A possible role for proinflammatory activation via cGAS-STING pathway in atherosclerosis induced by accumulation of DNA double-strand breaks.

DNA damage contributes to atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be established. Here, we investigated the role of DSBs in atherosclerosis using mice and vascular cells deficient in Ku80, a DSB repair protein. After 4 weeks of a high-fat diet, Ku80-deficient apolipoprotein E knockout mice (Ku80+/-ApoE-/-) displayed increased plaque size and DSBs in the aorta compared to those of ApoE-/- control. In the preatherosclerotic stages (two-week high-fat diet), the plaque size was similar in both the Ku80+/-ApoE-/- and ApoE-/- control mice, but the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80+/-ApoE-/- aortas. We further investigated molecular links between DSBs and inflammatory responses using vascular smooth muscle cells isolated from Ku80 wild-type and Ku80+/- mice. The Ku80+/- cells displayed senescent features and elevated levels of inflammatory cytokine mRNAs. Moreover, the cytosolic DNA-sensing cGAS-STING pathway was activated in the Ku80+/- cells. Inhibiting the cGAS-STING pathway reduced IL-6 mRNA level. Notably, interferon regulatory factor 3 (IRF3), a downstream effector of the cGAS-STING pathway, was activated, and the depletion of IRF3 also reduced IL-6 mRNA levels in the Ku80+/- cells. Finally, DSBs accumulation in normal cells also activated the cGAS-STING-IRF3 pathway. In addition, cGAS inhibition attenuated DNA damage-induced IL-6 expression and cellular senescence in these cells. These results suggest that DSBs accumulation promoted atherosclerosis by upregulating proinflammatory responses and cellular senescence via the cGAS-STING (-IRF3) pathway.

Klotho protects chromosomal DNA from radiation-induced damage.

Klotho is an anti-aging, single-pass transmembrane protein found mainly in the kidney. Although aging is likely to be associated with DNA damage, the involvement of Klotho in protecting cells from DNA damage is still unclear. In this study, we examined DNA damage in human kidney cells and mouse kidney tissue after ionizing radiation (IR). The depletion and overexpression of Klotho in human kidney cells reduced and increased the cell survival rates after IR, respectively. The formation of γ-H2AX foci, representing DNA damage, was significantly elevated immediately after IR in cells with Klotho depletion and decreased in cells overexpressing Klotho. These results were confirmed in mouse renal tissues after IR. Quantification of DNA damage by a comet assay revealed that the Klotho knockdown significantly increased the amount of DNA damage immediately after IR, suggesting that Klotho protects chromosomal DNA from the induction of damage, rather than facilitating DNA repair. Consistent with this notion, Klotho was detected in both the nucleus and cytoplasm. In the nucleus, Klotho may serve to protect chromosomal DNA from damage, leading to its anti-aging effects.

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway: application to a biomarker for atherosclerosis.

Cigarette smoking is a major risk factor for atherosclerosis. We previously reported that DNA damage was accumulated in atherosclerotic plaque, and was increased in human mononuclear cells by smoking. As vascular endothelial cells are known to modulate inflammation, we investigated the mechanism by which smoking activates innate immunity in endothelial cells focusing on DNA damage. Furthermore, we sought to characterize the plasma level of cell-free DNA (cfDNA), a result of mitochondrial and/or genomic DNA damage, as a biomarker for atherosclerosis. Cigarette smoke extract (CSE) increased DNA damage in the nucleus and mitochondria in human endothelial cells. Mitochondrial damage induced minority mitochondrial outer membrane permeabilization, which was insufficient for cell death but instead led to nuclear DNA damage. DNA fragments, derived from the nucleus and mitochondria, were accumulated in the cytosol, and caused a persistent increase in IL-6 mRNA expression via the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. cfDNA, quantified with quantitative PCR in culture medium was increased by CSE. Consistent with in vitro results, plasma mitochondrial cfDNA (mt-cfDNA) and nuclear cfDNA (n-cfDNA) were increased in young healthy smokers compared with age-matched nonsmokers. Additionally, both mt-cfDNA and n-cfDNA were significantly increased in patients with atherosclerosis compared with the normal controls. Our multivariate analysis revealed that only mt-cfDNA predicted the risk of atherosclerosis. In conclusion, accumulated cytosolic DNA caused by cigarette smoke and the resultant activation of the cGAS-STING pathway may be a mechanism of atherosclerosis development. The plasma level of mt-cfDNA, possibly as a result of DNA damage, may be a useful biomarker for atherosclerosis.

