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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

Non-NASA Center↗
NASA NTRS2001

The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

NASA Discipline Radiation Health↗
NASA NTRS2001

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Non-NASA Center↗
NASA NTRS2001

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.

Non-NASA Center↗
NASA NTRS2000

Chromosomal instability induced by heavy ion irradiation

PURPOSE: To establish the dose-response relationship for the induction of chromosomal instability in GM10115 cells exposed to high-energy iron ions (1 GeV/nucleon, mean LET 146 keV/microm) and gold ions (11 GeV/nucleon, mean LET 1450 keV/microm). Past work has established that sparsely ionizing X-rays can induce a long-lived destabilization of chromosomes in a dose-dependent manner at an incidence of approximately 3% per gray. The present investigation assesses the capacity of High-Z and High-energy (HZE) particles to elicit this same endpoint. MATERIALS AND METHODS: Clonal populations derived from single progenitor cells surviving heavy-ion irradiation were analyzed cytogenetically to identify those clones showing a persistent destablization of chromosomes. RESULTS: Dose-response data, with a particular emphasis at low dose (< 1.0 Gy), indicate a frequency of approximately 4% per gray for the induction of chromosomal instability in clones derived from single progenitor cells surviving exposure to iron ions. The induction of chromosomal instability by gold ions was, however, less responsive to applied dose, as the observed incidence of this phenotype varied from 0 to 10% over 1-8 Gy. Both iron and gold ions gave dose-dependent increases in the yield of chromosomal aberrations (both chromosome- and chromatid-type) measured at the first mitosis following irradiation, as well as shoulderless survival curves having D0=0.87 and 1.1 Gy respectively. CONCLUSIONS: Based on the present dose-response data, the relative biological effectiveness of iron ions is 1.3 for the induction of chromosomal instability, and this indicates that heavy ions are only slightly more efficient than X-rays at eliciting this delayed phenotype.

Non-NASA Center↗
NASA NTRS2000

IR spectroscopic characteristics of cell cycle and cell death probed by synchrotron radiation based Fourier transform IR spectromicroscopy

Synchrotron radiation based Fourier transform IR (SR-FTIR) spectromicroscopy allows the study of individual living cells with a high signal to noise ratio. Here we report the use of the SR-FTIR technique to investigate changes in IR spectral features from individual human lung fibroblast (IMR-90) cells in vitro at different points in their cell cycle. Clear changes are observed in the spectral regions corresponding to proteins, DNA, and RNA as a cell changes from the G(1)-phase to the S-phase and finally into mitosis. These spectral changes include markers for the changing secondary structure of proteins in the cell, as well as variations in DNA/RNA content and packing as the cell cycle progresses. We also observe spectral features that indicate that occasional cells are undergoing various steps in the process of cell death. The dying or dead cell has a shift in the protein amide I and II bands corresponding to changing protein morphologies, and a significant increase in the intensity of an ester carbonyl C===O peak at 1743 cm(-1) is observed. Copyright John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 57: 329-335, 2000.

Non-NASA Center↗
NASA NTRS2000

The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

Review, Tutorial↗
NASA NTRS1999

Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization

We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.

NASA Discipline Cell Biology↗
NASA NTRS1999

Centrosome and microtubule instability in aging Drosophila cells

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

Non-NASA Center↗
NASA NTRS1998

Rejoining and misrejoining of radiation-induced chromatin breaks. III. Hypertonic treatment

It has been shown that treatment in anisotonic medium modifies rejoining of radiation-induced breaks in interphase chromosomes. In previous work, we have demonstrated that formation of exchanges in human lymphocytes has a slow component (half-time of 1-2 h), but a fraction of exchanges are also observed in samples assayed soon after exposure. In this paper we studied the effect of hypertonic treatment on rejoining and misrejoining of radiation-induced breaks using fluorescence in situ hybridization of prematurely condensed chromosomes in human lymphocytes. Isolated lymphocytes were irradiated with 7 Gy gamma rays, fused to mitotic hamster cells and incubated in hypertonic solution (0.5 M NaCl) for the period normally allowed for interphase chromosome condensation to occur. The data from hypertonic treatment experiments indicate the presence of a class of interphase chromosome breaks that rejoin and misrejoin very quickly (half-time of 5-6 min). The fast misrejoining of these lesions is considered to be responsible for the initial level of exchanges which we reported previously. No significant effect of hypertonic treatment on the yield of chromosome aberrations scored at the first postirradiation mitosis was detected.

