Radiation-induced Chromosome Aberrations Research Articles

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging.

P53 -Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

Functional cell-cycle chromatin conformation changes in the presence of DNA damage result into chromatid breaks: A new insight in the formation of radiation-induced chromosomal aberrations based on the direct observation of interphase chromatin

Experiments were carried out to explore the correlation between chromatin conformation changes in the presence of DNA lesions and the formation of radiation-induced chromosomal aberrations. To modulate the onset and dynamics of chromatin conformation changes following irradiation, premature chromosome condensation (PCC) was induced by means of cell fusion. G2-check point abrogation by caffeine or elevated heat treatment was also applied. In addition, transfer of irradiated mitotic cells was employed either into depleted media to restrain them from proceeding through G1/S, or holding them further in colcemid to avoid M/G1 transition. To investigate the correlation between efficiency of chromosomal conformation changes and chromosomal breakage in irradiated G0 peripheral blood lymphocytes, cell fusion with different mitotic PCC-inducer cells was used. The experimental evidence supports the hypothesis that functional cell-cycle chromatin conformation changes in the presence of DNA damage are important determinants in the formation of radiation-induced chromosomal aberrations. Specifically, it is proposed here that following irradiation, chromatin structure may not be broken but instead it unfolds to a conformation that is more accessible to repair enzymes at the sites of DNA lesions. If subsequent chromosomal conformation changes occur while DNA is still being repaired, such changes will lead into an energetically unfavorable state, thus exerting mechanical stress on the unfolded chromatin at the damaged sites, which will in turn result into chromatid breaks that may not be able to restitute or mis-rejoin. Therefore, this biophysical conversion process of DNA damage into chromatid breaks as such is antagonistic to the DNA repair process. Alternatively, in the absence of chromosomal conformation changes, either DNA repair will take place efficiently or DNA misrepair will cause the formation of exchanges and chromosomal rearrangements. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell cycle stage will be the combined effect of the interaction, at that particular stage, of the DNA repair process and the proposed conversion process of DNA lesions into chromatid breaks.

Biphasic Effects of Nitric Oxide Radicals on Radiation-Induced Lethality and Chromosome Aberrations in Human Lung Cancer Cells Carrying Different p53 Gene Status

The aim of this study was to clarify the effects of nitric oxide (NO) on radiation-induced cell killing and chromosome aberrations in two human lung cancer cell lines with a different p53 gene status. We used wild-type (wt) p53 and mutated (m) p53 cell lines that were derived from the human lung cancer H1299 cell line, which is p53 null. The wtp53 and mp53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pretreated with different concentrations of isosorbide dinitrate (ISDN) (an NO donor) and/or 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (an NO scavenger) and then exposed to X-rays. Cell survival, apoptosis, and chromosome aberrations were scored by use of a colony-forming assay, Hoechst 33342 staining assay and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxyuridine triphosphate] nick end labeling) assay, and chromosomal banding techniques, respectively. In wtp53 cells the induction of radioresistance and the inhibition of apoptosis and chromosome aberrations were observed in the presence of ISDN at low 2- to 10-mumol/L concentrations before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. However, at high concentrations of ISDN (100-500 mumol/L), clear evidence of radiosensitization, enhancement of apoptosis, and chromosome aberrations was detected. However, these phenomena were not observed in mp53 cells at either concentration range with ISDN. These results indicate that low and high concentrations of NO radicals can choreograph inverse radiosensitivity, apoptosis, and chromosome aberrations in human lung cancer cells and that NO radicals can affect the fate of wtp53 cells.

Mechanisms of the formation of radiation-induced chromosomal aberrations

Although much is now known about the mechanisms of radiation-induction of DNA double-strand breaks (DSB), there is less known about the conversion of DSB into chromosomal aberrations. In particular the induction and ‘rejoining’ of chromatid breaks has been a controversial topic for many years. However, its importance becomes clear in the light of the wide variation in the chromatid break response of human peripheral blood lymphocytes from different individuals when exposed to ionizing radiation, and the elevation of the frequency of radiation-induced chromatid breaks in stimulated peripheral blood lymphocytes of around 40% of breast cancer cases. A common assumption has been that chromatid breaks are merely expansions of initiating DSB, although the classic ‘breakage-first’ hypothesis (Sax, Ref. 44) was already challenged in the 50's by Revell [30] who maintained that chromatid breaks were formed as a result of an incomplete exchange process initiated by two interacting lesions of an unspecified nature. Here we argue that both these models of chromatid break formation are flawed and we suggest an alternative hypothesis, namely that a radiation-induced DSB initiates an indirect mechanism leading to a chromatid break. This mechanism we suggest involves the nuclear enzyme topoisomerase IIα and we present evidence from topoisomerase IIα expression variant human cell lines and from siRNA treatment of human cells that supports this hypothesis.

