Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

Exposure Worry: The Psychological Impact of Perceived Ionizing Radiation Exposure in British Nuclear Test Veterans.

Potential psychological issues faced by British nuclear test veterans have been under-researched. This study assessed the prevalence of clinically relevant anxiety in British nuclear test veterans and aimed to explore experiences of worry and the broader psychological impact of the British nuclear weapons testing programme. The Geriatric Anxiety Inventory (Short-Form) was completed by 89 British nuclear test veterans (33.7% met the criteria for clinically relevant anxiety). Nineteen veterans then participated in semi-structured interviews. Thematic analysis of the data generated three themes. The first theme highlighted how worry was relevant only in a few cases (four) generally regarding their grandchildren's health, but the guilt in those who perceive responsibility for family health conditions also appeared to be a pertinent issue. The second theme highlighted the anger towards authorities resulting from perceived negligence and deception. The third theme highlighted the relevance of how certain life events across the life course influence the potential psychological impact. This study suggests that guilt must be considered in (potentially) exposed individuals whose family members experience health conditions, which may exacerbate distress. It also suggests the importance that authorities ensure transparency when dealing with any radiological exposure scenario to reduce the potential for anger.

The psychological consequences of (perceived) ionizing radiation exposure: a review on its role in radiation-induced cognitive dysfunction.

PURPOSE: Exposure to ionizing radiation following environmental contamination (e.g., the Chernobyl and Fukushima nuclear accidents), radiotherapy and diagnostics, occupational roles and space travel has been identified as a possible risk-factor for cognitive dysfunction. The deleterious effects of high doses (≥1.0 Gy) on cognitive functioning are fairly well-understood, while the consequences of low (≤0.1 Gy) and moderate doses (0.1-1.0 Gy) have been receiving more research interest over the past decade. In addition to any impact of actual exposure on cognitive functioning, the persistent psychological stress arising from perceived exposure, particularly following nuclear accidents, may itself impact cognitive functioning. In this review we offer a novel interdisciplinary stance on the cognitive impact of radiation exposure, considering psychological and epidemiological observations of different exposure scenarios such as atomic bombings, nuclear accidents, occupational and medical exposures while accounting for differences in dose, rate of exposure and exposure type. The purpose is to address the question that perceived radiation exposure - even where the actual absorbed dose is 0.0 Gy above background dose - can result in psychological stress, which could in turn lead to cognitive dysfunction. In addition, we highlight the interplay between the mechanisms of perceived exposure (i.e., stress) and actual exposure (i.e., radiation-induced cellular damage), in the generation of radiation-induced cognitive dysfunction. In all, we offer a comprehensive and objective review addressing the potential for cognitive defects in the context of low- and moderate-dose IR exposures. CONCLUSIONS: Overall the evidence shows prenatal exposure to low and moderate doses to be detrimental to brain development and subsequent cognitive functioning, however the evidence for adolescent and adult low- and moderate-dose exposure remains uncertain. The persistent psychological stress following accidental exposure to low-doses in adulthood may pose a greater threat to our cognitive functioning. Indeed, the psychological implications for instructed cohorts (e.g., astronauts and radiotherapy patients) is less clear and warrants further investigation. Nonetheless, the psychosocial consequences of low- and moderate-dose exposure must be carefully considered when evaluating radiation effects on cognitive functioning, and to avoid unnecessary harm when planning public health response strategies.

Meeting report on ICRR2019, the 16th International Congress on Radiation Research.

The 16th International Congress of Radiation Research (ICRR2019) was held in Manchester, UK, in August 2019. The Congress, which is held every four years, covered a wide spectrum of topics relevant for all aspects of radiation research including basic mechanisms, translational research, radiotherapy and health effects, and ecology. Here, we provide a report of the plenary and keynote talks presented at the meeting.

DNA Damage Processing is Perturbed in Both Proliferative and Non-Proliferative Cells of Increased Chronological Cellular Age.

p53BP1 forms discrete foci within minutes of radiation exposure, at sites of DNA double-strand breaks, which ordinarily decay to background levels within 24 h of induction. Longer lived, persisting 53BP1 foci are thought to mark unrepaired or misrepaired damage and potentially, to be associated with genomic instability. It is known that repair of DNA damage is impaired in senescent (permanently arrested) and aged cells. We examined this further by measuring the induction and persistence of 53BP1 foci in proliferating and non-proliferating mid-passage (non-aged) and late-passage (in vitro aged) normal human bronchial epithelial cells. Our results showed background levels of 53BP1 foci to be elevated in in vitro aged cultures as expected and induction of 53BP1 foci after radiation exposure to be independent of culture age or proliferative status. In terms of 53BP1 decay, more cells with persisting foci were seen in in vitro aged cultures compared to non-aged populations; furthermore, this was observed in both non-cycling (nominally senescent) cells, as well as in actively proliferating cells. In conclusion, perturbation in radiation-induced damage processing is a function of increasing chronological cellular age per se and should be considered when extrapolating experimental data for radiation risk modeling.

