Showing papers on "Chromosome breakage published in 1991"

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

Abstract: A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

Micronuclei and other nuclear anomalies in buccal smears: a field test in snuff users.

Abstract: A revised protocol for the exfoliated cell micronucleus assay was field-tested in a population exposed to a genotoxic agent, snuff, at levels associated with a significant increase in cancer risk. The standard assay involves examination of epithelial smears to determine the prevalence of micronucleated cells, an indication of chromosome breakage or mitotic interference. The assay was revised to increase specificity and to include separate scoring of other nuclear anomalies associated with cytotoxicity and genotoxicity. The modified assay was applied to buccal smears of 38 female snuff users and 15 female nonusers recruited from a North Carolina clinic in 1987. The prevalence of micronucleation was elevated in the snuff users as compared with the nonusers (prevalence ratio = 2.4, 95% confidence interval 1.1-5.2) and, to a lesser extent, at the usual contact site as compared with a distal buccal site in the snuff users (prevalence ratio = 1.5, 95% confidence interval 0.9-2.5). The pattern of relative frequencies of several nuclear anomalies provided strong evidence of a cytotoxic effect, the prevalence ratios ranging from 2 to 13. Nuclear degenerative phenomena can be difficult to distinguish from classical micronuclei; thus, the observed association of indicators of cytotoxicity with exposure introduces the possibility of bias away from the null in micronucleus findings due to differential misclassification. Until methods to better distinguish extranuclear bodies of different origins become available, investigators should use the revised protocol and should focus on agents not thought to be cytotoxic.

Functional reintroduction of human telomeres into mammalian cells.

Abstract: Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5'----3' toward the chromosomal end. The terminal repeats in yeast are the only elements necessary for telomere function in this organism. To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line. In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria. Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional. Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.

Factors contributing to chromosome damage in lymphocytes of cigarette smokers.

Abstract: Cigarette smoking is generally believed to be responsible for a substantial number of human health problems. However, the causal relationship between smoking, the induction of biological effects and the extent of health problems among smokers have not been fully documented. Using the recently developed lymphocyte micronucleus (MN) assay, we have evaluated the chromosome aberration frequencies in 67 cigarette smokers and 59 matched non-smoking control subjects. We found that the mean MN frequency (per 100 cells) in the smokers was slightly higher than that found in the non-smokers (0.71 ± 0.23 and 0.58 ± 0.05 respectively; p < 0.08). Factors which contribute to the expression of chromosome aberrations were also investigated. A significant age-dependent increase in MN frequencies was observed in both groups (p < 0.05). Linear regression analysis showed that the age-dependent effects among smokers (r = 0.54; p < 0.02) was further enhanced by cigarette consumption (r = 0.62; p < 0.005). Consumption of low potency ‘one-a-day’ type multivitamins had no effect on MN frequencies in either sex of non-smokers and in the 1 male smoker who took multivitamins but vitamin intake consistently reduced the MN frequencies among female smokers. Using a challenge assay, fidelity of DNA repair was evaluated. Lymphocytes from both smokers and non-smokers were irradiated with single doses of 0 or 100 cGy of X-rays or with double doses of 100 cGy of X-rays each separated by 15 or 60 min (100/15 or 100/60). Chromosome translocation frequencies were consistently higher after irradiation in lymphocytes from smokers than in those from non-smokers. Statistically significant differences were detected when the cells were irradiated with the double doses of 100 cGy X-rays each separated by 60 min (p < 0.05). These data suggest that lymphocytes from smokers made more mistakes in the repair of DNA damage than cells from non-smokers. Our studies provide new insights into the genotoxic effects of cigarette smoke and new information which may be useful for understanding the mechanisms for induction of health problems from smoking.

Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.

Abstract: Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.

The lethal(1)TW-6cs mutation of Drosophila melanogaster is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain.

