bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ DNAse I sensitivity assay indicates DNA conformation differences between CHO cells and the radiation-sensitive CHO mutant IRS-20
Authors: Marañon D.G. , Laudicina A.O. , Muhlmann M.
Published: May 2004

The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2–5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.
DNA repair factors and telomere-chromosome integrity in mammalian cells
Authors: Hande M.P.
Published: May 2004

Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/– cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53–/– cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/–PARP–/– mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.
Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells
Authors: Desmaze C. , Pirzio L.M. , Blaise R. , Mondello C. , Giulotto E. , Murnane J.P. , Sabatier L.
Published: May 2004

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.
Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells
Authors: Wong H.-P. , Mozdarani H. , Finnegan C. , McIlrath J. , Bryant P.E. , Slijepcevic P.
Published: May 2004

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells.
Cytological indications of the complex subtelomeric structure
Authors: Drets M.E.
Published: May 2004

Research on the subtelomeric region has considerably increased because this chromosome segment (1) keeps the chromosome number constant, (2) intervenes in cancer and cell senescence processes, (3) presents more crossovers than other regions of the genome and, (4) is the site of cryptic chromosome aberrations associated with mental retardation and congenital malformations. Quantitative microphotometrical scanning and computer graphic image analysis enables the detection of differentially distributed Giemsa-stained structures in T-banded subtelomeric segments of human and Chinese hamster ovary (CHO) chromosomes. The presence of high density stain patterns in the subtelomeric region was confirmed using endoreduplicated chromosomes as a model. Besides, prolonging the incubation in the T-buffer, specific holes were induced in subtelomeric segments. Hole specificity was confirmed inducing them in complex CHO chromosome aberrations obtained by AluI. The method was also used to detect minute sister chromatid exchanges in the T-banded subtelomeric area (t-SCEs). The presence of t-SCEs was suspected to reflect, at the microscope level, the high crossover activity prevailing in the region. Due to the fact that the fluorescent signals obtained with subtelomeric probes seem to be colocalized with subtelomeric high density areas, measurements on the position of both structures with respect to the diffraction and chromosome edges were carried out. Data obtained showed comparable values suggesting that the high density segments were located where telomeric probes usually fluoresce. The possible relationship of the high density patterns, the production of specific holes, the localization of fluorescent areas and the detection of minute SCEs in the subtelomeric segment observed in T-banded CHO and human chromosomes is briefly reviewed.
Quantitative analysis of radiation-induced chromosome aberrations
Authors: Sachs R.K. , Levy D. , Hahnfeldt P. , Hlatky L.
Published: May 2004

We review chromosome aberration modeling and its applications, especially to biodosimetry and to characterizing chromosome geometry. Standard results on aberration formation pathways, randomness, dose-response, proximity effects, transmissibility, kinetics, and relations to other radiobiological endpoints are summarized. We also outline recent work on graph-theoretical descriptions of aberrations, Monte-Carlo computer simulations of aberration spectra, software for quantifying aberration complexity, and systematic links of apparently incomplete with complete or truly incomplete aberrations.
Effect of DMSO on radiation-induced chromosome aberrations analysed by FISH
Authors: Cigarrán S. , Barrios L. , Caballín M.R. , Barquinero J.F.
Published: May 2004

The purpose of the present work was to determine if the described reduction in the frequency of radiation-induced chromosome aberrations by DMSO is homogeneous within different human chromosomes. Blood samples were irradiated with 4 Gy of X-rays in absence and presence of 0.5 M DMSO. FISH painting was carried out independently for human chromosomes 1, 2, 3, 4, 7, 11 and 12. The observed frequencies of apparently simple translocations and dicentrics for all these chromosomes, showed a homogeneous reduction when the irradiation was done in the presence of DMSO. Moreover, a better fit between the observed and expected frequencies was obtained when (DNA content)2/3 was used to calculate the expected frequencies, instead of just the DNA content. This result supports the idea that for exchange type aberrations, a better adjustment is obtained when the surface area of spherical chromosome territories is considered.
DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays
Authors: Güerci A.M. , Dulout F.N. , Seoane A.I.
Published: May 2004

