Chromatin mobility is increased at sites of DNA double-strand breaks.

DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the formation of chromosome rearrangements. Therefore, we analyzed the mobility of chromatin domains containing DSBs, marked by the fluorescently tagged DSB marker 53BP1, in living mammalian cells and compared it with the mobility of undamaged chromatin on a time-scale relevant for DSB repair. We found that chromatin domains containing DSBs are substantially more mobile than intact chromatin, and are capable of roaming a more than twofold larger area of the cell nucleus. Moreover, this increased DSB mobility, but not the mobility of undamaged chromatin, can be reduced by agents that affect higher-order chromatin organization.

Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins.

For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains.

Determination of the DNA content of a six-gene amplicon in the multidrug-resistant Chinese hamster cell line CHRB3 by flow karyotyping.

Acquired multidrug resistance in cultured cells is often due to amplification of pgp genes, which gives rise to overproduction of P-glycoproteins that confer resistance by reducing the intracellular drug accumulation. The size of these amplicons varies between multidrug resistant cell lines and is often much larger than the gene selected for. Amplicons of the multidrug resistant Chinese hamster ovary cell line CHRC5 and its progenitor CHRB3, for example, span at least five different genes besides the pgp genes. Linkage of these gene classes with pgp had been shown by in situ hybridization and by long distance mapping using pulsed field gradient gel electrophoresis. Because the boundaries of the larger amplicons could not be determined, the size of such amplicons is not yet known, even though the six genes span at least 1500 kilobases. In the present study we have determined the amplicon size in B3+, a subclone of CHRB3 with a homogeneously staining region on chromosome 7q+ that harbors the amplified genes. We estimated the amplicon size in revertant clones by correlating the decreased DNA content of the 7q+ homogeneously staining region with the number of lost amplicons. The reduction of the homogeneously staining region DNA that accompanied reversion was determined by flow cytometry of propidium iodide stained chromosome suspensions of the various cell lines. We found that about 107 megabase pairs were lost together with 11-24 P-glycoprotein gene copies, suggesting that the mean amplicon size is in the range of 4.5-10.1 megabase pairs.

A new immunocytochemical technique for ultrastructural analysis of DNA replication in proliferating cells after application of two halogenated deoxyuridines.

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

Micronuclei expression in tumors as a test for radiation sensitivity.

Dose-effect curves were determined for the frequency of micronuclei and impairment of cell clonogenicity from two types of tumours of different sensitivity irradiated in situ. Micronucleated cells were measured 24, 48 and 72 h after treatment. The quantitative relationships between cell reproductive death and the induction of micronuclei are the same for both tumours.

X-ray- and neutron-induced chromosome damage detected by flow cytometry compared to cell lethality and chromosome structural changes.

V79 Chinese hamster cells were irradiated in G0 phase with 200 kV X rays or 14 MeV neutrons, and dose-response curves were determined for three end points: chromosome damage detected by flow cytometric analysis of chromosomes isolated from metaphase cells in irradiated cultures; loss of clonogenic capacity; and induction of dicentric, tricentric, and ring chromosomes. The changes observed in the flow karyotypes from irradiated cultures were quantitatively evaluated by computer analysis. Estimates of the frequencies of chromosome lesions were derived from an analysis of the flow cytometric measurements by means of a comparison with model calculations simulating the effect of chromosome changes on flow karyotypes. The results indicate that lesions assayed by flow cytometry occur three times more frequently than lethal lesions, while the chromosomal structural changes detected by microscopic analysis were about 10 times less frequent than the lesions detected by flow cytometry. Dose-response curves for X rays and neutrons show that cell reproductive death and changes in flow karyotypes result from damage, induced with a similar relative biological effectiveness. Dose-effect relations derived from changes in flow karyotypes, which can be obtained within 24 h after irradiation, might be of value as a predictive test for the sensitivity of cells for loss of clonogenic capacity.

Measurement of co-localization of objects in dual-colour confocal images.

A method to measure the degree of co-localization of objects in confocal dual-colour images has been developed. This image analysis produced two coefficients that represent the fraction of co-localizing objects in each component of a dual-channel image. The generation of test objects with a Gaussian intensity distribution, at well-defined positions in both components of dual-channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co-localization coefficients were determined before degrading the image with background, cross-talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non-specific binding and cross-reactivity of fluorescent probes, optical cross-talk and photon noise. The degraded images were restored by filtering and cross-talk correction. The co-localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co-localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.