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells.

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.

TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription.

DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine.

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.

mFISH analysis reveals complexity of chromosome aberrations in individuals occupationally exposed to internal plutonium: a pilot study to assess the relevance of complex aberrations as biomarkers of exposure to high-LET alpha particles.

We recently demonstrated that a significant proportion of apparently stable insertions induced after exposure to a mean of one alpha particle/cell, detected using three-color FISH, were part of larger unstable complexes when visualized by 24-color FISH. Interestingly, regardless of the long-term persistence capability of the cell, the complexity of each alpha-particle-induced complex appeared to be specific to the nuclear traversal of a single alpha particle. To assess whether aberrations of a similar complexity are observed in vivo and also to examine the usefulness of detecting such aberrations as a biomarker of chronic exposure to alpha particles, we have carried out a limited pilot study of Russian workers with large body burdens of alpha-particle-emitting plutonium. We found unstable cells containing non-transmissible complex aberrations in all of the plutonium-exposed subjects analyzed by mFISH. In addition, all of the complexes seen were consistent with those previously observed in vitro. Non-transmissible complex aberrations were more common than transmissible-type complexes, consistent with ongoing/chronic exposure, and insertions were dominant features of both types of complex. Accordingly, this preliminary study supports the proposal that aberration complexity and non-transmissibility are the major cytogenetic features of alpha-particle exposure that could potentially be exploited as a specific indicator of chronic exposures to high-LET alpha particles.

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles.

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.

Reduced chromosome aberration complexity in normal human bronchial epithelial cells exposed to low-LET γ-rays and high-LET α-particles.

PURPOSE: Cells of the lung are at risk from exposure to low and moderate doses of ionizing radiation from a range of environmental and medical sources. To help assess human health risks from such exposures, a better understanding of the frequency and types of chromosome aberration initially-induced in human lung cell types is required to link initial DNA damage and rearrangements with transmission potential and, to assess how this varies with radiation quality. MATERIALS AND METHODS: We exposed normal human bronchial lung epithelial (NHBE) cells in vitro to 0.5 and 1 Gy low-linear energy transfer (LET) γ-rays and a low fluence of high-LET α-particles and assayed for chromosome aberrations in premature chromosome condensation (PCC) spreads by 24-color multiplex-fluorescence in situ hybridization (M-FISH). RESULTS: Both simple and complex aberrations were induced in a LET and dose-dependent manner; however, the frequency and complexity observed were reduced in comparison to that previously reported in spherical cell types after exposure to comparable doses or fluence of radiation. Approximately 1-2% of all exposed cells were categorized as being capable of transmitting radiation-induced chromosomal damage to future NHBE cell generations, irrespective of dose. CONCLUSION: One possible mechanistic explanation for this reduced complexity is the differing geometric organization of chromosome territories within ellipsoid nuclei compared to spherical nuclei. This study highlights the need to better understand the role of nuclear organization in the formation of exchange aberrations and, the influence three-dimensional (3D) tissue architecture may have on this in vivo.

Cellular Senescence - its role in cancer and the response to ionizing radiation.

Cellular senescence is a normal biological process that is initiated in response to a range of intrinsic and extrinsic factors that functions to remove irreparable damage and therefore potentially harmful cells, from the proliferative pool. Senescence can therefore be thought of in beneficial terms as a tumour suppressor. In contrast to this, there is a growing body of evidence suggesting that senescence is also associated with the disruption of the tissue microenvironment and development of a pro-oncogenic environment, principally via the secretion of senescence-associated pro-inflammatory factors. The fraction of cells in a senescent state is known to increase with cellular age and from exposure to various stressors including ionising radiation therefore, the implications of the detrimental effects of the senescent phenotype are important to understand within the context of the increasing human exposure to ionising radiation. This review will discuss what is currently understood about senescence, highlighting possible associations between senescence and cancer and, how exposure to ionising radiation may modify this.

M-FISH analysis shows that complex chromosome aberrations induced by alpha -particle tracks are cumulative products of localized rearrangements.

Complex chromosome aberrations are characteristically induced after exposure to low doses of densely ionizing radiation, but little is understood about their formation. To address this issue, we irradiated human peripheral blood lymphocytes in vitro with 0.5 Gy densely ionizing alpha-particles (mean of 1 alpha-particle/cell) and analyzed the chromosome aberrations produced by using 24-color multiplex fluorescence in situ hybridization (M-FISH). Our data suggest that complex formation is a consequence of direct nuclear alpha-particle traversal and show that the likely product of illegitimate repair of damage from a single alpha-particle is a single complex exchange. From an assessment of the "cycle structure" of each complex exchange we predict alpha-particle-induced damage to be repaired at specific localized sites, and complexes to be formed as cumulative products of this repair.