Candidate biomarkers and persistent transcriptional responses after low and high dose ionizing radiation at high dose rate.

PURPOSE: Development of an integrated time and dose model to explore the dynamics of gene expression alterations and identify biomarkers for biodosimetry following low- and high-dose irradiations at high dose rate. MATERIAL AND METHODS: We utilized multiple transcriptome datasets (GSE8917, GSE43151, and GSE23515) from Gene Expression Omnibus (GEO) for identifying candidate biological dosimeters. A linear mixed-effects model with random intercept was used to explore the dose-time dynamics of transcriptional responses and to functionally characterize the time- and dose-dependent changes in gene expression. RESULTS: We identified genes that are correlated with dose and time and discovered two clusters of genes that are either positively or negatively correlated with both dose and time based on the parameters of the model. Genes in these two clusters may have persistent transcriptional alterations. Twelve potential transcriptional markers for dosimetry-ARHGEF3, BAX, BBC3, CCDC109B, DCP1B, DDB2, F11R, GADD45A, GSS, PLK3, TNFRSF10B, and XPC were identified. Of these genes, BAX, GSS, and TNFRSF10B are positively associated with both dose and time course, have a persistent transcriptional response, and might be better biological dosimeters. CONCLUSIONS: With the proposed approach, we may identify candidate biomarkers that change monotonically in relation to dose, have a persistent transcriptional response, and are reliable over a wide dose range.

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells.

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

ATM-dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure.

Following radiation exposure, unrepaired DNA double-strand breaks (DSBs) persist to some extent in a subset of cells as residual damage; they can exert adverse effects, including late-onset diseases. In search of the factor(s) that characterize(s) cells bearing such damage, we discovered ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of the transcription factor chromodomain helicase DNA binding protein 7 (CHD7). CHD7 controls the morphogenesis of cell populations derived from neural crest cells during vertebrate early development. Indeed, malformations in various fetal bodies are attributable to CHD7 haploinsufficiency. Following radiation exposure, CHD7 becomes phosphorylated, ceases promoter/enhancer binding to target genes, and relocates to the DSB-repair protein complex, where it remains until the damage is repaired. Thus, ATM-dependent CHD7 phosphorylation appears to act as a functional switch. As such stress responses contribute to improved cell survival and canonical nonhomologous end joining, we conclude that CHD7 is involved in both morphogenetic and DSB-response functions. Thus, we propose that higher vertebrates have evolved intrinsic mechanisms underlying the morphogenesis-coupled DSB stress response. In fetal exposure, if the function of CHD7 becomes primarily shifted toward DNA repair, morphogenic activity is reduced, resulting in malformations.

The Association of Radiation Exposure with Stable Chromosome Aberrations in Atomic Bomb Survivors Based on DS02R1 Dosimetry and FISH Methods.

The frequency of stable chromosome aberrations (sCA) in lymphocytes is a recognized radiation biological dosimeter. Its analysis can provide insights into factors that affect individual susceptibility as well as into the adequacy of radiation dose estimates used in studies of atomic bomb survivors. We analyzed the relationship between atomic bomb radiation exposure using the most recent DS02R1 dose estimates and the frequency of sCA as determined by FISH in 1,868 atomic bomb survivors. We investigated factors that may affect the background sCA rate and the shape and magnitude of the dose response. As in previous analyses of sCA in atomic bomb survivors that were based on Giemsa staining methods and used older DS86 dose estimates, the relationship between radiation dose and sCA rate was significant (P < 0.0001) with a linear-quadratic relationship at lower doses that did not persist at higher doses. As before, age at the time of the bombing and type of radiation shielding were significant dose-effect modifiers (P < 0.0001), but in contrast the difference in dose response by city was not so pronounced (P = 0.026) with a city effect not evident at doses below 1.25Gy. Background sCA rate increased with age at the time of examination (P < 0.0001), but neither sex, city, nor smoking was significantly associated with background rate. Based on FISH methods and recent dosimetry, the relationship between radiation dose and sCA frequency is largely consistent with previous findings, although the lesser importance of city as an effect modifier may reflect better dosimetry as well as more reproducible scoring of sCA. The persisting difference in sCA dose response by shielding category points to remaining problems with the accuracy or precision of radiation dose estimates in some A-bomb survivors.

