A new method for the simultaneous detection of mammalian cells and ion tracks on a surface of CR-39

The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effects. We employed a contact microscopy technique, which was developed for boron imaging in boron neutron capture therapy to the irradiation mammalian cells by low-energy heavy ions. This method enables the simultaneous visualization of mammalian cells as a relief on a plastic track detector, CR-39, and the etch pits which indicate the positions of ion traversals. This technique provides visual geometric information about the cells and ion traversal, without any specially designed devices or microscopes. Only common laboratory equipment, such as a conventional optical microscope, a UV lamp, and commercially available CR-39 is required. To validate this method, CHO-K1 and HeLa cells were cultured on the CR-39 surface and then irradiated with low-energy Ar and Ne ions, respectively. The positions of induced DNA double strand breaks were detected as gamma-H2AX fluorescent spots, which coincided with the positions of the etch pits in the cell relief image.     

Evaluation of chemical phototoxicity,focusing on phosphorylated histone H2AX

Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX.     

Number of Fe ion traversals through a cell nucleus for mammalian cell inactivation near the bragg peak

HeLa and CHO-K1 cells were irradiated with Fe ions (1.14 MeV/nucleon) near the Bragg peak to determine how many ion traversals through a cell nucleus are necessary to induce cell inactivation. The ion traversals through a cell nucleus were visualized by immunostaining the phosphorylated histone H2AX (gamma-H2AX), as an indicator of DNA double strand breaks (DSBs), to confirm that DSBs are actually induced along every Fe ion traversal through the nucleus. The survival curves after irradiation with Fe ions decreased exponentially with the ion fluence without a shoulder. The inactivation cross sections calculated from the slope of the survival curves and the standard errors were 96.9 +/- 1.8 and 57.9 +/- 5.4 microm2 for HeLa and CHO-K1 cells, respectively, corresponding to 0.442 and 0.456 of the mean value of each cell nucleus area. Taking the distribution of the cell nucleus area into consideration with an equation proposed by Goodhead et al. (1980), which calculates the average number of lesions per single ion track through the average area of a sensitive organelle (mainly nucleus), these two ratios were converted to 0.705 and 0.659 for HeLa and CHO-K1 cells, respectively. These ratios were less than one, suggesting that the average numbers of lethal hits per cell produced by a single ion traversal were less than one. We thus considered two possible explanations for ion traversals of more than one, necessary for cell inactivation.     

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.     

辐射对沉默ATRX的H460细胞增殖以及DNA损伤修复的影响

目的 探讨辐射对沉默ATRX的肺癌H460细胞增殖和DNA损伤修复的影响及二者的关系。方法 靶向ATRX的3个慢病毒载体转染293T细胞后,慢病毒感染H460细胞,获得ATRX低/无表达的细胞株shATRX1-H460、shATRX2-H460和shATRX3-H460,并以shControl-H460作为对照,利用Western blot检测沉默效率。分别以克隆形成实验检测细胞增殖,免疫荧光技术检测γH2AX和Rad51焦点数,同时以Western blot检测PARP1、γH2AX和Rad51蛋白的表达。结果 shControl-H460细胞中可见ATRX表达,而shATRX1-H460、shATRX2-H460和shATRX3-H460细胞中ATRX表达均出现不同程度的降低。克隆形成实验显示,shATRX2-H460和shATRX3-H460细胞的存活分数（survival fraction,SF）均较shControl-H460细胞降低。shControl-H460和shATRX3-H460细胞经4 Gy照射后1 h,γH2AX焦点最多,而3 h时Rad51焦点最多,而后均降低,与shControl-H460细胞比较,在1和6 h时shATRX3-H460细胞γH2AX焦点,以及1、3和6 h时Rad51焦点显著增加（P<0.05,P<0.001）。而且shATRX3-H460细胞中PARP1、γH2AX和Rad51蛋白在3和6 h时均较shControl-H460细胞表达增加。结论 成功地获得靶向沉默ATRX的细胞模型,辐射后细胞增殖能力降低,可能与DNA损伤修复能力降低有关。     