DNA Damage Induced by Radiation Exposure from Cardiac Catheterization.

Almost 40% of medical radiation exposure is related to cardiac imaging or intervention. However, the biological effects of low-dose radiation from medical imaging remain largely unknown. This study aimed to evaluate the effects of ionized radiation from cardiac catheterization on genomic DNA integrity and inflammatory cytokines in patients and operators.Peripheral mononuclear cells (MNCs) were isolated from patients (n = 51) and operators (n = 35) before and after coronary angiography and/or percutaneous coronary intervention. The expression of γH2AX, a marker for DNA double-strand breaks, was measured by immunofluorescence. Dicentric chromosomes (DICs), a form of chromosome aberrations, were assayed using a fluorescent in situ hybridization technique.In the patient MNCs, the numbers of γH2AX foci and DICs increased after cardiac catheterization by 4.5 ± 9.4-fold and 71 ± 122%, respectively (P < 0.05 for both). The mRNA expressions of interleukin (IL)-1α, IL-1ß, leukemia inhibitory factor, and caspase-1 were significantly increased by radiation exposure from cardiac catheterization. The increase in IL-1ß was significantly correlated with that of γH2AX, but not with the dose area product. In the operators, neither γH2AX foci nor the DIC level was changed, but IL-1ß mRNA was significantly increased. The protein expression of IκBα was significantly decreased in both groups.DNA damage was increased in the MNCs of patients, but not of operators, who underwent cardiac catheterization. Inflammatory cytokines were increased in both the patients and operators, presumably through NF-κB activation. Further efforts to reduce radiation exposure from cardiac catheterization are necessary for both patients and operators.

Biological and internal dosimetry for radiation medicine: current status and future perspectives.

The International Atomic Energy Agency (IAEA) and Hiroshima International Council for Health Care of the Radiation-Exposed (HICARE) jointly organized two relevant workshops in Hiroshima, Japan, i.e. a Training Meeting 'Biodosimetry in the 21st century' (BIODOSE-21) on 10-14 June 2013 and a Workshop on 'Biological and internal dosimetry: recent advance and clinical applications' which took place between 17 and 21 February 2020. The main objective of the first meeting was to develop the ability of biodosimetry laboratories to use mature and novel techniques in biological dosimetry for the estimation of radiation doses received by individuals and populations. This meeting had a special focus on the Asia-Pacific region and was connected with the then on-going IAEA Coordinated Research Project (CRP) E35008 'Strengthening of "Biological dosimetry" in IAEA Member States: Improvement of current techniques and intensification of collaboration and networking among the different institutes' (2012-17). The meeting was attended by 25 participants, which included 11 lecturers. The 14 trainees for this meeting came from India, Indonesia, Japan, Malaysia, Philippines, Republic of Korea, Singapore, Thailand and Vietnam. During the meeting 13 lectures by HICARE and IAEA invited lecturers were delivered besides eight research reports presented by the IAEA CRP E35008 network centers from the Asia-Pacific region. Two laboratory exercises were also undertaken, one each at Hiroshima University and the Radiation Effects Research Foundation (RERF). The second training workshop aimed to discuss with the participants the use of mature and novel techniques in biological and internal dosimetry for the estimation of radiation effects by accidental, environmental and medical exposures. The workshop was attended by 19 participants from Indonesia, Jordan, Oman, Philippines, Singapore, Syrian Arab Republic, Thailand, UAE, USA and Yemen. The main outcome of both meetings was a review of the state-of-the-art of biodosimetry and internal dosimetry and their future perspectives in medical management. This report highlights the learning outcome of two meetings for the benefit of all stake-holders in the field of biological and internal dosimetry.