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90–95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.
The repair of γ-ray-induced chromosomal damage in human lymphocytes after exposure to extremely low frequency electromagnetic fields
Authors: Lloyd D. , Hone P. , Edwards A. , Cox R. , Halls J.
Published: May 2004

G₀ human blood lymphocytes were irradiated with 2.0 Gy γ-rays and cultured to metaphase whilst held in a 50-Hz power frequency magnetic field of 0.23, 0.47 or 0.7 mT. No differences were found in the frequencies of γ-induced chromosome aberrations observed in cells held in the EM fields compared with replicates held in a sham coil. Similar field conditions have been reported to increase the frequency of γ-induced HPRT mutations, leading to a suggestion that the EM fields alter the fidelity of repair of genomic lesions. This was not confirmed by the chromosome aberration assay described here.
Cytogenetic damage in lymphocytes for the purpose of dose reconstruction: a review of three recent radiation accidents
Authors: Wojcik A. , Gregoire E. , Hayata I. , Roy L. , Sommer S. , Stephan G. , Voisin P.
Published: May 2004

The analysis of chromosomal aberrations in peripheral blood of radiation accident victims is an established method of biological dosimetry. The dose estimate on the basis of an in vitro calibration curve is straightforward when the radiation exposure is homogeneous and the analysis not delayed. In recent years three radiation accidents occurred, where the irradiation or sampling conditions precluded a simple estimation of the dose. During the Georgian accident soldiers carried in their pockets small sources of 137Cs leading to partial and protracted body exposures. During the Tokai-mura accident, three employees involved in the process of 235U enrichment were exposed to very high doses of gamma rays and neutrons. During the Bialystok accident, five patients with breast cancer undergoing radiotherapy were exposed to a single dose of electrons which reached about 100 Gy. In the present paper the approaches chosen to estimate, by cytogenetic methods, the doses absorbed by the people involved in the accidents are described.
Complex chromatid-isochromatid exchanges following irradiation with heavy ions?
Authors: Loucas B.D. , Eberle R.L. , Durante M. , Cornforth M.N.
Published: May 2004

We describe a peculiar and relatively rare type of chromosomal rearrangement induced in human peripheral lymphocytes that were ostensibly irradiated in G₀ phase of the cell cycle by accelerated heavy ions, and which, to the best of our knowledge, have not been previously described. The novel rearrangements which were detected using mFISH following exposure to 500 MeV/nucleon and 5 GeV/n 56Fe particles, but were not induced by either 137Cs gamma rays or 238Pu alpha particles, can alternatively be described as either complex chromatid-isochromatid or complex chromatid-chromosome exchanges. Different mechanisms potentially responsible for their formation are discussed.
Chromosome aberrations induced by high-LET carbon ions in radiosensitive and radioresistant tumour cells
Authors: Virsik-Köpp P. , Hofman-Huether H.
Published: May 2004

Chromosome aberration formation was analysed in two human tumour cell lines displaying different radiosensitivity. Aberrations involving chromosomes 2, 4, and 5 were studied in one radioresistant cell line (WiDr) and in one radiosensitive cell line (MCF-7). Chromosome aberrations were studied by application of single-colour FISH. We studied the effects of monoenergetic 100 MeV/u carbon ions and carbon ions from extended Bragg peak. Chromosome aberrations induced by carbon ions were compared with aberrations induced by standard 200 kV X-rays. In both tumour cell lines, carbon ions induced aberrations more effectively than X-rays. The radioresistance and radiosensitivity of the corresponding cell lines, as observed for X-rays, were also found after carbon ion irradiation. In both cell lines, the typical effects of ion irradiation were an increased proportion of cells containing complex aberrations, and an increased complexity of these complex exchanges. However, comparable effects were induced in MCF-7 cells by a much lower dose than in WiDr cells. Insertions were also induced more efficiently in MCF-7 cells than in WiDr cells.
Induction of homologous recombination in the hprt gene of V79 Chinese hamster cells in response to low- and high-LET irradiation
Authors: Olsson G. , Czene S. , Jenssen D. , Harms-Ringdahl M.
Published: May 2004