Construction of mouse chromosome-specific DNA libraries and their use for the detection of X-ray-induced aberrations.

We describe here the development of mouse chromosome-specific DNA libraries and their use in the detection of radiation-induced chromosome aberrations by fluorescence in situ hybridization. Large metacentric chromosomes, resulting from a translocation involving chromosomes 1, 11 and 13, were flow-sorted. Using a slit-scan technique for morphometric analysis, metacentric chromosomes were separated from normal acrocentric chromosomes and their aggregates. DNA from the metacentric chromosomes was amplified by PCR using the linker/adaptor method. In this pilot study, mouse was whole-body irradiated with 1, 2 and 3 Gy and aberrations were scored in metaphase spreads of splenocytes cultured in vitro. The results indicate that directly after radiation exposure, stable and unstable aberrations are induced at about equal frequencies in the splenocytes. The availability of chromosome-specific probes for mouse may prove very useful when analysing the behaviour of stable aberrations, as well as the testing of many suspected mutagenic carcinogens and aneugens in vivo for induction of chromosomal translocations and non-disjunction, respectively.

Induction and detection of bystander effects after combined treatment of cells with 5-bromo-2'-deoxyurine, Hoechst 33 258 and ultraviolet A light.

PURPOSE: A combined treatment of cells with 5-bromo-2'-deoxyurine (BrdU), Hoechst 33 258 and ultraviolet A (UVA) light was used to introduce chromosomal aberrations in cells for the study of bystander effects in non-labelled cells. MATERIALS AND METHODS: Mixtures of BrdU-labelled and non-labelled Chinese hamster cells (V79) in S phase were exposed to Hoechst 33 258 and/or UVA light. Metaphase cells were collected and analysed for chromosomal aberrations by Giemsa staining. BrdU immunostaining was performed to verify BrdU incorporation in the cells. RESULTS: Combined treatment with BrdU, Hoechst dye and UVA light induced reduced cell survival and increased chromosomal aberrations, whereas treatment with Hoechst 33 258 and/or UVA light had no effect on cells. Elevated frequencies of chromosomal aberrations were found in non-labelled cells mixed with BrdU-labelled cells and exposed to Hoechst dye and UVA light, suggesting the induction of bystander effects by damaged BrdU-labelled cells. These bystander clastogenic effects were also observed in non-labelled cells mixed with dying cells, indicating a contribution of dying cells in the induction of the bystander effects. CONCLUSIONS: The combined treatment with BrdU, Hoechst 33 258 and UVA light is a valid method for the study of bystander effects as it enables both induction of DNA damage and discrimination of targeted cells and bystander cells.

Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus.

PURPOSE: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. MATERIALS AND METHODS: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain. RESULTS: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain. CONCLUSIONS: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations.

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.

Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm.

The most effective method to control the sex of offspring is by separating X- from Y-bearing sperm on the basis of their DNA content. Sperm can be stained with Hoechst 33342 and efficiently sexed using a flow cytometer/cell sorter. However, applying this established assay to cryopreserved bovine sperm presents specific problems, such as broad fluorescence distributions without a distinct X- and Y-peak. Our results indicate that these problems are mainly caused by the large amount of dead sperm normally present in a thawed sperm population. We showed that Percoll quenches the fluorescence of chromatin stained with Hoechst 33342 and that this quenching can be applied to reduce the fluorescence of dead sperm. We used this finding to exclude the dead sperm from the sorting window and thus obtained narrower fluorescence distributions and sorted X- and Y-bearing sperm populations containing up to 85 to 92% viable sperm. The viability of the sorted sperm was monitored by propidium iodide exclusion.

Slit scan flow cytometry of isolated chromosomes following fluorescence hybridization: an approach of online screening for specific chromosomes and chromosome translocations.

