Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma

Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 μM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.     

电离辐射诱导DNA链断裂的动力学研究

通过测试γ射线辐照下超螺旋ｐＢＲ３２２ ＤＮＡ分子单链断裂（ＳＳＢ）,双链剂量效应,得到ＳＳＢ、αＤＳＢ产额与ＤＮＡ现含一定浓度甘露醇的ＤＮＡ溶液体系中,Ｇ（ＳＳＢ）、Ｇ（αＤＳＢ）的例数与ｃ（ＤＮＡ）的倒数成线性关系,并以二级动力学描述了ＤＮＡ和甘露醇分子对．ＯＨ的竞争反应,得到．ＯＨ引起ＳＳαＤＳＢ的速率常数及其效率。     

Saturation of Dark Repair Synthesis: Accumulation of Strand Breaks

Reversal of ultraviolet light damage to DNA by the dark repair system is limited. Experiments utilizing density and radioactive labels demonstrated that repair synthesis is not proportional to dose at doses above 200 ergs/mm². In addition, the number of residual excision induced gaps in Escherichia coli B/r hcr⁺ DNA increases with higher UV doses. The extent of repair is apparently limited by saturation of the repair synthesis step.     

HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

Background

Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.

Results

Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.

Conclusions

This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.     

Radiation-Induced Breaks of DNA in Cultured Mammalian Cells

Mouse leukemic cells (L5178Y) in suspension culture were irradiated and the extent of single-strand breaks and double-strand cuts of DNA was estimated by sucrose gradient centrifugation. The radiation produced 3.0 single-strand breaks per cell (G(1) stage) per rad and approximately 0.3 double-strand breaks per cell (G(1) stage) per rad.     

Dinitrophenol Inhibits the Rejoining of Radiation-Induced DNA Breaks by L-Cells

The production and rejoining of X-ray-induced single-stranded DNA breaks was studied using the alkaline sucrose density gradient technique and by measuring the disappearance of both 5' termini and 3'-OH termini using polynucleotide kinase and DNA polymerase, respectively. All studies were conducted using L-cell suspensions irradiated both in the presence and absence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. Results show that the induction of single-stranded DNA breaks probably includes a nucleolytic component in addition to indirect free radical effects. A greater number of breaks were produced in the absence of DNP, suggesting that depressed adenosine triphosphate (ATP) levels reduce endogenous nucleolytic activity. The rejoining mechanism is enzymatic and requires an available ATP supply for operation. In the presence of DNP no DNA rejoining was observed following 30 min incubation after 10,000 rad. These results suggest that DNA breaks produced may be characterized by 5'-PO(4)-3'-OH termini and are rejoined by DNA ligase.     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

DNA双链断裂损伤反应及它的医学意义

DNA损伤应激反应是维持基因组稳定性的基石.细胞在长期进化中形成了由损伤监视、周期调控、损伤修复、凋亡诱导等在内的自稳平衡机制.一方面,借助感应、识别并启动精细而复杂的修复机制修复损伤;另一方面,通过DNA损伤应激活化的细胞周期检查点机制,延迟或阻断细胞周期进程,为损伤修复提供时间,使细胞能安全进入新一轮细胞周期;损伤无法修复时则诱导细胞凋亡.DNA双链断裂(double strand breaks,DSBs)是真核基因组后果最严重的损伤类型之一,其修复不利,同肿瘤等人类疾病的发生发展密切相关.新进展揭示:DSBs损伤反应信号分子ATM-Chk2-p53、H2AX等的组成性活化,是肿瘤形成早期所激活的细胞内可诱导的抗癌屏障,其信号网络的精确、精细调控在基因组稳定性维持中发挥重要作用.此外,HIV病毒整合进入宿主细胞基因组的过程也依赖于宿主细胞中ATM介导的DSBs损伤反应信号转导;ATM特异性的小分子抑制剂在抗HIV感染中显示重要的功能意义.文中重点讨论调控DSBs损伤应激反应信号网络的主要研究进展,及其在肿瘤发生、发展及抗HIV感染中的新医学意义.     

Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks

Following DNA double-strand breaks cells activate several DNA-damage response protein kinases, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as MDC1, p53-binding protein 1, and breast cancer gene 1 at the damage site to promote DNA double-strand breaks repair. We identified a novel biomarker, Bora (previously called C13orf34), that is associated with radiosensitivity. In the current study, we set out to investigate how Bora might be involved in response to irradiation. We found a novel function of Bora in DNA damage repair response. Bora down-regulation increased colony formation in cells exposed to irradiation. This increased resistance to irradiation in Bora-deficient cells is likely due to a faster rate of double-strand breaks repair. After irradiation, Bora-knockdown cells displayed increased G2-M cell cycle arrest and increased Chk2 phosphorylation. Furthermore, Bora specifically interacted with the tandem breast cancer gene 1 C-terminal domain of MDC1 in a phosphorylation dependent manner, and overexpression of Bora could abolish irradiation induced MDC1 foci formation. In summary, Bora may play a significant role in radiosensitivity through the regulation of MDC1 and DNA repair.     

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.     

Dynamics of Chromatin during the Repair of DNA Double-Strand Breaks

Double strand breaks (DSBs) are arguably the most deleterious DNAlesion that a cell can sustain, and defects in the ability to detect and repair thesebreaks result in increased genomic instability and have been causatively linked tocancer. The repair of DNA DSBs must occur in the context of chromatin, andthere is increasing evidence that the modulation of chromatin plays an integralrole in the DNA DSB repair process. Here, we summarize a number of keyfindings, largely from studies performed in budding yeast, highlighting the role ofchromatin in DNA DSB responses.     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.     

Volatile Gas Components Contribute to Cigarette Smoke-induced DNA Single Strand Breaks in Cultured Human Cells

Thermostable purine nucleoside phosphorylases, PUN PI and PUNPII, have been purified from Bacillus stearothermophilus JTS 859. The characterization of PUNPI was reported previously. [Hori et al.₉ Agric. Biol. Chem. 53, 2205 (1989)] PUNPII had a molecular weight of 113,000, consisting of 4 identical subunits (M_w 28,000). The isoelectric point was 5.3. The Michaelis constants for inosine, guanosine, and adenosine were 0.22, 0.34, and 0.075 mm, respectively. The optimal temperature of the reaction was 70°C. The enzyme was stable at 70°C. Although other reported purine nucleoside phosphorylases were SH-enzymes, PUNPII was not a SH-enzyme because the enzyme reaction was not inhibited by PCMB and iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to 11.0, and the enzyme did not contain cysteine.

PUNPII and PUNPI were different in several points. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. Specific activity of PUNPI and II for inosine were 405 and 50.6 μmol/min/mg protein at 60°C, respectively. PUNPI was stable at 80°C. PUNPII was stable at 70°C, but was denatured at 80°C.     

Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.