Translocations are induced in hematopoietic stem cells after irradiation of fetal mice.

Although mammalian fetuses have been suggested to be sensitive to radiation, an increased frequency of translocations was not observed in blood lymphocytes from atomic bomb (A-bomb) survivors who were exposed to the bomb in utero and examined as adults. Since experiments using hematopoietic cells of mice and rats confirmed this finding, it was hypothesized that either irradiated fetal hematopoietic stem cells (f-HSCs) cannot generate exchange-type chromosomal aberrations or cells bearing induced aberrations are eliminated before the animals reach adulthood. In the present study, pregnant mice (12.5-15.5 days post coitum [dpc]) were irradiated with 2 Gy of X-rays and long-term HSCs (LT-HSCs) were isolated 24 h later. Multicolor fluorescence in situ hybridization (mFISH) analysis of LT-HSC clones proliferated in vitro showed that nine out of 43 (21%) clones from fetuses and 21 out of 41 (51%) clones from mothers bore translocations. These results indicate that cells with translocations can arise in mouse f-HSCs but exist at a lower frequency than in the mothers 24 h after X-ray exposure. Thus, it seems likely that translocation-bearing f-HSCs are generated but subsequently disappear, so that the frequency of lymphocyte translocations may decrease and reach the control level by the time the animals reach adulthood.

Massive expansion of multiple clones in the mouse hematopoietic system long after whole-body X-irradiation.

Clonal hematopoiesis (CH) is prevalent in the elderly and associates with hematologic malignancy and cardiovascular disease. Although the risk of developing these diseases increases with radiation doses in atomic-bomb survivors, the causal relationship between radiation exposure and CH is unclear. This study investigated whether radiation exposure induces CH in mice 12-18 months after 3-Gy whole-body irradiation. We found radiation-associated increases in peripheral blood myeloid cells and red blood cell distribution width (RDW). Deep sequencing of bone marrow and non-hematopoietic tissue cells revealed recurrent somatic mutations specifically in the hematopoietic system in 11 of 12 irradiated mice but none in 6 non-irradiated mice. The irradiated mice possessed mutations with variant allele frequencies (VAFs) of > 0.02 on an average of 5.8 per mouse; mutations with VAFs of > 0.1 and/or deletion were prevalent. Examining hematopoietic stem/progenitor cells in two irradiated mice revealed several mutations co-existing in the same clones and multiple independent clones that deliver 60-80% of bone marrow nuclear cells. Our results indicate development of massive CH due to radiation exposure. Moreover, we have characterized mutations in radiation-induced CH.

Ethical, legal and social implications of human genome studies in radiation research: a workshop report for studies on atomic bomb survivors at the Radiation Effects Research Foundation.

The Radiation Effects Research Foundation (RERF) is the primary organization in Japan dedicated to studying the health consequences of the Hiroshima and Nagasaki atomic bombings in World War II. In December 2020, RERF held a virtual international workshop on the ethical, legal and social implications (ELSI) of genome studies. In this workshop, the ELSI considerations of future human genome studies on radiation research including atomic bomb survivors and their families were discussed. Since genome sequencing (GS) is now practical and affordable, RERF now plans GS of parents/child trios to examine genetic effects of atomic bomb radiation. As such studies may engender some novel risks and benefits, ethics review and engagement with families (including consent) need to be considered. These include protection of individual privacy, use of samples from deceased prior participants, return of results to the participants, public sharing of genome data and advance science and social welfare. Specifically with regard to social welfare, the results of such studies may have implications for public and government decision-making regarding social benefits of victims and other important questions. Based on these broad-ranging discussions we have developed the following concepts to guide this work: "trust," "compromise" and "relationship building," inclusive of the concerned stakeholders, scientific aims and Japanese society at large. We conclude that in order to realize, establish and maintain these concepts, it is essential to put procedures into place to ensure the successful, consensus-based implementation of the RERF studies.

Increased risk of skin cancer in Japanese heterozygotes of xeroderma pigmentosum group A.