Dense ionization tracks from high linear energy transfer (LET) radiations form multiple damaged sites (MDS), which involve several types of DNA lesions in close vicinity. The primary DNA damage triggers sensor proteins that activate repair processes, cell cycle control or eventually apoptosis in subsequent cellular responses. The question how homologous recombination (HR) and non-homologous end joining (NHEJ) interact in the repair of radiation-induced DNA damage of MDS type has been addressed in different model systems but several questions remain to be answered. We have therefore challenged cells with treatments of ionizing radiation of different qualities to investigate whether primary DNA damages of different complexity are reflected in the processes of repair by HR as well as cell survival. We used the V79 derived SPD8 cell line to determine the induction of HR in the hprt exon 7 and clonogenic assay for survival in response to radiation. SPD8 cells were irradiated with γ-rays (137Cs 0.5 keV/µm), boron ions (40 and 80 keV/µm) and nitrogen ions (140 keV/µm), with doses up to 5 Gy. Analysis of clonogenic survival showed that B-ions (80 keV/µm) and N-ions were more toxic than γ-rays, 4.1 and 5.0 times respectively, while B-ions at 40 keV/µm were 2.0 times as toxic as γ-rays. Homologous recombination in the cells exposed to B-ions (80 keV/µm) increased 2.9 times, a significant response as compared to cells exposed to γ-rays, while for B-ions (40 keV/µm) and N-ions a nonsignificant increase in HR of 1.2 and 1.4, respectively, was observed. We hypothesize that the high-LET generated formation of MDS is responsible for the enhanced cytotoxicity as well as for the mobilization of the HR machinery.
Effect of high-level natural radiation on chromosomes of residents in southern China
Authors: Hayata I. , Wang C. , Zhang W. , Chen D. , Minamihisamatsu M. , Morishima H. , Wei L. , Sugahara T.
Published: May 2004

To study the effect of low-dose (rate) radiation on human health, we analyzed chromosomes of peripheral lymphocytes of residents in a high background radiation area (HBRA) and compared the results with those obtained from residents in a control area (CA) in Guangdong Province, China. Unstable types of chromosome aberrations (dicentrics and rings) were studied in 22 members of eight families in HBRA and 17 members of five families in CA. Each family consists of three generations. On average 2,600 cells per subject were analyzed. 27 adults and six children in HBRA and 25 adults and eight children in CA were studied with respect to translocations. On average 4,741 cells per subject were examined. We found an increase of the frequency of dicentrics and rings in HBRA, where the natural radiation level is three to five times higher than in the control area. But the increase of translocations in HBRA was within the range of individual variation in the controls.
Contribution of chromosomal imbalance to sperm selection and pre-implantation loss in translocation-heterozygous Chinese hamsters
Authors: Sonta S.
Published: May 2004

Chinese hamster stocks with various structurally abnormal chromosomes have been produced by X irradiation. Among these stocks, 18 with various reciprocal translocations were used to investigate the participation of unbalanced gametes in fertilization and the development of unbalanced embryos. Among males as well as females heterozygous for the same translocation, there is no difference in the frequency of each disjunctional class. The participation of chromosomally unbalanced gametes in fertilization was investigated by chromosomal analysis of meiotic cells in heterozygotes for the 18 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from MII scoring, the frequencies of male pronuclei having a common deficiency of chromosome 1 (1q17→1q42) or chromosome 3 (3p23→3q31) decreased significantly in one-cell embryos. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same abnormalities as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results revealed clearly that most gametes with nullisomies as well as disomies for any chromosomal segments may participate in fertilization, whereas only male gametes nullisomic for certain segments of chromosomes 1 and 3 failed to participate in fertilization. The zygotic selection of chromosomal imbalance was also investigated by direct chromosomal and morphological analyses of preimplantation embryos from crosses between karyotypically normal females and male heterozygotes from the 18 stocks with various reciprocal translocations. These analyses revealed that some embryos were arrested in development at the two-cell stage. The karyotype of these two-cell embryos had a common deficiency in a segment of chromosome 1 or chromosome 2. Embryos with partial monosomy including chromosomes 1, 3, 4 and 5 showed arrested development at four- to eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of segments of chromosomes 1p, 1q, 2q, 5q, 7q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between Chinese hamster and mouse chromosomes relating to abnormal embryogenesis at early stages has been partially confirmed from reported maps of chromosomes. The Chinese hamster is useful for further cytogenetic studies during the stages of meiosis and early embryogenesis.
Heritable translocations induced by dermal exposure of male mice to acrylamide
Authors: Adler I.-D. , Gonda H. , de Angelis M. Hrabé , Jentsch I. , Otten I.S. , Speicher M.R.
Published: May 2004

Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It can be absorbed through the skin. AA was shown to be a germ cell clastogen that entails a genetic risk for exposed workers. The genetic risk calculation was based on mouse heritable translocation test data obtained after acute intraperitoneal (ip) exposure (Adler et al., 1994). To obtain a correction factor between ip and dermal exposure, dominant lethal and heritable translocation tests were carried out with dermal exposure of male mice to AA. In the dominant lethal test, male (102/El × C3H/El)F1 mice were exposed by dermal application to the shaved backs of 50 mg/kg AA per day on five consecutive days or to five daily ip injections of 50 mg/kg AA. One day after the end of exposure, the males were mated to untreated females of the same hybrid stock for four days and females were changed every four days for a total of five matings. Dominant lethal effects were found during matings 1–3. For ip exposure, these values were 81.7, 85.7 and 45.4%, respectively; for dermal exposure the corresponding values were 22.1, 30.6 and 16.5%, respectively. In the heritable translocation assay, male C3H/El mice were treated with five dermal exposures of 50 mg/kg AA and mated 1.5–8.5 days after the end of exposure to untreated female 102/El mice. Pregnant females were allowed to come to term and all offspring were raised to maturity. Translocation carriers among the F1 progeny were selected by a sequential fertility testing and cytogenetic analysis including G-band karyotyping and M-FISH. A total of 475 offspring were screened and 41 translocation carriers were identified. The observed translocation frequency after dermal exposure was 8.6% as compared to 21.9% after similar ip exposure (Adler, 1990). The calculated ratio of ip vs. dermal exposure of 0.39 can be applied to obtain a more realistic calculation of genetic risk for dermally exposed workers.
Chromosome banding in Amphibia
Authors: Schmid M. , Steinlein C. , Haaf T.
Published: May 2004

The present study reports for the first time on the numerical and structural chromosome anomalies that spontaneously arise in aging cultured fibroblast cells of Amphibia. The analyses were conducted on kidney fibroblasts of three anuran species with extremely divergent genome sizes (Bufo rubropunctatus, Scaphiopus holbrooki, Gastrotheca riobambae), in the sixth up to the 14th culture passage. The chromosomal rearrangements were identified by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. The aberrations can be either confined to a single chromosome, or else involve all chromosomes of the karyotype. The most frequent structural aberrations in the cell cultures of S. holbrooki and G. riobambae are tandem fusions between two or more chromosomes. These tandem fusions originating in vitro in long-termed cell cultures reflect the chromosome mutations which also took place during amphibian phylogenesis.
Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells
Authors: Satoh T. , Yamamoto K. , Miura K.F. , Sofuni T.
Published: May 2004

A human diploid lung fibroblast cell strain, TIG-7, has a heteromorphic chromosome 15 with an extra short arm carrying a homogeneously staining region (15p+hsr). We demonstrated previously that the 15p+hsr consists of an inactive and G+C-rich rDNA cluster characterized by fluorescence in situ hybridization (FISH) and various chromosome banding techniques. Thus, it was suggested that the region could contain highly methylated DNA. To observe methylation status on the target region directly under the microscope, we used a demethylating agent, 5-azacytidine (5-azaC), to induce decondensation of the chromatin containing methylated DNA. At 24 h after treatment with 0.5 µM 5-azaC, marked decondensation of the 15p+hsr was observed in almost all of the metaphases. Furthermore, we observed micronuclei, which were equivalent to the rDNA of the 15p+hsr demonstrated by FISH in the same preparation. In contrast, the DNA cross-linking agent mitomycin C (MMC) preferentially induced 15p+hsr-negative micronuclei. These findings indicated that chromatin decondensation and subsequent DNA strand breakage induced by the demethylating effect of 5-azaC led specifically to 15p+hsr-positive micronuclei.
Micronuclei in lymphocytes of uranium miners of the former Wismut SDAG
Authors: Müller W.-U. , Kryscio A. , Streffer C.
Published: May 2004

