Rearrangements between irradiated chromosomes in three-species radiation hybrid cell lines revealed by two-color in situ hybridization (original) (raw)
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Biology of the Cell, 1993
A human-hamster hybrid cell line containing only the human X chromosome (GM06318B) was exposed to 6,000-7,000 rad of X-rays and fused with a mouse cell line (CL1D,TK-). Three radiation hybrids, LXKC40, LXKC50, and LXKC56, were selected among 39 independent clones containing human material. Twocolor in situ hybridization with total genomic DNA probes (cotl human DNA and hamster total genomic DNA) was used to analyse the irradiated chromosome rearrangements. With this three-species model system (human-hamster-mouse) and the chromosome painting process it was possible to determine the origin of each chromosomal fragment in metaphase and interphase. The resuits obtained indicate preferential rearrangement between irradiated human and hamster chromosomes. Whole, apparently intact hamster chromosomes were observed in all the mitoses. We suggest that these chromosomes could be neoformated from random fragments after irradiation. Hamster and human "minichromosomes" were also detected. While the integration of human material into the mouse genome was exceptional, the integration of hamster material into mouse chromosomes was more frequent. During interphase the irradiated chromosome domains were often at the periphery of the nucleus. Irradiated material protruded at the periphery of the nuclei. Micronuclei containing hamster material were detected in the vicinity of these protrusions.
Radiation Research, 2012
Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A L) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1985
The quantitative analysis of the chromosome rearrangements detected in 2128 R-banded metaphases, obtained from T-irradiated human lymphocytes after 48 to 96 h in culture is reported. Depending on the culture time, and possibly on the dose of radiation (from 1 to 3 Gy), the most frequent type of rearrangement was either dicentrics or reciprocal translocations. In first generation mitoses, the frequency of cells without rearrangement ranged from 0.66 to 0.18, and the mean number of rearranged chromosomes per cell from 0.79 to 3.28. The dose-response curve follows a quadratic function for dicentric aberration yields, but not for other rearrangements.
Human Genetics, 1982
Unsynchronized cells of an essentially diploid strain of female Chinese hamster cells derived from lung tissue (CHL) were laser-UV-microirradiated (2 = 257 nm) in the nucleus either at its central part or at its periphery. After 7-9 h postincubation with 0.5 mM caffeine, chromosome preparations were made in situ. Twenty-one and 29 metaphase spreads, respectively, with partial chromosome shattering (PCS) obtained after microirradiation at these two nuclear sites, were Q-banded and analyzed in detail. A positive correlation was observed between the frequency of damage of chromosomes and both their DNA content and length at metaphase. No significant difference was observed between the frequencies of damage obtained for individual chromosomes at either site of microirradiation. The frequency of joint damage of homologous chromosomes was low as compared to nonhomologous ones. Considerable variation was noted in different cells in the combinations of jointly shattered chromosomes. Evidence which justifies an interpretation of these data in terms of an interphase arrangement of chromosome territories is discussed. Our data strongly argue against somatic pairing as a regular event, and suggest a considerable variability of chromosome positions in different nuclei. However, present data do not exclude the possibility of certain non-random chromosomal arrangements in CHL-nuclei. The interphase chromosome distribution revealed by these experiments is compared with centromere-centromere, centromere-center and angle analyses of metaphase spreads and the relationship between interphase and metaphase arrangements of chromosomes is discussed.
Cytometry, 1990
Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably...
Chromosome content and ultrastructure of radiation-induced micronuclei
Mutagenesis, 1996
Unrepaired or misrepaired radiation damage in mammalian chromosomes can result in micronucleus formation at the first cell division. This represents loss of genomic information which may cause cell death. To improve our understanding of the mechanism of radiation-induced micronucleus formation, we characterized micronucleus ultrastructure and identified the origin of micronucleus DNA. Immunofluorescence microscopy showed that micronuclei were structurally similar to main nuclei since they contained nuclear lamins A and C and were encapsulated by a network of vimentin intermediate filaments. The contents of radiation-induced micronuclei were characterized using fluorescence in situ hybridization to probe for DNA originating from chromosomes 2, 7, 11 and 16. We postulated that if incorporation of DNA into micronuclei were random, then the probability of chromosomal DNA in micronuclei would be related to the target, i.e. chromosome size. Our results demonstrated that incorporation of DNA from smaller chromosomes (11 and 16) was not different from expected values but incorporation of DNA from the larger chromosomes (2 and 7) was significantly greater than expected. Not all chromosomes in the human genome, therefore, were equally susceptible to genomic loss by micronucleus encapsulation. In conclusion, radiationinduced micronuclei have similar structural characteristics to main nuclei, chromosome damage and/or repair after ionizing radiation may be non-random, and micronucleus formation may reflect this variability.
International Journal of Radiation Biology, 2002
new experimental data. Six decades ago the classical Purpose : It is generally accepted that chromosom e exchanges in 'breakage-and-reunion' model for the formation of irradiated cells are formed through interactions between separate chromosome exchanges (e.g. interchanges) was DNA double-strand breaks (DSB). Here we tested whether nondeveloped based on the assumption that two separate irradiated DNA participates in the formation of chromosom e breaks on diVerent chromosomes interact (Sax 1941, aberrations when complex DNA DSB are induced elsewhere in the nucleus. Lea 1946). Later, these lesions were identi ed as Materials and methods: Synchronized Chinese hamster cells con-DNA double-strand breaks (DSB) (Bryant 1984, taining an X chromosome with a late replicating q arm (X q Natarajan 1990). It was assumed that chromosome domain) were labelled with 1 2 5 I-iododeoxyuridine (1 2 5 IdUrd) in aberrations result from errors in the DSB repair a period of S-phase when the vast majority of the X q domain was not replicating. DNA damage from 1 2 5 I decay was accumu-process. The classical model is interpreted in terms lated at the G1/S border while the cells were stored in liquid of the non-homologous end-joining repair of DSB nitrogen. Decay of 125 I induced DSB in the immediate vicinity (Jeggo 1998). Twenty years ago the classical model of the 125 I atom. Chromosome aberrations involving what is was challenged by an alternative model postulating essentially the 125 I-free X q domain were scored at the rst mitosis that chromosome exchanges were formed through after cell thawing. As a positive control, cells were treated with 125 IdUrd at a later period in S-phase when the X q domain interaction of a single DSB with undamaged DNA replicates, yielding a labelled X q domain. (Chadwick and Leenhouts 1978). In their model the Results : The 1 25 I-free X q domain exhibited chromosome aberraauthors introduced homologous recombination tions (exchanges and fragments). The frequency of these aberrarepair to explain the involvement undamaged DNA tions was linearly dependent on the number of 1 2 5 I decays elsewhere in the cell nucleus. The eYciency of formation of in the repair of DSB (Resnick 1976). chromosom e aberrations by the 1 2 5 I-free X q domain was approxi-Experimental evidence has been provided for the mately half of that observed in the 1 2 5 I-labelled Xq domain. formation of chromosome aberrations in a way pre-Conclusions : The involvement of the 1 2 5 I-free X q domain in dicted by the classical model. This was demonstrated chromosom e aberrations suggests that DNA not damaged by the decay of incorporated 1 2 5 I can interact with damaged DNA, for DSB induced by c-radiation (Cornforth 1990) indicating the existence of an alternative pathway for the formaand bleomycin (Wang et al. 1997). Several studies tion of chromosome aberrations.