Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.     

Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Delayed cell cycle progression in human lymphoblastoid cells after exposure to high-LET radiation correlates with extremely localized DNA damage

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G(2)/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.     

Cell cycle delay in murine pre-osteoblasts is more pronounced after exposure to high-LET compared to low-LET radiation

Space radiation contains a complex mixture of particles comprised primarily of protons and high-energy heavy ions. Radiation risk is considered one of the major health risks for astronauts who embark on both orbital and interplanetary space missions. Ionizing radiation dose-dependently kills cells, damages genetic material, and disturbs cell differentiation and function. The immediate response to ionizing radiation-induced DNA damage is stimulation of DNA repair machinery and activation of cell cycle regulatory checkpoints. To date, little is known about cell cycle regulation after exposure to space-relevant radiation, especially regarding bone-forming osteoblasts. Here, we assessed cell cycle regulation in the osteoblastic cell line OCT-1 after exposure to various types of space-relevant radiation. The relative biological effectiveness (RBE) of ionizing radiation was investigated regarding the biological endpoint of cellular survival ability. Cell cycle progression was examined following radiation exposure resulting in different RBE values calculated for a cellular survival level of 1 %. Our findings indicate that radiation with a linear energy transfer (LET) of 150 keV/μm was most effective in inducing reproductive cell killing by causing cell cycle arrest. Expression analyses indicated that cells exposed to ionizing radiation exhibited significantly up-regulated p21(CDKN1A) gene expression. In conclusion, our findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.     

Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation

Primary mouse ear and kidney cultures were established for determination of cytogenetic aberrations at short (3 days to 1 month) and long (12-23 months) times after exposure of their right sides to 7.5 Gy of (137)Cs gamma radiation. In every case, higher levels of aberrations were observed in primary cultures established from the irradiated tissues than in those established from the contralateral tissues. The most common aberrations in the contralateral tissues and those from nonirradiated mice were chromatid and isochromatid breaks and small chromatid fragments. Primary cells from irradiated tissues removed from animals within a month of exposure displayed a variety of unstable chromosome-type aberrations characteristic of recent exposure to ionizing radiation including rings, dicentrics, double minutes, and large acentric fragments. The percentages of cells exhibiting chromatid breaks and small chromatid fragments were also markedly elevated. Although the levels of chromosome-type aberrations found in primary cells from irradiated tissues dropped to near background levels a year or more after exposure, chromatid-type aberrations remained elevated. These results are consistent with long-term persistence of damage in the genomes of ionizing radiation-exposed cells in solid tissues and the induction of genomic instability in vivo.     

Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.     

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.     

mBAND analysis of chromosomal aberrations in human epithelial cells exposed to low- and high-LET radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.     

Retention of tumorigenicity in radioresistant progeny of cells surviving exposure to high doses of gamma irradiation

The cell tumorigenic ability and the cell clonogenicity in semi-solid medium of highly radioresistant variant cell line, PIC-20 (the progeny of djungarian hamster fibroblast cell line DX-TK- surviving acute exposure to 20 Gy of gamma-irradiation), were examined. In the absence of additional radiation, no differences between tested features of non-irradiated PIC-20 cells and parental DX-TK- cells were observed. On the contrary, after gamma-irradiation with high doses the essential differences in the properties of the examined cell lines were revealed. After exposure to 10 Gy the surviving fraction of PIC-20 cells was 20 times higher than that of the parental cells. Both irradiated and non-irradiated PIC-20 cells produced colonies of similar size. It is revealed that even after irradiation with doses of 5, 10 or 15 Gy, the PIC-20 cells kept their tumorigenicity as high as non-irradiated ones. In all these cases the 90-100% of animals had the tumour, with the average latent period of tumour appearance after inoculation being the same both for irradiated and non-irradiated PIC-20 cells. After irradiation of parental DX-TK- cells with the highest dose of 15 Gy, the amount animals with tumour decreased by 70% and the average latent period of tumour appearance increased fivefold as compared with that for non-irradiated DX-TK- cells. The data obtained indicate that PIC-20 is highly radioresistant cells, which are able to proliferate both in semi-solid medium and in an animal organism even after radiation exposure to high doses.     

