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At least 55 records · Page 3

The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

Non-NASA Center↗

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

Non-NASA Center↗

A model for interphase chromosomes and evaluation of radiation-induced aberrations

We have developed a theoretical model for evaluating radiation-induced chromosomal exchanges by explicitly taking into account interphase (G(0)/G(1)) chromosome structure, nuclear organization of chromosomes, the production of double-strand breaks (DSBs), and the subsequent rejoinings in a faithful or unfaithful manner. Each of the 46 chromosomes for human lymphocytes (40 chromosomes for mouse lymphocytes) is modeled as a random polymer inside a spherical volume. The chromosome spheres are packed randomly inside a spherical nucleus with an allowed overlap controlled by a parameter Omega. The rejoining of DSBs is determined by a Monte Carlo procedure using a Gaussian proximity function with an interaction range parameter sigma. Values of Omega and sigma have been found which yield calculated results of interchromosomal aberration frequencies that agree with a wide range of experimental data. Our preferred solution is one with an interaction range of 0.5 microm coupled with a relatively small overlap parameter of 0.675 microm, which more or less confirms previous estimates. We have used our model with these parameter values and with resolution or detectability limits to calculate yields of translocations and dicentrics for human lymphocytes exposed to low-LET radiation that agree with experiments in the dose range 0.09 to 4 Gy. Five different experimental data sets have been compared with the theoretical results. Essentially all of the experimental data fall between theoretical curves corresponding to resolution limits of 1 Mbp and 20 Mbp, which may reflect the fact that different investigators use different limits for sensitivity or detectability. Translocation yields for mouse lymphocytes have also been calculated and are in good agreement with experimental data from 1 cGy to 10 cGy. There is also good agreement with recent data on complex aberrations. Our model is expected to be applicable to both low- and high-LET radiation, and we include a sample prediction of the yield of interchromosomal rejoining in the dose range 0.22 Gy to 2 Gy of 1000 MeV/nucleon iron particles. This dose range corresponds to average particle traversals per nucleus ranging from 1.0 to 9.12.

Non-NASA Center↗

Microgravitational effects on chromosome behavior (7-IML-1)

The effects of the two major space-related conditions, microgravity and radiation, on the maintenance and transmission of genetic information have been partially documented in many organisms. Specifically, microgravity acts at the chromosomal level, primarily on the structure and segregation of chromosomes, in producing major abberations such as deletions, breaks, nondisjunction, and chromosome loss, and to a lesser degree, cosmic radiation appears to affect the genic level, producing point mutations and DNA damage. To distinguish between the effects from microgravity and from radiation, it is necessary to monitor both mitotic and meiotic genetic damage in the same organism. The yeast Saccharomyces cerevisiae is used to monitor at high resolution the frequency of chromosome loss, nondisjunction, intergenic recombination, and gene mutation in mitotic and meiotic cells, to a degree impossible in other organisms. Because the yeast chromosomes are small, sensitive measurements can be made that can be extrapolated to higher organisms and man. The objectives of the research are: (1) to quantitate the effects of microgravity and its synergism with cosmic radiation on chromosomal integrity and transmission during mitosis and meiosis; (2) to discriminate between chromosomal processes sensitive to microgravity and/or radiation during mitosis and meiosis; and (3) to relate these findings to anomalous mitotic mating type switching and ascosporogenesis following meiosis.

Bruschi, Carlo↗

The Influence of Shielding on the Biological Effectiveness of Accelerated Particles for the Induction of Chromosome Damage

Chromosome damage was assessed in human peripheral blood lymphocytes after in vitro exposure to the either Si-28 (490 or 600 MeV/n), Ti-48 (1000 MeV/n), or Fe-56 (600, 1000, or 5000 MeV/n). LET values for these ions ranged from 51 to 184 keV/micron and doses ranged from 10 to 200 cGy. The effect of either aluminum or polyethylene shielding on the induction of chromosome aberrations was investigated for each ion. Chromosome exchanges were measured using fluorescence in situ hybridization (FISH) with whole chromosome probes in cells collected at G2 and mitosis in first division post irradiation after chromosomes were prematurely condensed using calyculin-A. The yield of chromosomal aberrations increased linearly with dose and the relative biological effectiveness (RBE) for the primary beams, estimated from the initial slope of the dose response curve for total chromosomal exchanges with respect to gamma-rays, ranged from 9 to 35. The RBE values increased with LET, reaching a maximum for the 600 MeV/n Fe ions with LET of 184 keV/micron. When the LET of the primary beam was below approximately 100 keV/micron, the addition of shielding material increased the effectiveness per unit dose. Whereas shielding decreased the effectiveness per unit dose when the LET of primary beams was higher than 100 keV/micron. The yield of aberrations correlated with the dose-average LET of the beam after traversal through the shielding.