Rad51基因沉默对铅诱导TK6细胞DNA双链断裂损伤修复的影响

目的 研究Rad51基因沉默后对醋酸铅染毒致人淋巴母细胞（TK6细胞）DNA双链断裂损伤的修复作用的影响。方法 构建Rad51沉默慢病毒载体及阴性对照,感染对数期TK6细胞,荧光定量PCR和Western blot验证感染效果。运用480μmol/L的醋酸铅染毒TK6细胞24 h(Control组、shRNA-NC组和shRNA-Rad51组),采用免疫荧光法检测TK6细胞的磷酸化组蛋白H2AX（γ-H2AX）的表达,Western Blot检测TK6细胞的Rad51、BRCA1、53BP1蛋白的表达。结果 shRNA-Rad51组的Rad51 mRNA表达水平和Rad51蛋白表达水平均低于Control组及shRNA-NC组（P <0.01）;shRNA-Rad51组的γ-H2AX阳性率为（27.48±1.66）%,与Control组的（14.77±1.21）%及shRNA-NC组的（14.04±1.31）%比较,差异均有统计学意义（P <0.01）;shRNA-Rad51组的BRCA1蛋白表达水平为（0.25±0.03）,与Control组的（0.55±0.04）及sh...     

A laser-plasma–produced soft X-ray laser at 89 eV generates DNA double-strand breaks in human cancer cells

While it has been expected that X-ray laser will be widely applied to biomedical studies, this has not been achieved to date and its biological effects such as DNA damage have not been evaluated. As a first step for its biological application, we developed a culture cell irradiation system, particularly designed for a plasma-driven soft X-ray laser pulse, to investigate whether the soft X-ray laser is able to induce DNA double strand breaks (DSBs) in living cells or not. The human adenocarcimona cell line A549 was irradiated with the soft X-ray laser at a photon energy of 89 eV and the repair focus formation of the DSBs was assessed by immunofluorescence staining with antiphosphorylated DNA-PKcs (p-DNA-PKcs), ATM (p-ATM) and γ-H2AX antibody. The p-DNA-PKcs, ATM, and γ-H2AX foci were clearly identified after soft X-ray laser irradiation. Furthermore, the increase in the X-ray laser shot number, even from a single shot, results in the increase in p-DNA-PKcs foci. These results are the first evidence that the 89 eV soft X-ray laser is able to induce DSB in living cells. Our study demonstrated that this irradiation system is a useful tool for investigating the radiobiological effect of soft X-ray laser.     

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm⁻¹, respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process.     

1 - 硝基芘对GC - 2细胞的损伤效应及AhR信号通路的活化

目的 研究1 - 硝基芘（1 - nitropyrene, 1 - NP）对小鼠精母细胞株GC - 2的损伤效应及芳香烃受体（aryl hydrocarbon receptor, AhR）信号通路的影响。方法 建立不同剂量1 - NP染毒的GC - 2细胞模型，采用CCK - 8法检测细胞活性，流式细胞术检测细胞周期，Western blot分析各组细胞DNA双链断裂损伤标志物γ - H2AX、DNA损伤反应通路蛋白以及AhR通路蛋白的表达。实时荧光定量PCR检测细胞色素450 (CYP)1a1和1b1 mRNA表达水平。结果 1 - NP处理可导致GC - 2细胞活性降低及G2/M期细胞比例升高，同时诱导γ - H2AX蛋白表达升高，DNA损伤反应及细胞周期G2/M期检测点蛋白ATM、Chk1、Cdc25c、Cdc2的磷酸化表达水平升高，Cyclin B1表达降低。1 - NP染毒也提高了GC - 2细胞中AhR核蛋白及下游靶基因CYP1A1的蛋白表达，提高了Cyp1a1和Cyp1b1的转录表达水平。结论 1 - NP暴露造成小鼠生精细胞的DNA损伤、细胞周期阻滞和细胞活性抑制，其效应可能与AhR信号通路的活化及对化学物的代谢活化有关。     