Abstract: The l(1)TW-6cs mutation is a cold-sensitive recessive lethal mutation in Drosophila melanogaster, that affects both meiotic and mitotic chromosome segregation. We report the isolation of three revertants of this mutation. All three revert both the meiotic and mitotic effects as well as the cold sensitivity, demonstrating that all three phenotypes are due to a single lesion. We further show that these revertants fail to complement an amorphic allele of the nod (no distributive disjunction) locus, which encodes a kinesin-like protein. These experiments demonstrate that l(1)TW-6cs is an antimorphic allele of nod, and we rename it nodDTW. Sequencing of the nod locus on a nodDTW-bearing chromosome reveals a single base change in the putative ATP-binding region of the motor domain of nod. Recessive, loss-of-function mutations at the nod locus specifically disrupt the segregation of nonexchange chromosomes in female meiosis. We demonstrate that, at 23.5 degrees, the meiotic defects in nodDTW/+ females are similar to those observed in nod/nod females; that is, the segregation of nonexchange chromosomes is abnormal. However, in nodDTW/nodDTW females, or in nodDTW/+ females at 18 degrees, we observe a more severe meiotic defect that apparently affects the segregation of both exchange and nonexchange chromosomes. In addition, nodDTW homozygotes and hemizygous males have previously been shown to exhibit mitotic defects including somatic chromosome breakage and loss. We propose that the defective protein encoded by the nodDTW allele interferes with proper chromosome movement during both meiosis and mitosis, perhaps by binding irreversibly to microtubules.

Sectors of liguleless-1 tissue interrupt an inductive signal during maize leaf development.

Abstract: The ligule and auricles separate the blade and sheath of normal maize leaves and are absent in liguleless-1 (lg1) mutant leaves. We induced chromosome breakage using X-rays to create plants genetically mosaic for lg1. In genetically mosaic leaves, when an lg1 mutant sector interrupts the normal ligule, the ligule is often displaced basipetally on the marginal side of the sector. Therefore, lg1 mutant sectors not only fail to induce ligule and auricle, but are also disrupting some form of intercellular communication that is necessary for the normally coordinated development of the ligular region. Our data are consistent with a model in which an inductive signal originates near the midvein, cannot traverse the lg1 mutant sector, and reinitiates in the wild-type tissue across the sector toward the leaf margin. The lg1 gene product, therefore, appears to be required for the transmission of this signal and could be involved with reception.

A quantitative model of DNA fragments generated by ionizing radiation, and possible experimental applications.

Abstract: We derive an equation for observed frequencies of DNA fragments as a function of size. In this derivation, we consider an experimental system where fragments are generated by random, independent double-strand breaks on chromosomes (or other large DNA molecules) and then separated by size on agarose gels. When visualizing these fragments using Southern hybridization techniques (employing a site-specific probe), we predict an intensity distribution that has unusual properties. In particular, peaks in the fragment size distribution depend not only on standard breakage parameters, but also on the location of the hybridization site. Our model is consistent with experimental and theoretical results reported elsewhere, where measurements of peaks are used for the physical mapping of genes. Further, we propose that similar experiments might be suitable for precise measurements of the parameters of double-strand breakage (as an alternative to neutral filter elutions and neutral sucrose gradients) and for testing the assumption of random, independent breakage for different types of radiation.

Chromosome breakage by pairs of closely linked transposable elements of the Ac-Ds family in maize.

Abstract: Chromosome breaks and hence chromosomal rearrangements often occur in maize stocks harboring transposable elements (TEs), yet it is not clear what types of TE structures promote breakage. We have shown previously that chromosomes containing a complex transposon structure consisting of an Ac (Activator) element closely linked in direct orientation to a terminally deleted or fractured Ac (fAc) element have a strong tendency to break during endosperm development. Here we show that pairs of closely linked transposons with intact ends, either two Ac elements--a common product of Ac transposition--or an Ac and a Ds (Dissociation) element, can constitute chromosome-breaking structures, and that the frequency of breakage is inversely related to intertransposon distance. Similar structures may also be implicated in chromosome breaks in other eukaryotic TE systems known to produce chromosomal rearrangements. The present findings are discussed in light of a model of chromosome breakage that is based on the transposition of a partially replicated macrotransposon delimited by the outside ends of the two linked TEs.

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C.

Abstract: We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.

A simple diagnostic test for Fanconi anemia by flow cytometry.

Abstract: A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.

