimental distributions have low frequency of SCE in centromeric and telomeric regions. It can be explained by specific structural organization of the chromosome. However, there are some differences between experimental distributions. Distribution of SCE induced by crosslinks differs from that of spontaneous SCE. Distribution of SCE induced by monoadducts, unlike other distributions, has an increased frequency of exchanges in the q11 region. This region contains several narrow closely disposed G+ bands. It is possible that monoadducts lead to increasing SCE frequency on G+-G- junctions. Distribution of SCE induced by crosslinks resembles random distribution, except centromeric and telomeric regions. These results lead to conclusion that the mechanisms of formation of spontaneous, induced by monoadducts and induced by crosslinks SCE differ from each other.     

Variations in the radiosensitivity of oocyte chromosomes during meiosis in Drosophila melanogaster

The synaptic stages of meiosis in Drosophila melanogaster females are very resistant to the induction of dominant lethal mutations by ionizing radiation. It is assumed that dominant lethals result from interstitial chromatid deletions, and that almost all potential chromatid breaks are repaired in synaptic cells. The type of dose response curve shown by oocytes at later developmental stages is a function of the degree of chromatid coiling and the presence or absence of an investing nuclear envelope.     

Competence for assembly of sister chromatid cores is progressively acquired during S phase in mammalian cells.

Condensed sister chromatids possess a protein scaffold or axial core to which loops of chromatin are attached. The sister cores are believed to be dynamic frameworks that function in the organization and condensation of chromatids. Chromosome structural proteins are implicated in the establishment of sister chromatid cohesion and in the maintenance of epigenetic phenomena. Both processes of templating are tightly linked to DNA replication itself. It is a question whether the structural basis of sister chromatid cores is templated during S phase. As cells proceed through the cell cycle, chromatid cores undergo changes in their protein composition. Cytologically, cores are first visualized at the start of prometaphase. Still, core assembly can be induced in G1 and G2 when interphase cells are fused with mitotic cells. In this study, we asked if chromatid cores are similarly able to assemble in S-phase cells. We find that the ability to assemble cores is transiently lost during local replication, then regained in chromosome regions shortly after they have been replicated. We propose that core templating occurs coincident with DNA replication and that the competence for the assembly of the sister chromatid cores is acquired shortly after passage of replication forks.     

Long-term effects of irradiation in the progeny of mammalian cells

It is shown that gamma-irradiation has remote consequences for mammalian cells cultivated in vitro. Many generations in the progeny of cells surviving acute and chronic irradiation at high and low doses are characterized by a number of abnormalities, including delayed cell death, the formation of micronuclei and giant cells, an increased frequency of sister chromatid exchanges, a reduced potential for repair, the loss of adaptive response, and increased radiosensitivity. These phenomena are regarded as manifestations of genomic instability induced by ionizing radiation.     

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

Background and Aims Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Methods Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Key Results Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 μg mL(-1) MCY-LR, accelerated cell cycle at 10 μg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. Conclusions MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.     

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.     

Inhibitory effect of rat immunization with tularemia vaccine on the in vivo clastogenicity of 4 anthracycline antibiotics

We studied the in vivo effect of rat immunization with tularemia live vaccine (TLV) on chromosomal aberrations (CA) induced in bone marrow cells by 4 anthracycline antibiotics. CA induced by adriamycin (ADR) and 4'-epiadriamycin (EADR) in rat bone marrow cells consisted mainly of chromatid breaks (approximately 90%), whereas lesions induced by aclacur (AC) and aclarubicin (ACR) consisted only of chromatid breaks. Preliminary cutaneous immunization of rats with TLV revealed significant suppression of CA induced by all 4 antibiotics. The present and previous results suggest that TLV may be a potent anticlastogenic factor.     

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used.