Goeorge, Kerry↗

The Influence of Shielding on the Biological Effectiveness of Accelerated Particles for the Induction of Chromosome Damages

Chromosome damage was assessed in human peripheral blood lymphocytes after in vitro exposure to the either Si-28 (490 or 600 MeV/n), Ti-48 (1000 MeV/n), or Fe-56 (600, 1000, or 5000 MeV/n). LET values for these ions ranged from approximately 50 to 174 keV/micrometers and doses ranged from 10 to 200 cGy. The effect of either aluminum or polyethylene shielding on the induction of chromosome aberrations was investigated for each ion. Chromosome exchanges were measured using fluorescence in situ hybridization (FISH) with whole chromosome probes in cells collected 48-56 hours after irradiation using a chemical-induced premature chromosome condensation (PCC) technique. The yield of chromosomal aberrations increased linearly with dose and the relative biological effectiveness (RBE) for the primary beams, estimated from the initial slope of the dose response curve for total chromosomal exchanges with respect to gamma-rays, ranged from 14 to 35. The RBE values increased with LET, reaching a maximum for the 1 GeV/n Fe ions with LET of 150 keV/micrometers, and decreased with further increases in LET. When LET of the primary beam was in the region of increasing RBE (i.e. below approximately 100 keV/micrometers), the addition of shielding material increased the effectiveness per unit dose. Whereas shielding decreased the effectiveness per unit dose when the LET of the primary particle beam was higher than 150 keV/micrometers.

George, K.↗

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further investigate the sensitivity differences for low and low high doses, we performed chronic low dose-rate irradiation, and have begun studies with ATM and Nibrin inhibitors and siRNA knockout of these proteins. Results support the conclusion that for the endpoint of simple chromosomal aberrations (translocation or dicentrics), the increased radiation sensitivity of AT cells found at high doses (>1 Gy) does not carry over to low doses or doserates, while NBS cells show increased sensitivity for both high and low dose exposures.

George, K. A.↗

A Monte-Carlo Model for the Formation of Radiation-induced Chromosomal Aberrations

Purpose: To simulate radiation-induced chromosome aberrations in mammalian cells (e.g., rings, translocations, and dicentrics) and to calculate their frequency distributions following exposure to DNA double strand breaks (DSBs) produced by high-LET ions. Methods: The interphase genome was assumed to be comprised of a collection of 2 kbp rigid-block monomers following the random-walk geometry. Additional details for the modeling of chromosomal structure, such as chromosomal domains and chromosomal loops, were included. A radial energy profile for heavy ion tracks was used to simulate the high-LET pattern of induced DSBs. The induced DSB pattern depended on the ion charge and kinetic energy, but always corresponded to the DSB yield of 25 DSBs/cell/Gy. The sum of all energy contributions from Poisson-distributed particle tracks was taken to account for all possible one-track and multi-track effects. The relevant output of the model was DNA fragments produced by DSBs. The DSBs, or breakpoints, were defined by (x, y, z, l) positions, where x, y, z were the Euclidian coordinates of a DSB, and where l was the relative position along the genome. Results: The code was used to carry out Monte Carlo simulations for DSB rejoinings at low doses. The resulting fragments were analyzed to estimate the frequencies of specific types of chromosomal aberrations. Histograms for relative frequencies of chromosomal aberrations and P.D.F.s (probability density functions) of a given aberration type were produced. The relative frequency of dicentrics to rings was compared to empirical data to calibrate rejoining probabilities. Of particular interest was the predicted distribution of ring sizes, irrespective of their frequencies relative to other aberrations. Simulated ring sizes were . 4 kbp, which are far too small to be observed experimentally (i.e., by microscopy) but which, nevertheless, are conjectured to exist. Other aberrations, for example, inversions, translocations, as well as multi-centrics were also recorded. Conclusion: High-LET DNA damage affects the frequencies of chromosomal aberrations. The ratio of rings to dicentrics is correct for the genomic size cut-offs corresponding to available experimental data. The present work predicts a relative abundance of small rings following irradiation by heavy ions.