Variations of chromosome radiation sensitivity in fetal and adult mice during gestation

Abstract: Mice were exposed to 2 Gy of gamma-rays at various days of pregnancy, and just before and after gestation. Chromosomes were analyzed 4 h after irradiation in spontaneously dividing hematopoietic cells from liver for fetuses and bone marrow for mothers. On average, there was significantly less chromosome damage in fetuses than in mothers. A very strong increase of chromosome breakage was observed in mothers at days 16-19 of gestation. This increase parallels that of gestation hormones, suggesting a direct relationship. The differences between fetuses and mothers in relation to gestation age result from the increase in the rate of chromatid and chromosome breaks but not of chromatid exchanges, which remained stable. This suggests that a DNA repair step involved in joining broken extremities is the cause. More experiments are needed to understand the origin of these variations of radiation sensitivity and the possible extrapolation of these observations to other species.

Implications for genetic toxicology of the chromosomal breakage syndromes.

Abstract: The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produced genetic changes in a tissue without producing cancer tere, if the types of genetic chagne do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberration should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half cases the other sex does not give th e same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuiune sex-specific carcinogenesis are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarely be expected to do better even if they are better.

Is Shwachman syndrome (McKusick 26040) a chromosome breakage syndrome

Abstract: Tada et al. (1987) reported on increased spontaneous chromosome breakage in a female affected by Shwachman syndrome studied when 1, 3, and 10 months old. Fraccaro et al. (1988) observed a higher number of aberrations per cell in two sisters with this syndrome (10 and 8 months old) when compared to control cultures. The difference, however, was not statistically significant. The authors suggested that there might be an age-dependent influence. We had the chance to study two Caucasian Brazilian brothers, aged 46 and 25 months, affected by Shwachman syndrome, born to nonconsanguineous healthy parents. They have been followed at the Instituto da Crianga where their diagnosis was established. Since birth they have had chronic diarrhea with recurrent steatorrhea and several respiratory infections. The older had many episodes of otites and four episodes of single fracture of the left leg, soon after starting to walk. Both presented a protruding abdomen, abnormal pro-

The mutagenicity of 2-amino-N6-hydroxyadenine to L5178Y tk +/- 3.7.2C mouse lymphoma cells: measurement of mutations to ouabain, 6-thioguanine and trifluorothymidine resistance, and the induction of micronuclei.

Abstract: 2-Amino-N6-hydroxyadenine (AHA) was tested in the mouse lymphoma L5178Y tk+/− assay using the microtitre cloning technique over concentrations from 0.005 μg/ml−1 (100% viability) to 6 μg/ml (10% viability) as measured by cloning efficiency immediately after treatment. At low, non-toxic concentrations (0.005–0.25 μg/ml) a dose-related linear increase in the frequency of ouabain-resistant mutants was seen, in addition to an increase in 6-thioguanine- and trifluorothymidine-resistant mutants. No consistent induction of micronucleated cells was observed in this concentration range. Toxic concentrations (20–90% kill) induced a dose-related increase in micronuclei, while the frequency of ouabain-resistant mutants fell (although it was still highly significantly above the control value). These results suggest that the mechanism of action of AHA depends on the concentration, with point mutations being induced at low, non-toxic doses and detectable chromosome breakage occurring only at higher doses. Both large-colony and small-colony trifluorothymidine-resistant mutants were induced at all concentrations. The utility of using multiple genetic end-points in one cell line and the importance of dose range selection for risk assessment and an understanding of the mode of action of test substances is underlined.

Fanconi anemia: a pleotropic mutation with multiple cellular and developmental abnormalities.

Abstract: Fanconi anemia (FA), an autosomal recessive disorder of children, is characterized by congenital or childhood aplastic anemia, multiple developmental anomalies, increased incidence of myeloid leukemia, increased spontaneous chromosome breakage, and cellular and chromosomal hypersensitivity to DNA bifunctional crosslinking and alkylating agents. Attempts to understand the biochemical basis of the disorder over the past two and a half decades have resulted in a number of descriptive studies pertaining to cytogenetic and biochemical abnormalities, especially of DNA repair proficiency following treatment with DNA bifunctional crosslinking agents. More recent approaches such as DNA transfection offer the potential for isolation and molecular characterization of the gene. The FA gene is postulated to belong to the family of genes that regulate the development of hematopoietic and other cell types.

Cytological changes produced by red pepper in mitotic cells of Vicia faba L.