A Combination of Iohexol Treatment and Ionizing Radiation Exposure Enhances Kidney Injury in Contrast-Induced Nephropathy by Increasing DNA Damage.

Contrast media has been shown to induce nephropathy (i.e., contrast-induced nephropathy) after various types of radiological examinations. The molecular mechanism of contrast-induced nephropathy has been unclear. In this study, we investigated the mechanism of contrast-induced nephropathy by examining the effects of combined treatment of contrast medium and ionizing radiation on kidney cells in vitro and kidney tissue in vivo. In human renal tubular epithelium cells, immunofluorescence analysis revealed that iohexol increased the numbers of radiation-induced γH2AX nuclear foci. The numbers of γH2AX nuclear foci remained high at 24 h, suggesting that some radiation-induced double-strand breaks remain unrepaired in the presence of iohexol. We established a mouse model of contrast-induced nephropathy, then showed that iohexol and ionizing radiation synergistically reduced renal function and induced double-strand breaks. Importantly, iohexol induced significant macrophage accumulation and oxidative DNA damage in the kidneys of contrast-induced nephropathy model mice in the absence of ionizing radiation; these effects were amplified by ionizing radiation. The results suggest that underlying inflammation and oxidative DNA damage caused by iohexol contribute to the enhancement of radiation-induced double-strand breaks, leading to contrast-induced nephropathy.

Replication-stress-associated DSBs induced by ionizing radiation risk genomic destabilization and associated clonal evolution.

Exposure to ionizing radiation is associated with cancer risk. Although multiple types of DNA damage are caused by radiation, it remains unknown how this damage is associated with cancer risk. Here, we show that after repair of double-strand breaks (DSBs) directly caused by radiation (dir-DSBs), irradiated cells enter a state at higher risk of genomic destabilization due to accumulation of replication-stress-associated DSBs (rs-DSBs), ultimately resulting in clonal evolution of cells with abrogated defense systems. These effects were observed over broad ranges of radiation doses (0.25-2 Gy) and dose rates (1.39-909 mGy/min), but not upon high-dose irradiation, which caused permanent cell-cycle arrest. The resultant genomic destabilization also increased the risk of induction of single-nucleotide variants (SNVs), including radiation-associated SNVs, as well as structural alterations in chromosomes. Thus, the radiation-associated risk can be attributed to rs-DSB accumulation and resultant genomic destabilization.

Evaluating Individual Radiosensitivity for the Prediction of Acute Toxicities of Chemoradiotherapy in Esophageal Cancer Patients.

In this work, individual radiosensitivity was evaluated using DNA damage response and chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) for the prediction of acute toxicities of chemoradiotherapy (CRT) in esophageal cancer patients. Eighteen patients with esophageal cancer were enrolled in this prospective study. Prescribed doses were 60 Gy in 11 patients and 50 Gy in seven patients. Patients received 2 Gy radiotherapy five days a week. PBLs were obtained during treatment just before and 15 min after 2 Gy radiation therapy on the days when the cumulative dose reached 2, 20, 40 Gy and 50 or 60 Gy. PBLs were also obtained four weeks and six months after radiotherapy in all and 13 patients, respectively. Dicentric and ring chromosomes in PBLs were counted to evaluate the number of CAs. Gamma-H2AX foci per cell were scored to assess DNA double-strand breaks. We analyzed the association between these factors and adverse events. The number of γ-H2AX foci before radiotherapy showed no significant increase during CRT, while their increment was significantly reduced with the accumulation of radiation dose. The mean number of CAs increased during CRT up to 1.04 per metaphase, and gradually decreased to approximately 60% six months after CRT. Five patients showed grade 3 toxicities during or after CRT (overreactors: OR), while 13 had grade 2 or less toxicities (non-overreactors: NOR). The number of CAs was significantly higher in the OR group than in the NOR group at a cumulative dose of 20 Gy (mean value: 0.63 vs. 0.34, P = 0.02), 40 Gy (mean value: 0.90 vs. 0.52, P = 0.04), and the final day of radiotherapy (mean value: 1.49 vs. 0.84, P = 0.005). These findings suggest that number of CAs could be an index for predicting acute toxicities of CRT for esophageal cancer.