Ponomarev, Artem L.↗

Distributions of Low- and High-LET Radiation-Induced Breaks in Chromosomes are Associated with Inter- and Intrachromosome Exchanges

To study the breakpoint along the length of the chromosome induced by low- and high-LET radiations, we exposed human epithelial cells in vitro to Cs-137 rays at both low and high dose rates, secondary neutrons at a low dose rate, and 600 MeV/u Fe ions at a high dose rate. The location of the breaks was identified using the multicolor banding in situ hybridization (mBAND) that paints Chromosome 3 in 23 different colored bands. The breakpoint distributions were found to be similar between rays of low and high dose rates and between the two high-LET radiation types. Detailed analysis of the chromosome break ends involved in inter- and intrachromosome exchanges revealed that only the break ends participating in interchromosome exchanges contributed to the hot spots found for low-LET. For break ends participating in intrachromosome exchanges, the distributions for all four radiation scenarios were similar with clusters of breaks found in three regions. Analysis of the locations of the two break ends in Chromosome 3 that joined to form an intrachromosome exchange demonstrated that two breaks with a greater genomic separation may be more likely to rejoin than two closer breaks, indicating that chromatin folding can play an important role in the rejoining of chromosome breaks. Our study demonstrated that the gene-rich regions do not necessarily contain more breaks. The breakpoint distribution depends more on the likelihood that a break will join with another break in the same chromosome or in a different chromosome.

Hada, Megumi↗

Dose Response for Chromosome Aberrations in Human Lymphocytes and Fibroblasts After Exposure to Very Low Dose of High Let Radiation

The relationship between biological effects and low doses of absorbed radiation is still uncertain, especially for high LET radiation exposure. Estimates of risks from low-dose and low-dose-rates are often extrapolated using data from Japanese atomic bomb survivor with either linear or linear quadratic models of fit. In this study, chromosome aberrations were measured in human peripheral blood lymphocytes and normal skin fibroblasts cells after exposure to very low dose (0.01 - 0.20 Gy) of 170 MeV/u Si-28 ions or 600 MeV/u Fe-56 ions, including doses where on average less than one direct ion traversal per cell nucleus occurs. Chromosomes were analyzed using the whole-chromosome fluorescence in situ hybridization (FISH) technique during the first cell division after irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). The responses for doses above 0.1 Gy (more than one ion traverses a cell) showed linear dose responses. However, for doses less than 0.1 Gy, both Si-28 ions and Fe-56 ions showed a dose independent response above background chromosome aberrations frequencies. Possible explanations for our results are non-targeted effects due to aberrant cell signaling [1], or delta-ray dose fluctuations [2] where a fraction of cells receive significant delta-ray doses due to the contributions of multiple ion tracks that do not directly traverse cell nuclei where chromosome aberrations are scored.

Hada, M.↗

Simulation of the Formation of DNA Double Strand Breaks and Chromosome Aberrations in Irradiated Cells

The formation of DNA double-strand breaks (DSBs) and chromosome aberrations is an important consequence of ionizing radiation. To simulate DNA double-strand breaks and the formation of chromosome aberrations, we have recently merged the codes RITRACKS (Relativistic Ion Tracks) and NASARTI (NASA Radiation Track Image). The program RITRACKS is a stochastic code developed to simulate detailed event-by-event radiation track structure: [1] This code is used to calculate the dose in voxels of 20 nm, in a volume containing simulated chromosomes, [2] The number of tracks in the volume is calculated for each simulation by sampling a Poisson distribution, with the distribution parameter obtained from the irradiation dose, ion type and energy. The program NASARTI generates the chromosomes present in a cell nucleus by random walks of 20 nm, corresponding to the size of the dose voxels, [3] The generated chromosomes are located within domains which may intertwine, and [4] Each segment of the random walks corresponds to approx. 2,000 DNA base pairs. NASARTI uses pre-calculated dose at each voxel to calculate the probability of DNA damage at each random walk segment. Using the location of double-strand breaks, possible rejoining between damaged segments is evaluated. This yields various types of chromosomes aberrations, including deletions, inversions, exchanges, etc. By performing the calculations using various types of radiations, it will be possible to obtain relative biological effectiveness (RBE) values for several types of chromosome aberrations.