Biphasic effects of an NO donor, ISDN on radiation-induced cell lethal and chromosomal aberration

Biphasic effects of an NO donor, ISDN on radiation-induced cell lethal and chromosomal aberration

Radioprotective and In-Vitro Cytotoxic Sapogenin from Euphorbia neriifolia (Euphorbiaceae) Leaf

Purpose: Euphorbia neriifolia Linn. (Euphorbiaceae) plant is traditionally used in the treatment of abdominal troubles, bronchitis, tumours, leucoderma, piles, inflammation, and enlargement of spleen. The objective of this study was to evaluate the antioxidant and anticancer activities of a sapogenin isolate of this plant. Methods: Euphol was isolated as a major constituent from the triterpenoidal sapogenin fraction of E. neriifolia leaf. Its in-vitro antioxidant activity was evaluated by reducing power assay, 1,1 – diphenyl –2- picryl hydrazyl (DPPH) assay, as well as hydroxyl radical and superoxide radical scavenging activities. Radioprotective activity was assessed against radiation-induced chromosomal aberrations and cytotoxicity on murine F1 B16 melanoma.Results: The sapogenin exerted moderate antioxidant activity with highly significant (p < 0.001) reduction of gamma radiation-induced chromosomal aberrations (33.5 % compared to 71.5 % for radiation treatment alone at 4 Gy). It also exhibited cytotoxic activity on melanoma cell lines (IC50 = 173.78 μg/ml). Conclusion: The sapogenin fraction showed antioxidant, radioprotective and cytotoxic activities. This study provides a scientific basis for the claimed traditional anticarcinogenic potentials of E. neriifolia.Keywords: Euphorbia neriifolia; Euphol; Sapogenin; Antioxidant; Radioprotective; Melanoma; Chromosomal aberration

Cytogenetic Effects of Low Doses of Energetic Carbon Ions on Rice After Exposures of Dry Seeds, Wet Seeds and Seedlings

In order to investigate the biological effects of heavy ion radiation at low doses and the different radiosensitivities of growing and non-growing plants, rice at different lift stages (dry seed, wet seed and seedling) were exposed to carbon ions at doses of 0.02, 0.2, 2 and 20 Gy. Radiobiological effects on survival, root growth and mitotic activity, as well as the induction of chromosome aberrations in root meristem, were observed. The results show that radiation exposure induces a stimulatory response at lower dose and an inhibitory response at higher dose on the mitotic activity of wet seeds and seedlings. Cytogenetic damages are induced in both seeds and seedlings by carbon ion radiation at doses as low as 0.02 Gy. Compared with seedlings, seeds are more resistant to the lethal damage and the growth rate damage by high doses of carbon ions, but are more sensitive to cytogenetic damage by low doses of irradiation. Different types of radiation induced chromosome aberrations are observed between seeds and seedlings. Based on these results, the relationships between low dose heavy ion-induced biological effects and the biological materials are discussed.

Strong association between cancer and genomic instability

After a first wave of radiation-induced chromosomal aberrations, a second wave appears 20-30 cell generations after radiation exposure and persists thereafter. This late effect is usually termed "genomic instability". A better term is "increased genomic instability". This effect has been observed in many cell systems in vitro and in vivo for quite a number of biological endpoints. The radiation-induced increase in genomic instability is apparently a general phenomenon. In the development of cancer, several mutations are involved. With increasing genomic instability, the probability for further mutations is enhanced. Several studies show that genomic instability is increased not only in the cancer cells but also in "normal" cells of cancer patients e.g. peripheral lymphocytes. This has for example been shown in uranium miners with bronchial carcinomas, but also in untreated head and neck cancer patients. The association between cancer and genomic instability is also found in individuals with a genetic predisposition for increased radiosensitivity. Several such syndromes have been found. In all cases, an increased genomic instability, cancer proneness and increased radiosensitivity coincide. In these syndromes, deficiencies in certain DNA-repair pathways occur as well as deregulations of the cell cycle. Especially, mutations are seen in genes encoding proteins, which are involved in the G(1)/S-phase checkpoint. Genomic instability apparently promotes cancer development. In this context, it is interesting that hypoxia, increased genomic instability and cancer are also associated. All these processes are energy dependent. Some strong evidence exists that the structure and length of telomeres is connected to the development of genomic instability.