The recently developed methods of non radioactive in situ hybridization of chromosomes offer new aspects for chromosome analysis. Fluorescent labelling of hybridized chromosomes or chromosomal subregions allows to facilitate considerably the detection of specific chromosomal abnormalities. For many biomedical applications (e.g. biological dosimetry in the low dose range), a fast scoring for aberrations (e.g. dicentrics or translocations) in required. Here, we present an approach depending on fluorescence in situ hybridization of isolated suspension chromosomes that indicates the feasibility of a rapid screening for specific chromosomes or translocations by slit scan flow cytometry. Chromosomes of a Chinese hamster x human hybrid cell line were hybridized in suspension with biotinylated human genomic DNA. This DNA was decorated with FITC by a double antibody system against biotin. For flow cytometry the chromosomes were stabilized with ethanol and counterstained with DAPI or propidium iodide (PI). An experimental data set of several hundred double profiles was obtained by two parameter slit scan flow cytometry and evaluated automatically. The evaluation algorithm developed allowed a classification of chromosomes according to the number of centromeres and their chromosomal positions in less than 1 msec per individual profile. Approximately 20% of the measured DAPI profiles showed a bimodal distribution with a significant centromeric dip indicating a "normal" chromosomal morphology and a correct alignment in the flow system. In many cases, profiles of a "normal" bimodal fluorescence distribution of the DNA stain (DAPI, PI) were correlated with a "normal" FITC profile. Due to their centromeric indices these profiles agreed well to the expected human chromosomes of the cell line. In some cases of "normal" DAPI (PI) profiles, "aberrant" FITC profiles were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Instrument for real-time pulse-shape analysis of slit-scan flow cytometry signals.

An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse-shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse-profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 microseconds from the onset of the pulse-profile and particles with a specified morphology of interest were selected for on-line registration of their profiles as digitized pulse-shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.

Comparison of radiation sensitivity for three cell lines as measured by the cloning assay and the micro-nucleus test.

The correlation between cell killing and the induction of micro-nuclei was studied for three cell lines after treatment with gamma radiation to investigate whether the frequency of micro-nucleated cells can be used to determine the radiation sensitivity of a cell type. R1 rat rhabdomyosarcoma cells showed a higher sensitivity for the induction of proliferative death than RUC rat ureter carcinoma cells and V79 Chinese hamster cells which had a similar radiation sensitivity. The frequencies of micro-nucleated cells were measured at 48 hours after the treatment. It was determined by time-lapse cinematography that almost all the cells in the treated cultures had divided at that time. Our results indicate that for these cell lines the correlation between the effectiveness for cell killing and the induction of micro-nuclei was the same, within the experimental errors.

Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei.

Fluorescence in situ hybridization has demonstrated that chromosomes form individual territories in interphase nuclei. However, this technique is not suitable to determine whether territories are mutually exclusive or interwoven. This notion, however, is essential for understanding functional organizations in the cell nucleus. Here, we analyze boundary areas of individual chromosomes during interphase using a sensitive method based on replication labeling and immunocytochemistry. Thymidine analogues IdUrd and CldUrd were incorporated during S-phase into DNA of Chinese Hamster fibroblasts. Cells labeled with IdUrd were fused with cells labeled with CldUrd. Fused nuclei contained both IdUrd or CldUrd labeled chromosomes. Alternatively, the two labels were incorporated sequentially during successive S-phases and segregated to separate chromosomes by culturing the cells one more cell cycle. Metaphase spreads showed IdUrd-, CldUrd- and unlabeled chromosomes. Some chromatids were divided sharply in differently labeled subdomains by sister chromatid exchanges. With both methods, confocal imaging of interphase nuclei revealed labeled chromosomal domains containing fiber-like structures and unlabeled areas. At various sites, fiber-like structures were embedded in other territories. Even so, essentially no overlap between chromosome territories or between subdomains within a chromosome was observed. These observations indicate that chromosome territories and chromosomal subdomains in G(1)-phase are mutually exclusive at the resolution of the light microscope.

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.

Nuclear and cell division in Bacillus subtilis. Antibiotic-induced morphological changes.

Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division. Septation is then only partially uncoupled from the normal division cycle. Observations on location and development of mesosomes in the presence of the antibiotics, made in three-dimensional cell reconstructions, suggest that the mesosome plays a role in the normal coordination between nuclear and cell division, and may explain the partial independence between these two processes in B. subtilis.

Effect of aniosmotic media on the volume of the T-lymphocyte nucleus.

Time-resolved measurements of the nuclear volume response of human peripheral T-lymphocytes, under aniosmotic conditions, are presented. In the experiments slit scanning FlowCytometry methods were used. We propose an extension to the standard solid viscoelastic model to interpret the observed dynamical behavior of the nucleus. It is shown that experimental and theoretical evidence indicates a passive nuclear response merely induced by a mechanical link (i.e., the cytoskeleton) between the cell membrane and the nuclear envelope. Implications of this work to the field of cellular mechanics and cytoskeletal rheology are surveyed.

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping.

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.