Bach1 plays an important role in angiogenesis through regulation of oxidative stress.

Bach1 is a known transcriptional repressor of the heme oxygenase-1 (HO-1) gene. The purpose of this study was to determine whether angiogenesis is accelerated by genetic ablation of Bach1 in a mouse ischemic hindlimb model. Hindlimb ischemia was surgically induced in wild-type (WT) mice, Bach1-deficient (Bach1-/-) mice, apolipoprotein E-deficient (ApoE-/-) mice, and Bach1/ApoE double-knockout (Bach1-/-/ApoE-/-) mice. Blood flow recovery after hindlimb ischemia showed significant improvement in Bach1-/- mice compared with that in WT mice. Bach1-/-/ApoE-/- mice showed significantly improved blood flow recovery compared with that in ApoE-/- mice to the level of that in WT mice. Migration of endothelial cells in ApoE-/- mice was significantly decreased compared with that in WT mice. Migration of endothelial cells significantly increased in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice to the level of that in WT mice. The expression levels of HO-1, peroxisome proliferator-activated receptor γ co-activator-1α, angiopoietin 1, and fibroblast growth factor 2 in endothelial cells isolated from Bach1-/-/ApoE-/- mice were significantly higher than those in ApoE-/- mice. Oxidative stress assessed by anti-acrolein antibody staining in ischemic tissues and urinary 8-isoPGF2α excretion were significantly increased in ApoE-/- mice compared with those in WT and Bach1-/- mice. Oxidative stress was reduced in Bach1-/-/ApoE-/- mice compared with that in ApoE-/- mice. These findings suggest that genetic ablation of Bach1 plays an important role in ischemia-induced angiogenesis under the condition of increased oxidative stress. Bach1 could be a potential therapeutic target to reduce oxidative stress and potentially improve angiogenesis for patients with peripheral arterial disease.

Biological Effects of Low-Dose Chest CT on Chromosomal DNA.

Background Although the National Lung Screening Trial reported a significant reduction in lung cancer mortality when low-dose (LD) CT chest examinations are used for a diagnosis, their biologic effects from radiation exposure remain unclear. Purpose To compare LD CT and standard-dose (SD) CT for DNA double-strand breaks and chromosome aberrations (CAs) in peripheral blood lymphocytes. Materials and Methods Between March 2016 and June 2018, 209 participants who were referred to a respiratory surgery department for chest CT studies were prospectively enrolled in this study. Individuals were excluded if they had undergone radiography examinations within the last 3 days or had undergone chemotherapy or radiation therapy. Peripheral blood samples were obtained before and 15 minutes after CT. The number of γ-H2AX foci and unstable CAs in lymphocytes was quantified by immunofluorescent staining of γ-H2AX and by fluorescence in situ hybridization by using peptide nucleic acid probes for centromeres and telomeres, respectively. The Wilcoxon signed rank test was used for statistical analysis. Bonferroni correction was applied for multiple comparisons. Results Of the 209 participants (105 women, 104 men; mean age, 67.0 years ± 11.3 [standard deviation]), 107 underwent chest LD CT and 102 underwent chest SD CT. Sex distribution, age, and body size metrics were similar between the two groups. The median effective dose of LD CT and SD CT was 1.5 and 5.0 mSv, respectively. The number of double-strand breaks and CAs increased after a SD CT examination (γ-H2AX, P < .001; CAs, P = .003); the number of double-strand breaks and CAs before and after LD CT was not different (γ-H2AX, P = .45; CAs, P = .69). Conclusion No effect of low-dose CT on human DNA was detected. In the same setting, DNA double-strand breaks and chromosome aberrations increased after standard-dose CT. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Brenner in this issue.

Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

Chromosomal Abnormalities in Human Lymphocytes after Computed Tomography Scan Procedure.