Effect of storage conditions of blood on radiation-induced chromosomal aberrations and apoptosis in human lymphocytes

To evaluate the effect of storage conditions of blood on the direct relationship between radiation-induced chromosome aberrations and apoptosis in human peripheral blood lymphocytes, whole blood was irradiated with 3Gy X-rays. Directly after irradiation, a sample of blood was analyzed for chromosome damage and proliferation index, after phytohaemagglutinin stimulation and incubation at 37°C for 56h. Blood samples were stored for 48h at 4 and 20°C with or without phytohaemagglutinin and analyzed for cell viability and apoptosis at 0, 24 and 48h storage time. After 48h of storage, unstimulated cultures were stimulated to proliferate. These samples and cultures stimulated immediately before storage were incubated at 37°C for 56h and analyzed for chromosome damage and proliferation index. Metaphases were examined for the presence of dicentrics, excess acentrics, and rings. Storage at 20°C without phytohaemagglutinin for 48h increases significantly the yield of apoptosis and decreases significantly the yield of dicentrics. During 48h of storage time the presence of phytohaemagglutinin and the temperature of 4°C protected the irradiated lymphocytes from apoptosis allowing accurate estimation of the real yield of radiation-induced chromosome damage. Therefore these blood-storage conditions enable analysis in metaphase and may offer some advantages for biodosimetry of absorbed radiation dose.

PCR-based Analysis of Radiation-induced Chromosome Aberrations

The majority of radiation-induced toxicity in carcinoma is linked to aberrant mitoses, a “mitotic catastrophe” driven by chromosomal aberrations, such as dicentrics. Cell survival analysis modeled the production of these rearrangements using a linear quadratic equation with two coefficients, α and β. In this widely used interpretation of cell survival curves, the α (linear) coefficient relates to damage affecting chromosomes simultaneously while the β (square) term describes chromosome damage separated in time.

Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.

Distribution of Breakpoints on Chromatid-type Aberration Induced by Three Different Radiations, in Relation to Fragile Sites

Based on experimental evidence using human blood samples we able to suggest that 'common fragile sites' can be targets for different radiation sources. For that we used peripheral blood samples from three healthy adult donors and exposed them to 60 Co γ-rays (2, 4 Gy) or neutrons (0.8, 2 Gy) at the G 2 stage. In another set, normal bone marrow cells from 19 adult donors were also exposed to 60 Co γ-rays, tritiated water β-rays, and 252 Cf neutrons and sampled 24 hr later. Chromatid breakpoint were identified using Wright's stain G-banding. Analyses of 912 breakpoint detected in lymphocytes and 545 breakpoint detected in bone marrow cells were within the same bands as 113 reported: (1) 41% - 52% of chromatid breakage had a chromosome band of relative fragile sites in both bone marrow cells and lymphocytes; (2) the breakage induced by different radiation sources were distributed in a similar pattern; (3) significantly higher numbers of breakpoint were found at 4q31, 2q35, 3p21, 5q31, 1q32, 2q31, 15q24 and 13q22 in lymphocytes, and at 3p21, 14q24, 17p13, 1q42, 7q22 and 9p11/q11 in bone marrow cells; (4) distribution of breakage was similar between lymphocytes and bone marrow cells except for certain breakpoints. Present study also revealed that a B-cell line established from lymphocytes with a history of 60 Co γ-ray irradiation had more chromosome breakpoints at telomere regions than no-irradiated cell line, which indicating telomere protein might be associated with radiation-induced chromosome instability. This study provides information that will be useful for increasing our understanding of the mechanisms that underlie radiationinduced chromosome aberrations and will aid in assessing genetic and cancer risks in radiation-exposed human populations.

Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of γ-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics

BackgroundThe objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations.ResultsThe relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%.ConclusionNew sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.

Analysis of chromosome aberrations in atomic bomb survivors for dose assessment: studies at the Radiation Effects Research Foundation from 1968 to 1993.

Exposure to ionizing radiation causes damage to living cells, especially to DNA in the cell nucleus. The degree of this cellular damage depends on the amount of radiation administered. This review discusses current findings concerning radiation-induced chromosome aberrations that were produced in 1945 and that can still be observed in the somatic cells of atomic bomb survivors in Hiroshima and Nagasaki. The scoring methods of G-banding and fluorescence in situ hybridization are compared. In addition, some findings concerning chromosomal aberrations in citizens of the former Soviet Union affected by the Chernobyl accident are presented.