Abstract: SUMMARYRed pepper is an important spice widely used for seasoning food. During the present study, the cytological changes produced by the aqueous extract of red pepper were investigated in detail in the root meristems of Vicia faba. The treated roots revealed a wide spectrum of abnormalities including chromosome breakage, achromatic lesions, c-mitoses, multipolar divisions, laggards, micronuclei etc. Statistically significant differences from controls were observed. Experiments to evaluate the clastogenic potential of red pepper in mammalian system are in progress.

Bone Marrow Failure Syndromes

Abstract: Laboratory diagnosis of inherited bone marrow failure syndromes includes general evaluations, such as blood counts, examination of the peripheral blood smear for morphology, and bone marrow aspirates and biopsies, which may help the clinician classify the patient, particularly if there are no characteristic physical anomalies. Specific diagnoses require unique tests that are only available for a few of the diagnoses. The most useful is chromosome breakage in the diagnosis of FA, with gene mutation analysis or mapping about to become the gold standard when all of the FA genes have been cloned. The diagnosis of DC remains clinical at this time, although linkage to Xq28 and skewed maternal X inactivation may be helpful in some families. Laboratory proof of SD may be provided by decreased serum trypsinogen or other evidence of exocrine pancreatic insufficiency. CHH is substantiated when absent central pigment in hair is found and when it is mapped to 9p21-p13. The only mitochondrial syndrome, PS, is proved with demonstration of deleted mitochondrial DNA. RD is diagnosed from blood and marrow studies that demonstrate lack of lymphoid as well as myeloid activity. Amega requires absent or abnormal marrow megakaryocytes; if radii are also absent, the diagnosis is TAR. DBA usually has elevated red-cell ADA, and the DBA locus may map to 19q13. KS is diagnosed in patients who have congenital nonimmune severe neutropenia. Clinical suspicion of particular diagnoses can often be substantiated by laboratory tests of varying specificity.

Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts

Abstract: Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.

Sister chromatid exchange (SCE) frequencies differ between directly prepared cytotrophoblasts and cultured mesenchymal core cells.

Abstract: Analysis of sister chromatid exchange (SCE) in chorionic villus cells may become useful in measuring the response of fetal tissues to clastogens or mutagens or for prenatal diagnosis of chromosome breakage syndromes such as Bloom syndrome. Previous studies have failed to analyze cytotrophoblastic cells and mesenchymal core cells, or have found no difference between SCE frequencies in directly prepared and cultured cells. Our data indicate significant differences in SCE frequencies between the two cell types: SCE frequency in directly prepared cytotrophoblasts was 6.73 SCE/cell ± 1.6, whereas SCE frequency in cultured mesenchymal core cells was 10.31 SCE/cell ± 0.49 (P < 0.001). SCE analyses involving chorionic villi must take into account cell type.

Cytogenetic characterization of eight human lymphoblastoid cell lines.

Abstract: Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.

Automatic counting of chromosome fragments for the determination of radiation dose

Abstract: A correlation exists between chromosome breakage and radiation exposure so that counting chromosome fragments can provide a measure of the radiation dose to an organism. We describe our method for automatically counting prematurely condensed chromosome fragments from microscope images of the exploded nucleus. An alternative perhaps more promising non-imaging approach using flow karyotyping is also discussed. 1 .© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Discussion: Cellular DNA Strand Breakage

Abstract: Many techniques are now available for measuring DNA single- and double-strand breaks by ionizing radiation. As we are reminded by the results of Van Loon and co-workers, single-strand break measurements are far simpler to perform than double-strand break assays and are sensitive to damage by lower radiation doses. However, while the number of single-strand breaks is linearly dependent on dose, it is not predictive for cell killing by ionizing radiation. Conversely, the number of initial double-strand breaks has been shown to correlate with cell killing. Double-strand break damage, if unrepaired or inaccurately repaired, is believed to be the likely “lethal lesion”, leading first to chromosome breakage and then to loss of reproductive capacity, mutation and perhaps transformation. While there is little doubt that initial DNA damage is responsible for the final outcome of cell death, time and multiple DNA repair events separate these two processes. It is therefore somewhat unexpected that such good predictions for cell killing across a variety of cell lines can be obtained with the filter elution assay based only on initial numbers of DNA double-strand breaks.