rAAV-hBMP2-GFP载体感染滴度测定方法的建立

目的建立携骨形态发生蛋白2基因的重组腺相关病毒载体（rAAV-hBMP2-GFP）感染滴度测定的方法,并制备高滴度的rAAV-hBMP2-GFP病毒液,为进一步实验hBMP2诱导大鼠牙槽骨骨缺损修复的基因治疗提供有利的工具。方法通过分析腺相关病毒-HT1080（AAV-HT1080）细胞接种量、培养时间及病毒吸附时间等参数,建立应用荧光计数法测定rAAV-hBMP2-GFP病毒感染滴度的方法,并分析其精密性及检测病毒的稳定性。结果 AAV-HT1080细胞的最佳接种浓度、培养时间及病毒吸附时间分别为3×105个/孔、24h及48h,在此条件下收获的病毒液的感染滴度最高可达3.82×1011/ml,所建立的荧光计数检测方法精密性较好。制备的病毒液的平均感染滴度为3.60×1011/ml,4℃和37℃保存7d及反复冻融5次,稳定性良好。结论建立了rAAV-hBMP2-GFP感染滴度的测定方法,并制备了高滴度及滴度稳定的rAAV-hBMP2-GFP病毒液。     

Mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer

Sorafenib, an orally available multikinase inhibitor, combined with radiation has shown potential as an anticancer treatment in an in vitro and in vivo colon cancer model. In this study, we investigated the mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer. The effects of sorafenib on radiation-induced cytotoxicity of DLD-1 and HT-29 were evaluated via clonogenic assay. The impact of sorafenib on radiation-induced cell cycle kinetics and on apoptosis was analyzed using flow cytometry. Cyclin B1 was examined by western blot. As a measure of DNA damage after treatment, γ-H2AX foci and nuclear fragmentation were determined as a function of time after irradiation plus sorafenib combination. Tumor growth delay was used to evaluate the effects of sorafenib on in vivo radiation-induced cytotoxicity. Exposure of each cell line to sorafenib combined with irradiation resulted in an increased radiation-induced cytotoxicity with dose enhancement factors at a surviving fraction of 0.37 ranging from 1.13 to 1.76. Sorafenib strengthened radiation-induced accumulation of tumor cells in the G2-M phase with attenuated expression of cyclin B1, but had no effect on radiation-induced apoptosis. Exposure to sorafenib and radiation resulted in a greater number of remaining γ-H2AX foci and fragmented nuclei than radiation alone. In vivo tumor xenograft study confirmed that administration of sorafenib results in significant tumor growth inhibition when combined with radiation. These results indicate that sorafenib enhances radiation-induced cytotoxicity in colorectal cancer and suggest that the mechanism is associated with delaying repair of radiation-induced DNA damage and down-regulation of cyclin B1.     

SPICE-NIRS Microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research

The Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) is a focused vertical microbeam system designed to irradiate the nuclei of adhesive mammalian cells with a defined number of 3.4 MeV protons. The approximately 2-μm diameter proton beam is focused with a magnetic quadrupole triplet lens and traverses the cells contained in dishes from bottom to top. All procedures for irradiation, such as cell image capturing, cell recognition and position calculation, are automated. The most distinctive characteristic of the system is its stability and high throughput; i.e. 3000 cells in a 5 mm × 5 mm area in a single dish can be routinely irradiated by the 2-μm beam within 15 min (the maximum irradiation speed is 400 cells/min). The number of protons can be set as low as one, at a precision measured by CR-39 detectors to be 99.0%. A variety of targeting modes such as fractional population targeting mode, multi-position targeting mode for nucleus irradiation and cytoplasm targeting mode are available. As an example of multi-position targeting irradiation of mammalian cells, five fluorescent spots in a cell nucleus were demonstrated using the γ-H2AX immune-staining technique. The SPICE performance modes described in this paper are in routine use. SPICE is a joint-use research facility of NIRS and its beam times are distributed for collaborative research.     