This study was designed to learn if asymptomatic heterozygotes with mutations in a DNA repair gene are at an increased risk for cancer. To examine this, we focused on carriers of an XPA founder mutation because the frequency of xeroderma pigmentosum (XP) patients is much greater among Japanese than Caucasians, more than half of Japanese XP patients are affected at the XPA gene, and the majority of XP-A patients carry the same founder mutation in the XPA gene. Here we show that the frequency of XPA heterozygote was 14/1698 (0.8%) in cancer-free controls, and the corresponding frequency in patients with nonmelanocytic skin cancer that developed in sun-exposed areas was 11/440 (2.5%, OR = 3.08, p = 0.0097) for basal cell carcinoma, and 3/272 (1.1%, OR = 1.34, p = 0.72) for squamous cell carcinoma. These results suggest a moderately elevated risk for skin cancer among XPA heterozygotes.

Radiation-induced unrepairable DSBs: their role in the late effects of radiation and possible applications to biodosimetry.

Although the vast majority of DNA damage induced by radiation exposure disappears rapidly, some lesions remain in the cell nucleus in very small quantities for days to months. These lesions may cause a considerable threat to an organism and include certain types of DNA double-strand breaks (DSBs) called 'unrepairable DSBs'. Unrepairable DSBs are thought to cause persistent malfunctioning of cells and tissues or cause late effects of radiation, especially the induction of delayed cell death, mutation, senescence, or carcinogenesis. Moreover, the measurement of unrepairable DSBs could potentially be used for retrospective biodosimetry or for identifying individuals at greater risk for developing the adverse effects associated with radiotherapy or chemotherapy. This review summarizes the concept of unrepairable DSBs in the context of persistent repair foci formed at DSBs.

Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

RAD18 activates the G2/M checkpoint through DNA damage signaling to maintain genome integrity after ionizing radiation exposure.

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.

Progerin, the protein responsible for the Hutchinson-Gilford progeria syndrome, increases the unrepaired DNA damages following exposure to ionizing radiation.

INTRODUCTION: Progerin, the protein responsible for the Hutchinson-Gilford Progeria Syndrome (HGPS), is a partially deleted form of nuclear lamin A, and its expression has been suggested as a cause for dysfunctional nuclear membrane and premature senescence. To examine the role of nuclear envelop architecture in regulating cellular aging and DNA repair, we used ionizing radiation to increase the number of DNA double strand breaks (DSBs) in normal and HGPS cells, and analyzed possible relationship between unrepaired DSBs and cellular aging. RESULTS: We found that HGPS cells are normal in repairing a major fraction of radiation-induced double strand breaks (M-DSBs)but abnormal to show increased amount of residual unrepaired DSBs (R-DSBs). Such unrepaired DSBs were 2.6 times (CI 95 %: 2.2-3.2) higher than that in normal cells one week after the irradiation, and 1.6 times (CI 95 %: 1.3-1.9) higher even one month after the irradiation. These damages tend to increase as the nuclear envelope become abnormal, a characteristic of both HGPS and normal human cells which undergo replicative senescence. The artificial, enforced over-expression of progerin further impaired the repair of M-DSBs, implying lamin A-associated nuclear membrane has an important role for DNA DSB repair. Introduction of telomerase gene function in HGPS cells reversed such aging phenotypes along with upregulation of lamin B1 and downregulation of progerin, which is a hallmark of young cells. CONCLUSION: We suggest that lamin A- or progerin-associated nuclear envelope is involved in cellular aging associated with DNA damage repair.

Fetal irradiation of rats induces persistent translocations in mammary epithelial cells similar to the level after adult irradiation, but not in hematolymphoid cells.

In both humans and mice, fetal exposure to radiation fails to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level response to radiation exposure is counterintuitive in view of the generally accepted belief that a fetus is sensitive to radiation. To determine if this is a general phenomenon, both mammary epithelial cells and spleen cells were studied in rats. Fetuses of 17.5 days postcoitus were irradiated with 2 Gy of gamma rays, and mammary tissues were removed 6-45 weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH method. The results showed that translocation frequencies were not only elevated in rats irradiated as fetuses, but were also almost as high as those in rats that were irradiated as adults (12 weeks old, pregnant mothers or young virgins) and examined 6-45 weeks later. There was no evidence of higher sensitivity in fetal cells with respect to the induction of translocations. In contrast, translocation frequencies in spleen cells were not elevated in adult rats irradiated as fetuses but were increased after irradiation of adults as previously seen in mouse spleen cells and human T lymphocytes. In the case of irradiation of adult rats, the induced translocation frequencies were similar between spleen cells and mammary epithelial cells. If we take translocation frequency as a surrogate marker of potential carcinogenic effect of radiation, the current results suggest that fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but this does not contribute to the increased risk of cancer since it has been reported that irradiation of fetal rats of the SD strain does not increase the risk of mammary cancers. Possible reasons for this discrepancy are discussed.