The incidence of chromosomal abnormalities and cancer risk correlates well with the radiation dose after exposure to moderate- to high-dose ionizing radiation. However, the biological effects and health risks at less than 100 mGy, e.g., from computed tomography (CT) have not been ascertained. To investigate the biological effects of low-dose exposure from a CT procedure, we examined chromosomal aberrations, dicentric and ring chromosomes (dic+ring), in peripheral blood lymphocytes (PBLs), using FISH assays with telomere and centromere PNA probes. In 60 non-cancer patients exposed to CT scans, the numbers of dicentric and ring chromosomes were significantly increased with individual variation. The individual variations in the increment of dicentric and ring chromosomes after CT procedures were confirmed using PNA-FISH analysis of PBLs from 15 healthy volunteers after in vitro low-dose exposure using a 137Cs radiation device. These findings strongly suggest that appropriate medical use of low-dose radiation should consider individual differences in radiation sensitivity.

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.

XRCC3 polymorphism is associated with hypertension-induced left ventricular hypertrophy.

Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and the 241Met variant of XRCC3 have been reported to increase endoreduplication, which induces polyploidy. The aims of this study were to determine the impact of the XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and to investigate the mechanisms underlying any potential relationship. Patients undergoing chronic hemodialysis (n = 77) were genotyped to assess for the XRCC3 Thr241Met polymorphism. The XRCC3 241Thr/Met genotype was more frequent in the LVH (+) group than in the LVH (-) group (42.3 vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate possible mechanisms underlying these observations, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured CHO cells. The surface area of CHO cells expressing XRCC3 241Met was larger than that expressing 241Thr. Spontaneous DNA double-strand breaks accumulated to a greater degree in NIH3T3 cells expressing 241Met (3T3-241Met) than in those expressing 241Thr (3T3-241Thr). DNA damage caused by radiation induced cell senescence more frequently in 3T3-241Met. The levels of basal and TNF-α-stimulated MCP-1 mRNA and protein secretion were higher in 3T3-241Met. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly higher in 3T3-241Met than in 3T3-241Thr. Furthermore, the basal level of MCP-1 mRNA positively correlated with the cell percentage in G2/M phase and polyploidy. These data suggest that the XRCC3 241Met increases the risk of LVH via accumulation of DNA damage, thereby altering cell cycle progression and inducing cell senescence and a proinflammatory phenotype.

Estimation of the effects of medical diagnostic radiation exposure based on DNA damage.

X-rays are widely applied in the medical field for the diagnosis and treatment of diseases. Among the uses of X-rays in diagnosis, computed tomography (CT) has been established as one of the most informative diagnostic radiology examinations. Moreover, recent advances in CT scan technology have made this examination much easier and more informative and increased its application, especially in Japan. However, the radiation dose of CT scans is higher than that of simple X-ray examinations. Therefore, the health risk of a CT scan has been discussed in various studies, but is still controversial. Consequently, the biological and cytogenetic effects of CT scans are being analyzed. Here, we summarize the recent findings concerning the biological and cytogenetic effects of ionizing radiation from a CT scan, by focusing on DNA damage and chromosome aberrations.

Exploration of genetic basis underlying individual differences in radiosensitivity within human populations using genome editing technology.

DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) are the initial and critical step in major alteration of genetic information and cell death. To prevent deleterious effects, DNA repair systems recognize and re-join DNA DSBs in human cells. It has been suggested that there are individual differences in radiosensitivity within human populations, and that variations in DNA repair genes might contribute to this heterogeneity. Because confounding factors, including age, gender, smoking, and diverse genetic backgrounds within human populations, also influence the cellular radiosensitivity, to accurately measure the effect of candidate genetic variations on radiosensitivity, it is necessary to use human cultured cells with a uniform genetic background. However, a reverse genetics approach in human cultured cells is difficult because of their low level of homologous recombination. Engineered endonucleases used in genome editing technology, however, can enable the local activation of DNA repair pathways at the human genome target site to efficiently introduce genetic variations of interest into human cultured cells. Recently, we used this technology to demonstrate that heterozygous mutations of the ATM gene, which is responsible for a hyper-radiosensitive genetic disorder, ataxia-telangiectasia, increased the number of chromosomal aberrations after IR. Thus, understanding the heterozygous mutations of radiosensitive disorders should shed light on the genetic basis underlying individual differences in radiosensitivity within human populations.