Brain signaling and behavioral responses induced by exposure to (56)Fe-particle radiation

Previous experiments have demonstrated that exposure to 56Fe-particle irradiation (1.5 Gy, 1 GeV) produced aging-like accelerations in neuronal and behavioral deficits. Astronauts on long-term space flights will be exposed to similar heavy-particle radiations that might have similar deleterious effects on neuronal signaling and cognitive behavior. Therefore, the present study evaluated whether radiation-induced spatial learning and memory behavioral deficits are associated with region-specific brain signaling deficits by measuring signaling molecules previously found to be essential for behavior [pre-synaptic vesicle proteins, synaptobrevin and synaptophysin, and protein kinases, calcium-dependent PRKCs (also known as PKCs) and PRKA (PRKA RIIbeta)]. The results demonstrated a significant radiation-induced increase in reference memory errors. The increases in reference memory errors were significantly negatively correlated with striatal synaptobrevin and frontal cortical synaptophysin expression. Both synaptophysin and synaptobrevin are synaptic vesicle proteins that are important in cognition. Striatal PRKA, a memory signaling molecule, was also significantly negatively correlated with reference memory errors. Overall, our findings suggest that radiation-induced pre-synaptic facilitation may contribute to some previously reported radiation-induced decrease in striatal dopamine release and for the disruption of the central dopaminergic system integrity and dopamine-mediated behavior.     

An analysis of the distribution and dose response of chromosome aberrations in human lymphocytes after in vitro exposure to137Cesium gamma radiation

The chromosome aberration yield for human lymphocytes exposed in vitro to various doses of 137Cesium has been studied. Dicentric, total acentric, and excess acentric data were seen to follow a Possion distribution. Calculated total hits demonstrated over-dispersion which could possibly be accounted for by a greater occurrence of single-hit phenomena being repaired than two-hit exchange processes. The resulting distribution generally contained an under-representation of cells with odd numbers of hits and an over-representation of zero- and even-hit classes as compared with Poisson predicted values. The relationship between dicentric yield and dose received in rads was fitted to the linear-quadratic formula Y = alpha D + beta D2 for dicentrics, yielding values of (20.1 +/- 3.8) X 10(-4) (aberrations/cell)/rad and (1.89 +/- 0.75) X 10(-6) (aberrations/cell)/rad2 for alpha and beta respectively. A plot of percent 'normal' cells versus the dose in rads resembled cell survival curves and was fitted to the relation P(D) = 100 e-Y where Y = alpha D + beta D2 with alpha = (23 +/- 11) X 10(-4) rad-1 and beta = (8.3 +/- 2.5) X 10(-6) rad-2. A possible use of scoring 'normal' cells for purposes of biological dosimetry is presented.     

Effects of exposure to low-dose-rate (60)co gamma rays on human tumor cells in vitro

Cells of three asynchronously growing human tumor cell lines, PC3 (human prostate carcinoma), T98G and A7 (human glioblastomas), which have been shown previously to demonstrate low-dose hyper-radiosensitivity to low acute single doses, were irradiated with (60)Co gamma rays at low dose rates (2 cGy-1 Gy h(-1)). Instead of a dose-rate sparing response, these cell lines demonstrated an inverse dose-rate effect on cell survival at dose rates below 1 Gy h(-1), whereby a decrease in dose rate resulted in an increase in cell killing per unit dose. A hyper-radiosensitivity-negative cell line, U373MG, did not demonstrate an inverse dose-rate effect. Analysis of the cell cycle indicated that this inverse dose-rate effect was not due to accumulation of cells in G(2)/M phase or to other cell cycle perturbations. T98G cells in reversible G(1)-phase arrest also showed an inverse dose-rate effect at dose rates below 30 cGy h(-1) but a sparing effect as the dose rate was reduced from 60 to 30 cGy h(-1). We conclude that this inverse dose-rate effect in continuous exposures reflects the hyper-radiosensitivity seen in the same cell lines in response to very small acute single doses.     

Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.     

Accumulation of the cell cycle regulators TP53 and CDKN1A (p21) in human fibroblasts after exposure to low- and high-LET radiation

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV microm(-1)) and xenon, bismuth and uranium ions (8900-15,000 keV microm(-1)) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900-15,000 keV microm(-1)) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.     

Sensitizing human cervical cancer cells In vitro to ionizing radiation with interferon beta or gamma.

Human cervical cancer is often associated with human papilloma virus (HPV). HPV products, such as the oncoproteins E6 and E7, are known to disrupt the function of TP53 (formerly known as p53). The protein encoded by the TP53 gene plays a central role in managing cellular damage. Interferons are known to down-regulate E6/E7 and may therefore restore TP53 function and influence radiation sensitivity. We investigated whether IFNB or IFNG, at various concentrations (2- 300 IU/ml) and for a range of durations of exposure (from 48 h before to 8 h after irradiation), were able to modify the radiation response of HeLa, C4-1, Me-180, C33-A and SiHa cells. In parallel to the clonogenic assays, we analyzed the effect on the mRNA that encodes IFNB and E6 by Northern blotting in the same experimental conditions. A significant change in the initial slope of the dose-response curve was observed more consistently with IFNB than with IFNG. No changes in the mRNA or protein level of TP53 and E6 could be detected. Thus other mechanisms of action need to be investigated to explain radiosensitization with recombinant IFNB in cells of human cervical cancer cell lines.