NASA Discipline Radiation Health↗
NASA NTRS1997

Induction of chromosome aberrations in human cells by charged particles

Chromosome aberrations induced by high-energy charged particles in normal human lymphocytes and human fibroblasts have been investigated. The charged particles included 250 MeV/nucleon protons, 290 MeV/nucleon carbon ions and 1 GeV/nucleon iron ions. The energies of the charged particles were higher than in most of the studies reported in the literature. Lymphocytes were stimulated to grow immediately after irradiation, while fibroblasts were incubated at 37 degrees C for 24 h for repair. Chromosomes were collected at the first mitosis after irradiation and chromosome aberrations were scored using the fluorescence in situ hybridization (FISH) technique with a whole-chromosome 4 probe. Chromosome aberrations were classified as reciprocal exchanges, incomplete exchanges, deletions and complex exchanges. The relative biological effectiveness (RBE) for each type of aberration was calculated by dividing a dose of 4 Gy by the dose of the charged particles producing the same effect as 4 Gy of gamma rays. Results of this study showed that complex aberrations have the highest RBE for radiation of high linear energy transfer (LET) for human lymphocytes, but for fibroblasts, the greatest effect was for incomplete exchanges. For both lymphocytes and fibroblasts, iron ions induced a similar fraction of aberrant cells.

NASA Center JSC↗
NASA NTRS1997

Biodosimetry of ionizing radiation by selective painting of prematurely condensed chromosomes in human lymphocytes

Painting of interphase chromosomes can be useful for biodosimetric purposes in particular cases such as radiation therapy, accidental exposure to very high radiation doses and exposure to densely ionizing radiation, for example during space missions. Biodosimetry of charged-particle radiation is analyzed in the present paper. Target cells were human peripheral blood lymphocytes irradiated in vitro with gamma rays, protons and iron ions. After exposure, lymphocytes were incubated for different times to allow repair of radiation-induced damage and then fused to mitotic hamster cells to promote premature condensation in the interphase chromosomes. Chromosome spreads were then hybridized with whole-chromosome DNA probes labeled with fluorescent stains. Dose-response curves for the induction of chromatin fragments shortly after exposure, as well as the kinetics of rejoining and misrejoining, were not markedly dependent on linear energy transfer. However, after exposure to heavy ions, more aberrations were scored in the interphase cells after incubation for repair than in metaphase samples harvested at the first postirradiation mitosis. On the other hand, no significant differences were observed in the two samples after exposure to sparsely ionizing radiation. These results suggest that interphase chromosome painting can be a useful tool for biodosimetry of particle radiation.

NASA Center JSC↗
NASA NTRS1997

Biodosimetry results from space flight Mir-18

Astronauts are classified as radiation workers due to the presence of ionizing radiation in space. For the assessment of health risks, physical dosimetry has been indispensable. However, the change of the location of dosimeters on the crew members, the variation in dose rate with location inside the spacecraft and the unknown biological effects of microgravity can introduce significant uncertainties in estimating exposure. To circumvent such uncertainty, a study on the cytogenetic effects of space radiation in human lymphocytes was proposed and conducted for Mir-18, a 115-day mission. This study used fluorescence in situ hybridization (FISH) with whole-chromosome painting probes to score chromosomal exchanges and the Giemsa staining method to determine the frequency of dicentrics. The growth kinetics of cells and sister chromatid exchanges (SCEs) were examined to ensure that chromosomal aberrations were scored in the first mitosis and were induced primarily by space radiation. Our results showed that the frequency of chromosomal aberrations increased significantly in postflight samples compared to samples drawn prior to flight, and that the frequency of SCEs was similar for both pre- and postflight samples. Based on a dose-response curve for preflight samples exposed to gamma rays, the absorbed dose received by crew members during the mission was estimated to be about 14.75 cSv. Because the absorbed dose measured by physical dosimeters is 5.2 cGy for the entire mission, the RBE is about 2.8.