RELATIVE BIOLOGICAL EFFECTIVENESS OF NEUTRONS DERIVED FROM THE EXCESS RELATIVE RISK MODEL WITH THE ATOMIC BOMB SURVIVORS DATA MANAGED BY HIROSHIMA UNIVERSITY.

According to an analysis of the Life Span Study cohort data conducted by the Radiation Effects Research Foundation in Hiroshima and Nagasaki, the sex-averaged excess relative risk (ERR) of all solid cancers was 0.42 Gy-Eq-1. On the other hand, analysis of the atomic bomb survivors (ABS) cohort data at Hiroshima University indicated the ERR value was 0.28 Gy-Eq-1 in Hiroshima. In both cases, initial radiation doses were derived from the dosimetry system DS02, in which the relative biological effectiveness (RBE) of neutrons was assumed to be a constant value of 10. To clarify the validity of the RBE, the authors investigated the possibility of different contributions of neutrons by using the ABS. Although there were no statistically significant differences among the estimated value of RBE (=65) and the ordinal value (=10), the corresponding ERR decreased by 30%, which might affect the interpretation of radiation health assessments.

Evaluation of ATM heterozygous mutations underlying individual differences in radiosensitivity using genome editing in human cultured cells.

Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity. To precisely quantify the effect of a genetic variation on radiosensitivity, we here used the CRISPR-ObLiGaRe (Obligate Ligation-Gated Recombination) method combined with the CRISPR/Cas9 system and a nonhomologous end joining (NHEJ)-mediated knock-in technique in human cultured cells with a uniform genetic background. We generated ATM heterozygous knock-out (ATM +/-) cell clones as a carrier model of a radiation-hypersensitive autosomal-recessive disorder, ataxia-telangiectasia (A-T). Cytokinesis-blocked micronucleus assay and chromosome aberration assay showed that the radiosensitivity of ATM +/- cell clones was significantly higher than that of ATM +/+ cells, suggesting that ATM gene variants are indeed involved in determining individual radiosensitivity. Importantly, the differences in radiosensitivity among the same genotype clones were small, unlike the individual differences in fibroblasts derived from A-T-affected family members.

Antagonizing effect of CLPABP on the function of HuR as a regulator of ARE-containing leptin mRNA stability and the effect of its depletion on obesity in old male mouse.

Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.

Cytogenetic effects of radioiodine therapy: a 20-year follow-up study.

The purpose of this study was to compare cytogenetic data in a patient before and after treatment with radioiodine to evaluate the assays in the context of biological dosimetry. We studied a 34-year-old male patient who underwent a total thyroidectomy followed by ablation therapy with (131)I (19.28 GBq) for a papillary thyroid carcinoma. The patient provided blood samples before treatment and then serial samples at monthly intervals during the first year period and quarterly intervals for 5 years and finally 20 years after treatment. A micronucleus assay, dicentric assay, FISH method and G-banding were used to detect and measure DNA damage in circulating peripheral blood lymphocytes of the patient. The results showed that radiation-induced cytogenetic effects persisted for many years after treatment as shown by elevated micronuclei and chromosome aberrations as a result of exposure to (131)I. At 5 years after treatment, the micronucleus count was tenfold higher than the pre-exposure frequency. Shortly after the treatment, micronucleus counts produced a dose estimate of 0.47 ± 0.09 Gy. The dose to the patient evaluated retrospectively using FISH-measured translocations was 0.70 ± 0.16 Gy. Overall, our results show that the micronucleus assay is a retrospective biomarker of low-dose radiation exposure. However, this method is not able to determine local dose to the target tissue which in this case was any residual thyroid cells plus metastases of thyroidal origin.