Forskolin Decreases Phosphorylation of Histone H2AX in Human Cells Induced by Ionizing Radiation

Forskolin is a natural compound found in the coleus herb that activates the enzyme adenylate cyclase and increases the concentration of intracellular cyclic AMP (cAMP). This chemical is widely used as a stimulating food additive. It is unknown whether forskolin can effect cellular responses to ionizing radiation, such as induction of phosphorylation of histone H2AX (gamma-H2AX) in megabase chromatin domains near DNA double-strand breaks (DSBs). Here we report that treatment with forskolin decreases H2AX phosphorylation after irradiation detected by immunoblotting or by analysis of the overall gamma-H2AX-associated fluorescence in the nuclei. However, this chemical does not affect the number of gamma-H2AX foci, the frequency of radiation-induced chromosome aberrations, or cell survival after X irradiation, which is consistent with the view that it does not change the induction of repair of DSBs. We suggest that the overall decrease of H2AX phosphorylation after treatment with forskolin in irradiated cells reflects a lesser extent of apparent H2AX modification at individual DSBs that may be caused by inhibition of the initial spread of gamma-H2AX and/or by stimulation of elimination of gamma-H2AX from chromatin after DSB rejoining.

Persistence of Radiation-Induced Chromosome Aberrations in a Long-Term Cell Culture

The aim of the present study was to evaluate the persistence of chromosome aberrations induced by X rays. FISH painting and mFISH techniques were applied to long-term cultures of irradiated cells. With painting, at 2 Gy the frequency of apparently simple translocations remained almost invariable during all the culture, whereas at 4 Gy a rapid decline was observed between the first and the second samples, followed by a slight decrease until the end of the culture. Apparently simple dicentrics and complex aberrations disappeared after the first sample at 2 and 4 Gy. By mFISH, at 2 Gy the frequency of complete plus one-way translocations remained invariable between the first and last sample, but at 4 Gy a 60% decline was observed. True incomplete simple translocations disappeared at 2 and 4 Gy, indicating that incompleteness could be a factor to consider when the persistence of translocations is analyzed. The analysis by mFISH showed that the frequency of complex aberrations and their complexity increased with dose and tended to disappear in the last sample. Our results indicate that the influence of dose on the decrease in the frequency of simple translocations with time postirradiation cannot be fully explained by the disappearance of true incomplete translocations and complex aberrations. The chromosome involvement was random for radiation-induced exchange aberrations and non-random for total aberrations. Chromosome 7 showed the highest deviations from expected, being less and more involved than expected in the first and last samples, respectively. Some preferential chromosome-chromosome associations were observed, including a coincidence with a cluster from radiogenic chromosome aberrations described in other studies.

Effects of superoxide dismutase and catalase on radiation-induced chromosome aberrations: dose and cell cycle dependence.

The frequency of chromosome aberrations induced by radiation in G0 and G2 was significantly reduced by adding the enzymes superoxide dismutase and/or catalase to lymphocytes either before or after their irradiation. A concentration of 5 times 10-2 mg enzyme/ml medium had a maximal effect. Heat-inactivated enzymes had no effect, nor had Cu2+-ions.

Influence of glutathione levels on radiation-induced chromosomal DNA damage and repair in human peripheral lymphocytes

Endogenous thiols, especially the tripeptide-reduced glutathione (GSH), are known to play an important role in cellular defense against radiation. However, there are evidences that suggest that GSH may not be an efficient protector of DNA. The present study will determine whether modulation of endogenous GSH levels protects or potentiates the amount of chromosomal damage induced by ionizing radiation (IR). Human lymphocytes were isolated and then treated with GSH (for 1 h) or buthionine sulfoximine (BSO; GSH-depleting agent for 5 h) before X-irradiation. DNA damage was analyzed by scoring chromosome aberrations (CAs) and by comet assay. The level of endogenous GSH was measured in lymphocytes treated with GSH, BSO or X-rays. A roughly 20% increase in endogenous GSH level was observed after a 3-h treatment with exogenous GSH and this reduced the frequency of all types of CA and aberrant metaphase chromosomes induced by 1 and 2 Gy of X-rays and also decreased the tail moment as determined by comet assay, suggesting radiation protection. Such uniform protection by GSH pretreatment was not visible while cells were exposed to 3 Gy or higher. Interestingly, in GSH-depleted lymphocytes, the frequency of radiation-induced CA was increased in a non-uniform manner. Therefore, an increase in the level of endogenous GSH in lymphocytes was unable to reduce chromosomal damage induced by 3 Gy or above, whereas decrease in the level of GSH enhanced the frequency of CA at all radiation doses in a non-uniform manner. It seems that GSH did not act as a radioprotector against DNA damage induced by higher dose X-rays rather it acts as a modulator of DNA repair activity.