We studied micronucleus frequencies in former German uranium miners of the Wismut SDAG (Sowjetisch-Deutsche Aktiengesellschaft). Various other groups were analyzed for comparison (individuals with lung tumors or lung fibrosis, controls). We had shown previously that micronucleus frequencies were not different among the various groups. Differences were observed, however, when centromere-positive and -negative micronuclei were distinguished. In the analyses presented here, we looked for the effects of smoking habits, alcohol consumption, vitamin uptake, chronic diseases, allergies, doing sports, γ-GT (gamma-glutamyltranspeptidase), lymphocyte numbers, CEA (carcinoembryonic antigen), X-ray diagnostics, computer tomographies, and scintigraphies. With the exception of more than one scintigraphy carried out during the last four months before micronucleus analysis, none of the factors mentioned above significantly affected micronucleus numbers. One result deserves specific attention: individuals with low percentages of binucleated lymphocytes after in vitro cytochalasin B exposure showed higher micronucleus frequencies than those individuals with high percentages of binucleated cells. The same result was obtained for various other populations that we monitored in the past.
SCE analysis in G2 lymphocyte prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action
Authors: Malik S.I. , Terzoudi G.I. , Pantelias G.E.
Published: May 2004

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a “possible human carcinogen”. The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used.
Chromosomal mutagen sensitivity associated with mutations in BRCA genes
Authors: Speit G. , Trenz K.
Published: May 2004

Chromosomal mutagen sensitivity is a common feature of cells from patients with different kinds of cancer. A portion of breast cancer patients also shows an elevated sensitivity to the induction of chromosome damage in cells exposed to ionizing radiation or chemical mutagens. Segregation analysis in families of patients with breast cancer indicated heritability of mutagen sensitivity. It has therefore been suggested that mutations in low-penetrance genes which are possibly involved in DNA repair predispose a substantial portion of breast cancer patients. Chromosomal mutagen sensitivity has been determined with the G2 chromosome aberration test and the G₀ micronucleus test (MNT). However, there seems to be no clear correlation between the results from the two tests, indicating that the inherited defect leading to enhanced G₀ sensitivity is different from that causing G2 sensitivity. Less than 5% of breast cancer patients have a familial form of the disease due to inherited mutations in the breast cancer susceptibility genes BRCA1 or BRCA2. Heterozygous mutations in BRCA1 or BRCA2 in lymphocytes from women with familial breast cancer are also associated with mutagen sensitivity. Differentiation between mutation carriers and controls seems to be much better with the MNT than with the G2 assay. Mutagen sensitivity was detected with the MNT not only after irradiation but also after treatment with chemical mutagens including various cytostatics. The enhanced formation of micronuclei after exposure of lymphocytes to these substances suggests that different DNA repair pathways are affected by a BRCA1 mutation in accordance with the proposed central role of BRCA1 in maintaining genomic integrity. Mutations in BRCA1 and BRCA2 seem to predispose cells to an increased risk of mutagenesis and transformation after exposure to radiation or cytostatics. This raises a question about potentially increased risks by mammography and cancer therapy in women carrying a mutation in one of the BRCA genes. Lymphoblastoid cell lines (LCLs) from breast cancer patients have been used to study the mechanisms and genetic changes associated with tumorigenesis. With respect to mutagen sensitivity, conflicting results have been reported. In particular enhanced induction of micronuclei does not seem to be a general feature of LCLs with a BRCA1 mutation in contrast to lymphocytes with the same mutation. Therefore, LCLs are of limited utility for studying the mechanisms underlying chromosomal mutagen sensitivity.
Possible causes of chromosome instability: comparison of chromosomal abnormalities in cancer cell lines with mutations in BRCA1, BRCA2, CHK2 and BUB1
Authors: Grigorova M. , Staines J.M. , Ozdag H. , Caldas C. , Edwards P.A.W.
Published: May 2004