NASA Discipline Radiation Health↗
NASA NTRS1997

Cytogenetic effects of space radiation in lymphocytes of MIR-18 crews

For assessing health risk, the measurement of physical dose received during a space mission, as well as the LETs, energies and charges of particles is important. It is also important to obtain quantitative information regarding the effectiveness of space radiation in causing damage to critical biological targets, e.g., chromosomes, since at present the estimated uncertainty of biological effects of space radiation is more than a factor of two. Such large uncertainty makes accurate health risk assessment very difficult. For this very reason, a study on cytogenetic effects of space radiation in human lymphocytes was proposed and done for MIR-18 mission. This study used FISH technique to score chromosomal translocations and C-banding method to determine dicentrics. Growth kinetics of cells and SCE were examined to ensure that chromosomal aberrations were scored in first mitosis and were induced not by chemical mutagens. Our results showed that chromosomal aberration frequency of post-flight samples was significantly higher than that of pre-flight ones and that SCE frequency was similar between pre- and post-flight samples. Based on a dose-response curve of preflight samples exposed to gamma rays, the absorbed dose received by crews during the mission was estimated to be about 14.5 cSv. Because the absorbed dose measured by physical dosimeters is 4.16 cGy for the entire mission, the RBE is about 3.5.

Mir Project↗
NASA NTRS1994

Radiation-induced genomic instability

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

NASA Discipline Radiation Health↗
NASA NTRS1992

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

NASA Discipline Plant Biology↗
NASA NTRS2007

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↗
NASA NTRS2009

Analysis of Chromosomal Aberrations after Low and High Dose Rate Gamma Irradiation in ATM or NBS Suppressed Human Fibroblast Cells

A detailed understanding of the biological effects of heavy nuclei is needed for space radiation protection and for cancer therapy. High-LET radiation produces more complex DNA lesions that may be non-repairable or that may require additional processing steps compared to endogenous DSBs, increasing the possibility of misrepair. Interplay between radiation sensitivity, dose, and radiation quality has not been studied extensively. Previously we studied chromosome aberrations induced by low- and high- LET radiation in several cell lines deficient in ATM (ataxia telangactasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; 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. We found that the yields of both simple and complex chromosomal aberrations were significantly increased in the DSB repair defective cells compared to normal cells. The increased aberrations observed for the ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher in NBS cells only for simple exchanges. These results point to the importance of the functions of ATM and NBS in chromatin modifications that function to facilitate correct DSB repair and minimize aberration formation. To further understand the sensitivity differences that were observed in ATM and NBS deficient cells, in this study, chromosomal aberration analysis was performed in normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, or Mirin, an MRN complex inhibitor involved in activation of ATM. We are also testing siRNA knockdown of these proteins. Normal and ATM or NBS suppressed cells were irradiated with gamma-rays and chromosomes were collected with a premature chromosome condensation (PCC) technique at the first mitosis post-irradiation. Chromosomes were analyzed using a multicolor fluorescence in-situ hybridization (mFISH) chromosome painting method. Preliminary analysis showed that chromosomal exchanges were increased in the cells treated with the specific ATM inhibitor. Possible cytogenetic signatures of acute and low dose-rate gamma irradiation in ATM or Nibrin deficient and suppressed cells will be discussed.

Hada, M.↗