Plante, Ianik↗

RBE of Energetic Iron Ions for the Induction of Early and Late Chromosome Aberrations in Different Cell Types

Numerous published studies have reported the Relative Biological Effectiveness (RBE) values for chromosome aberrations induced by charged particles of different LET. The RBE for chromosome aberrations in human lymphocytes exposed ex vivo has been suggested to show a similar relationship as the quality factor for cancer induction. Therefore, increased chromosome aberrations in the astronauts' white blood cells post long-duration missions are used to determine the biological doses from exposures to space radiation. However, the RBE value is known to be very different for different types of cancer. Previously, we reported that, even though the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions, the RBE was significantly reduced after multiple cell divisions post irradiation. To test the hypothesis that RBE values for chromosome aberrations are cell type dependent, and different between early and late damages, we exposed human lymphocytes ex vivo, and human mammary epithelial cells in vitro to various charged particles. Chromosome aberrations were quantified using the samples collected at first mitosis post irradiation for initial damages, and the samples collected after multiple generations for the remaining or late arising aberrations. Results of the study suggested that the effectiveness of high-LET charged particles for late chromosome aberrations may be cell type dependent, even though the RBE values are similar for early damages.

Zhang, Ye↗

Biological Effectiveness of Accelerated Particles for the Induction of Chromosome Damage Measured in Metaphase and Interphase Human Lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.

NASA Discipline Radiation Health↗

Characteristics of chromosome instability in the human lymphoblast cell line WTK1

The characteristics of spontaneous and radiation-induced chromosome instability were determined in each of 50 individual clones isolated from control populations of human lymphoblasts (WTK1), as well as from populations of these cells previously exposed to two different types of ionizing radiation, Fe-56 and Cs-137. The types of chromosome instability did not appear to change in clones surviving radiation exposure. Aneuploidy, polyploidy, chromosome dicentrics and translocations, and chromatid breaks and gaps were found in both control and irradiated clones. The primary effect of radiation exposure was to increase the number of cells within any one clone that had chromosome alterations. Chromosome instability was associated with telomere shortening and elevated levels of apoptosis. The results suggest that the proximal cause of chromosome instability is telomere shortening.

NASA Discipline Radiation Health↗

A role for chromosomal instability in the development of and selection for radioresistant cell variants

Chromosome instability is a common occurrence in tumour cells. We examined the hypothesis that the elevated rate of mutation formation in unstable cells can lead to the development of clones of cells that are resistant to the cancer therapy. To test this hypothesis, we compared chromosome instability to radiation sensitivity in 30 independently isolated clones of GM10115 human-hamster hybrid cells. There was a broader distribution of radiosensitivity and a higher mean SF(2)in chromosomally unstable clones. Cytogenetic and DNA double-strand break rejoining assays suggest that sensitivity was a function of DNA repair efficiency. In the unstable population, the more radioresistant clones also had significantly lower plating efficiencies. These observations suggest that chromosome instability in GM10115 cells can lead to the development of cell variants that are more resistant to radiation. In addition, these results suggest that the process of chromosome breakage and recombination that accompanies chromosome instability might provide some selective pressure for more radioresistant variants. Copyright 2001 Cancer Research Campaign.

NASA Discipline Radiation Health↗

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

NASA Center JSC↗

Radiation-induced chromosomal instability in human mammary epithelial cells

Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

NASA Center JSC↗

SCHIP: Statistics for Chromosome Interphase Positioning Based on Interchange Data

he position of chromosomes in the interphase nucleus is believed to be associated with a number of biological processes. Here, we present a web-based application that helps analyze the relative position of chromosomes during interphase in human cells, based on observed radiogenic chromosome aberrations. The inputs of the program are a table of yields of pairwise chromosome interchanges and a proposed chromosome geometric cluster. Each can either be uploaded or selected from provided datasets. The main outputs are P-values for the proposed chromosome clusters. SCHIP is designed to be used by a number of scientific communities interested in nuclear architecture, including cancer and cell biologists, radiation biologists and mathematical/computational biologists.

Vives, Sergi↗