A large proportion of epithelial cancers show the chromosome-instability phenotype, in which they have many chromosome abnormalities. This is thought to be the result of mutations that disrupt chromosome maintenance, but the causative mutations are not known. We identified cell lines known to have mutations that might cause chromosome instability, and examined their karyotypes. Two cell lines, the breast cancer line HCC1937 and the pancreatic cancer line CAPAN-1, that have mutations respectively in BRCA1 and BRCA2, had very abnormal karyotypes, with many structural and numerical chromosome changes and substantial variation between metaphases. However, two colorectal cancer lines with mutations in BUB1, a spindle checkpoint protein involved in chromosome segregation, had rather simple near-tetraploid karyotypes, with minimal loss or gain of chromosomes other than the endoreduplication event, and minimal structural change. Apart from tetraploidy, these karyotypes were typical of colorectal lines considered to be chromosomally stable. Two lines derived from the same tumour, DLD-1 and HCT-15, with bi-allelic mutation of CHK2, had karyotypes that were typical of near-diploid colorectal lines considered chromosomally stable. The karyotypes observed supported the proposed role for BRCA1 and BRCA2 mutations in chromosomal instability, but showed that the tested mutations in BUB1 and CHK2 did not result in karyotypes that would have been predicted if they were sufficient for chromosomal instability.
Chromosomal radiosensitivity and low penetrance predisposition to cancer
Authors: Scott D.
Published: May 2004

This mini-review summarises studies in this Institute on the sensitivity of cells of patients with common cancers to the chromosome-damaging effects of ionising radiation, in the context of related studies. Using the 90th percentile of healthy controls (n >200) as the cut-off point between a normal and a sensitive response, 40% of patients with breast cancer (n = 166) were sensitive when cells were irradiated in the G2 phase of the cell cycle. Smaller studies showed that patients with colorectal, head and neck (at < 45 years) and childhood cancers also exhibited degrees of enhanced sensitivity, whereas cervical and lung cancer cases did not. Cells from breast and head and neck cases irradiated in G₀ also showed increased sensitivity. We propose that such elevated sensitivity is a marker of low penetrance predisposition to cancer. The strongest support for this hypothesis was our demonstration of the Mendelian heritability of chromosomal radiosensitivity in 95 family members of breast cancer cases. Challenges for the future include more heritability studies, identification of the underlying genetic determinants, assessment of the associated cancer risk (spontaneous and radiogenic) and population screening for cancer prevention strategies.
New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals
Authors: Schunck C. , Johannes T. , Varga D. , Lörch T. , Plesch A.
Published: May 2004

The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.
Expression of a Human SOCS Protein, HSOCP-1, in Peripheral Blood Eosinophils from Patients with Atopic Dermatitis
Authors: Ogawa Kaoru , Itoh Mikito , Miyagawa Masami , Nagasu Takeshi , Sugita Yuji , Katsunuma Toshio , Akasawa Akira , Matsumoto Kenji , Tsujimoto Gozoh , Saito Hirohisa , Hashida Ryoichi
Published: May 2004

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34SEI-1 was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16INK4, suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.
Correlation of Allergen-Induced IL-5 and IL-13 Production by Peripheral Blood T Cells of Asthma Patients
Authors: Hashimoto Tomomi , Akiyama Kazuo , Kawaguchi Hiroshi , Maeda Yuji , Taniguchi Masami , Kobayashi Noriaki , Mori Akio
Published: May 2004