Effects of GSM 1800 MHz radiofrequency electromagnetic fields on DNA damage in Chinese hamster lung cells

OBJECTIVE: To study the effects of GSM 1800 MHz radiofrequency electromagnetic fields (RF EMF) on DNA damage in Chinese hamster lung (CHL) cells. METHODS: The cells were intermittently exposed or sham-exposed to GSM 1800 MHz RF EMF (5 minutes on/10 minutes off) at a special absorption rate (SAR) of 3.0 W/kg for 1 hour or 24 hours. Meanwhile, cells exposed to 2-acetaminofluorene, a DNA damage agent, at a final concentration of 20 mg/L for 2 hours were used as positive control. After exposure, cells were fixed by using 4% paraformaldehyde and processed for phosphorylated form of H2AX (gammaH2AX) immunofluorescence measurement. The primary antibody used for immunofluorescence was mouse monoclonal antibody against gammaH2AX and the secondary antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The gammaH2AX foci and nuclei were visualized with an Olympus AX70 fluorescent microscope. Image Pro-Plus software was used to count the gammaH2AX foci in each cell. For each exposure condition, at least 50 cells were selected to detect gammaH2AX foci. Cells were classified as positive when more than five foci were detected. The percentage of gammaH2AX foci positive cells was adopted as the index of DNA damage. RESULTS: The percentage of gammaH2AX foci positive cell of 1800 MHz RF EMF exposure for 24 hours (37.9 +/- 8.6)% or 2-acetylaminofluorene exposure (50.9 +/- 9.4)% was significantly higher compared with the sham-exposure (28.0 +/- 8.4)%. However, there was no significant difference between the sham-exposure and RF EMF exposure for 1 hour (31.8 +/- 8.7)%. CONCLUSION: 1800 MHz RF EMF (SAR, 3.0 W/kg) for 24 hours might induce DNA damage in CHL cells.     

IFN-γ诱导小鼠颗粒细胞DNA损伤及凋亡的体外研究

目的初步探讨IFN-γ诱导小鼠卵泡颗粒细胞DNA损伤及凋亡的机制。方法体外分离培养小鼠原代颗粒细胞,分为正常对照组、IFN-γ(1 000 U/ml)处理组及NAC(20 mM)+IFN-γ(1 000 U/ml)联合处理组,处理5d后,采用免疫荧光染色、Western blot和流式细胞术检测颗粒细胞的ATM/ATR信号通路中DNA损伤相关蛋白,ROS水平及凋亡相关蛋白的变化。结果 IFN-γ组颗粒细胞表达γ-H2AX,而NAC组未见明显γ-H2AX表达;Western blot发现IFN-γ组颗粒细胞γ-H2AX、Chk2和p-Chk2蛋白以及p53和p-p53、Caspase-3等凋亡相关蛋白水平均明显上调,NAC组中以上蛋白均被抑制;流式细胞术检测发现IFN-γ可诱导颗粒细胞活性氧簇(reactive oxygen species,ROS)水平上调,而NAC组颗粒细胞的ROS下降至对照组水平。结论 IFN-γ可通过DNA氧化损伤信号引起颗粒细胞凋亡。     

Conjugation of substituted ferrocenyl to thiadiazine as apoptosis-inducing agents targeting the Bax/Bcl-2 pathway

Ferrocene compounds are a class of biologically active compounds that has antitumour and antifungal properties. This study investigated the induction of apoptosis in human fibrosarcoma cells (HT1080) after treatment with a series of 6-ferrocenyl-3-subsituted7H-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazine (FTFs). We found that FTFs could suppress the viability of HT1080 cells. Cell cycle analysis showed that proliferative inhibition of HT1080 cells occurred through apoptosis, as the cells were blocked in G1 phase. Moreover, mitochondrial membrane staining assay demonstrated that FTFs exposure significantly decreased mitochondrial membrane potential. Finally, under the stress of FTFs, Bax/Bcl-2 ratio in HT1080 cells was significantly increased. These results suggested that FTFs-induced apoptosis in HT1080 cells may work dependent on a Bax/Bcl-2 pathway.     

Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model

The microdosimetric-kinetic (MK) model is one of the models that can describe the fraction of cells surviving after exposure to ionizing radiation. In the MK model, there are specific parameters, k and y_D, where k is an inherent parameter to represent the number of potentially lethal lesions (PLLs) and y_D indicates the dose-mean lineal energy in keV/μm. Assuming the PLLs to be DNA double-strand breaks (DSBs), the rate equations are derived for evaluating the DSB number in the cell nucleus. In this study, we estimated the ratio of DSBs for two types of photon irradiation (6 MV and 200 kVp X-rays) in Chinese hamster ovary (CHO-K1) cells and human non-small cell lung cancer (H1299) cells by observing the surviving fraction. The estimated ratio was then compared with the ratio of γ-H2AX foci using immunofluorescent staining. For making a comparison of the number of DSBs among a variety of radiation energy cases, we next utilized the survival data in the literature for both cells exposed to other photon types, such as ⁶⁰Co γ-rays, ¹³⁷Cs γ-rays and 100 kVp X-rays. The ratio of DSBs based on the MK model with conventional data was consistent with the ratio of γ-H2AX foci numbers, confirming that the γ-H2AX focus is indicative of DSBs. It was also shown that the larger y_D is, the larger the DSB number is. These results suggest that k and y_D represent the characteristics of the surviving fraction and the biological effects for photon irradiation.     

α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku–DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku–DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin–proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.     

γ-H2AX 在辐射生物剂量估算中的研究进展

γ-H2AX对受照人员DNA双链断裂损伤修复具有重要作用,利用γ-H2AX焦点计数可以估算照射剂量,因其检测周期短、特异性与灵敏度相对较高,近几年被国内外广泛应用于辐射剂量生物估算研究。本文对γ-H2AX在生物剂量估算中的应用与研究进展做一综述,对其在核与辐射事故批量受照人员的快速剂量估算以及伤员分类的应用前景上进行展望。     

NNK-Induced DNA Methyltransferase 1 in Lung Tumorigenesis in A/J Mice and Inhibitory Effects of (−)-Epigallocatechin-3-Gallate

DNA methyltransferase 1 (DNMT1), a key enzyme mediating DNA methylation, is known to be elevated in various cancers, including the mouse lung tumors induced by the tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). However, it is not known whether DNMT1 expression is induced right after NNK treatment and how DNMT1 expression varies throughout lung tumorigenesis. In the present study, we found that administration of NNK to A/J mice caused elevation of DNMT1 in bronchial epithelial cells at Days 1, 3, and 14 after NNK treatment. DNMT1 elevation at Day 1 was accompanied by an increase in phospho-histone H2AX (γ-H2AX) and phospho-AKT (p-AKT). At Weeks 5 to 20, NNK-induced DNMT1 in lung tissues was in lower levels than the early stages, but was highly elevated in lung tumors at Week 20. In addition, the early induction of p-AKT and γ-H2AX as well as cleaved caspase-3 in NNK-treated lung tissues was not detected at Weeks 5 to 20 but was elevated in lung tumors. In concordance with DNMT1 elevation, promoter hypermethylation of tumor suppressor genes Cdh13, Prdm2, and Runx3 was observed in lung tissues at Day 3 and in lung tumors. Treatment by EGCG attenuated DNMT1, p-AKT, and γ-H2AX inductions at Days 1 and 3 and inhibited lung tumorigenesis.     

Current topics in DNA double-strand break repair

DNA double strand break (DSB) is one of the most critical types of damage which is induced by ionizing radiation. In this review, we summarize current progress in investigations on the function of DSB repair-related proteins. We focused on recent findings in the analysis of the function of proteins such as 53BP1, histone H2AX, Mus81-Eme1, Fanc complex, and UBC13, which are found to be related to homologous recombination repair or to non-homologous end joining. In addition to the function of these proteins in DSB repair, the biological function of nuclear foci formation following DSB induction is discussed.