Radiation effects on human heredity.

In experimental organisms such as fruit flies and mice, increased frequencies in germ cell mutations have been detected following exposure to ionizing radiation. In contrast, there has been no clear evidence for radiation-induced germ cell mutations in humans that lead to birth defects, chromosome aberrations, Mendelian disorders, etc. This situation exists partly because no sensitive and practical genetic marker is available for human studies and also because the number of people exposed to large doses of radiation and subsequently having offspring was small until childhood cancer survivors became an important study population. In addition, the genome of apparently normal individuals seems to contain large numbers of alterations, including dozens to hundreds of nonfunctional alleles. With the number of mutational events in protein-coding genes estimated as less than one per genome after 1 gray (Gy) exposure, it is unsurprising that genetic effects from radiation have not yet been detected conclusively in humans.

Unrepairable DNA double-strand breaks that are generated by ionising radiation determine the fate of normal human cells.

After an exposure to ionising radiation, cells can quickly repair damage to their genomes; however, a few unrepairable DNA double-strand breaks (DSBs) emerge in the nucleus in a prolonged culture and perpetuate as long as the culture continues. These DSBs may be retained forever in cells such as non-dividing ageing tissues, which are resistant to apoptosis. We show that such unrepairable DSBs, which had been advocated by the classical target theory as the 'radiation hit', could account for permanent growth arrest and premature senescence. The unrepairable DSBs build up with repeated irradiation, which accounts for an accumulated dose. Because these DSBs tend to be paired, we propose that the untethered and 'torn-off' molecular structures at the broken ends of the DNA result in an alteration of chromatin structure, which protects the ends of the DNA from genomic catastrophe. Such biochemical responses are important for cell survival but may cause gradual tissue malfunction, which could lead to the late effects of radiation exposure. Thus, understanding the biology of unrepairable damage will provide new insights into the long-term effects of radiation.

International workshop: radiation effects on mutation in somatic and germline stem cells.

PURPOSE: New developments in knowledge of radiation effects on tissue stem cells were discussed in a Workshop held at the Radiation Effects Research Foundation (RERF) in Hiroshima, Japan, 18-19 January 2012. RESULTS: Stem cells and their niche in intestinal mucosa, haemopoietic tissue, hair follicles, and spermatogenesis were discussed variously with regard to radiosensitivity, repair, regeneration, age-dependency of effects, genetic effects, and protection aspects. These tissues all possess a common basic template, but there are structural and hierarchical differences between tissues which continue to be elucidated in terms of a stem-cell age structure and niche regulatory signals which together govern radiation responses. CONCLUSIONS: Stem cells and their niche have become much better characterized in recent years, and their radiation response can be elucidated in detail in experimental systems to help underpin both protection and therapeutic recommendations established from human epidemiological evidence. This report summarizes the presentations at the meeting, and concludes with some remaining questions which may be answered with the help of this type of research.

Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells.

We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene (TetO-EGFP) and tetracycline repressor (TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8-3.4×10(-6)/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.

Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population.

Individuals who are homozygotes for mutations in DNA repair genes are at high risk for cancer. It is not well documented, however, if the heterozygous carriers of the mutation are also predisposed to cancer. To address the issue, xeroderma pigmentosum (XP) in Japan is an interesting candidate because of three major reasons: XP is an autosomal recessive disorder with an enormously elevated risk of skin cancer, the frequency of XP patients is higher in Japan than in other parts of the world, and more than half of Japanese XP patients are homozygous for the same founder mutation in the XPA gene. We screened archival blood samples from Japanese individuals who resided in Hiroshima or Nagasaki. A simple PCR-RFLP method was developed that is highly specific for detection of XPA heterozygotes carrying the founder mutation. We identified nine XPA heterozygotes among 1,020 individuals screened for a prevalence of 0.88%. This rate, if representative, implies that there are about 1 million carriers of the XPA founder mutation in the Japanese population. Thus, investigation of their cancer risk may be warranted.