Background: Helper T cells and T cell cytokines are implicated in allergic disorders such as asthma and atopic dermatitis (AD). We reported enhanced interleukin-5 (IL-5) production by peripheral blood T cells of asthmatic patients. Production of cytokines, particularly IL-5 and IL-13, by peripheral blood mononuclear cells (PBMC) obtained from bronchial asthma (BA) and AD patients was investigated in the present study. Methods: PBMC were cultured in the presence of either polyclonal activator, phorbol ester plus Ca2+ ionophore or Dermatophagoidesfarinae (Df) antigen. The resulting supernatants were assayed for IL-4, IL-5, and IL-13 by specific ELISAs. Results: IL-5 and IL-13 production in response to Df antigen is significantly higher in allergic groups compared to control subjects. IL-5 responses induced by Df antigen were strongly correlated with IL-13 responses. Conclusion: In spite of IL-13 gene proximity to IL-4 gene, IL-5 production was more strongly correlated to IL-13 production in the BA group, suggesting a common control mechanism that regulates the IL-5 and IL-13 gene.
Molecular Mechanisms of Repression of Eotaxin Expression with Fluticasone Propionate in Airway Epithelial Cells
Authors: Matsukura Satoshi , Kokubu Fumio , Kurokawa Masatsugu , Kawaguchi Mio , Kuga Hideki , Ieki Koushi , Odaka Miho , Suzuki Shintaro , Watanabe Shin , Takeuchi Hiroko , Schleimer Robert P. , Schindler Ulrike , Adachi Mitsuru
Published: May 2004

Background: Glucocorticoids are known to repress the expression of CC chemokine eotaxin in airway epithelial cells. We focused our study on the molecular mechanisms of the glucocorticoid, fluticasone, in the inhibition of the expression of the eotaxin gene in the cells. Methods: The airway epithelial cell line, BEAS-2B, was stably transfected with signal transducers and activators of transcription 6 (STAT6)-expressing vector and used in the following experiments to clarify the function of STAT6. Levels of eotaxin mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by the electrophoretic mobility shift assay and dual luciferase assay using eotaxin promoter-luciferase reporter plasmids. Results: Fluticasone significantly inhibited the induction of eotaxin protein stimulated with TNF-α and IL-4 in the cells. Fluticasone also repressed the induction of eotaxin mRNA with these stimuli. It partially inhibited the activity of eotaxin promoter; however, it did not inhibit the nuclear translocation and binding of transcription factors, nuclear factor-kappa B (NF-ĸB) or STAT6, to the DNA derived from the proximal promoter region of the eotaxin gene. Moreover, the inhibitory effect was also conserved in the experiments using the reporter plasmid of which the putative glucocorticoid-responsive element was mutated. Conclusions: Fluticasone inhibits the expression of eotaxin gene in airway epithelial cells in part through repression of the transcription. However, the mechanisms depend neither on the inhibition of transcription factors’ translocation into nuclei nor the function of the putative glucocorticoid-responsive element in the promoter, indicating that other mechanisms would be related to the transcriptional repression of the eotaxin gene in airway epithelial cells.
Immunotherapy Attenuates Eosinophil Transendothelial Migration Induced by the Supernatants of Antigen-Stimulated Mononuclear Cells from Atopic Asthmatics
Authors: Nagata Makoto , Saito Keiko , Kikuchi Izumi , Tabe Kazuaki , Hagiwara Koichi , Kanazawa Minoru , Sakamoto Yoshio
Published: May 2004

Background: Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. There is increasing evidence that specific immunotherapy (SIT) modulates the production of inflammatory molecules from mononuclear cells. Objective: The present study was undertaken to examine whether SIT modifies eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics. Methods:Dermatophagoides farinae(Df)-sensitive mild persistent asthmatics were divided into a SIT-treated group and a control group. Peripheral blood mononuclear cells (PBMC) were isolated before and after SIT using the rush protocol, and cultured for 96 h at 37°C in the presence or absence of Df antigen. Eosinophils were isolated from the blood of healthy subjects, and put on transwell filters coated with pulmonary microvascular endothelial cell monolayers stimulated with IL-4 plus TNF-α. The supernatants of PBMC were applied to the lower compartment and the transmigration of eosinophils was examined. Results:Df stimulation of PBMC resulted in an augmentation of eosinophil transendothelial migration. This enhancement was abrogated following SIT. In the control group, the antigen-induced effect on eosinophil transmigration did not show an interval change. Conclusion: SIT attenuates eosinophil transendothelial migration induced by antigen-stimulated mononuclear cells.
Tyk2 Is Essential for IFN-α-Induced Gene Expression in Mast Cells
Authors: Mori Yumiko , Hirose Koichi , Suzuki Kotaro , Nakajima Hiroshi , Seto Yohei , Ikeda Kei , Shimoda Kazuya , Nakayama Kei-ichi , Saito Yasushi , Iwamoto Itsuo
Published: May 2004

Mast cells are recognized not only as the major effector cells of type I hypersensitivity reactions but also as an important player of innate immune response against bacterial infection. Type I IFNs are also involved in the response against bacterial infection. However, the role of type I IFNs and their associated Janus kinase Tyk2 in mast cell functions remains to be determined. In this study, we addressed this issue using Tyk2-deficient (Tyk2–/–) bone marrow-derived mast cells (BMMCs). When BMMCs from wild-type (WT) mice were stimulated with IFN-α, they expressed mRNA for IFN-γ-inducible protein 10 (IP-10) and monocyte chemoattractant protein-5 (MCP-5). Interestingly, IFN-α-induced expression of IP-10 and MCP-5 was severely decreased in Tyk2–/– BMMCs. In addition, IFN-α-induced Stat1 phosphorylation was decreased in Tyk2–/– BMMCs. On the other hand, IFN-α-induced Stat1 phosphorylation and IP-10 and MCP-5 expression were normal in Tyk2–/– fibroblasts. These results indicate that IFN-α induces the expression of TNF-α and the chemokines IP-10 and MCP-5 in mast cells and thatTyk2 plays a nonredundant role in IFN-α signaling in mast cells.
Dual Signaling and Effector Pathways Mediate Human Eosinophil Activation by Platelet-Activating Factor
Authors: Kato Masahiko , Kita Hirohito , Tachibana Atsushi , Hayashi Yasuhide , Tsuchida Yoshiaki , Kimura Hirokazu
Published: May 2004

Platelet-activating factor (PAF) induces various cellular functions in eosinophils including chemotaxis, adhesion, superoxide anion (O2–) production, and degranulation. While PAF shares many biological effects with other chemotactic factors such as N-formyl-methionyl-leucyl-phenylalanine, complement fragments, and lipid mediators, PAF is unique in that its action is relatively resistant to pertussis toxin (PTX), and in activating eosinophils more strongly than neutrophils. In this review we consider how PAF might activate human eosinophils in preference to neutrophils, and discuss possible mechanisms of PAF-induced activation of human eosinophils via two distinct signaling and effector pathways. Recently we analyzed O2– production by eosinophils using a sensitive, real-time chemiluminescence method. Our results showed that in human eosinophils PAF activates two distinct signaling and effector pathways coupled to the PAF receptor: one linked to PTX-sensitive G protein(s) and another to PTX-resistant G protein(s), phosphatidylinositol 3-kinase, and cellular adhesion. This activation of two different G proteins by the eosinophil PAF receptor may explain the strong and diverse biological responses of human eosinophils to PAF.
Rapidly Prototyped Temporal Bone Model for Otological Education
Authors: Suzuki Mamoru , Ogawa Yasuo , Hagiwara Akira , Yamaguchi Hiroya , Ono Hidenori
Published: May 2004

The anatomy of the temporal bone is extremely complicated. If a three-dimensional model could be simulated, it would greatly contribute to the stereoscopic understanding of anatomy and surgery. A simulated three-dimensional model of a human temporal bone was prototyped using the selective laser sintering method. The model could be shaved using a surgical drill in the same way as in real surgery. A magnified model was particularly useful for the instruction of anatomy and surgery. When a translucent area was selected, a bony labyrinth could be created together with an internal auditory meatus and facial nerve, which also contributed to the easy understanding of the inner ear structure. The three-dimensional prototyped model using selective laser sintering serves as a good educational material for middle ear anatomy and surgery.