Nampt is involved in DNA double-strand break repair

DNA double-strand break(DSB) is the most severe form of DNA damage,which is repaired mainly through high-fidelity homologous recombination(HR) or error-prone non-homologous end joining(NHEJ).Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer.Nicotinamide phosphoribosyltransferase(Nampt),which is involved in nicotinamide adenine dinucleotide metabolism,is overexpressed in a variety of tumors.In this report,we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80,which are key factors in HR and NHEJ,respectively.Depletion of Nampt by small interfering RNA(siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair.Furthermore,the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining,indicating a delay in the onset of cellular senescence in normal human fibroblasts.Taken together,our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair,contributing to the acceleration of cellular senescence.     

DNA double-strand break repair and development

Normal development of an organism requires the ability to respond to DNA damage. A particularly deleterious lesion is a DNA double-strand break (DSB). The cellular response to DNA DSBs occurs via an integrated sensing and signaling network that maintains genomic stability. The outcomes of defective DNA DSB repair are related to the developmental stage of an organism, and often show striking tissue specificity. Many human diseases are associated with deficiencies in DNA DSB repair and can be characterized by neuropathology, immune deficiency, growth retardation or predisposition to cancer. This review will focus on the requirements of the DNA DSB response that function to maintain homeostasis during mammalian development.     

DNA双链断裂修复与卵巢癌的相关性研究

DNA双链断裂修复(DSBR)是人类最主要的修复途径之一,其中修复基因可以修复损伤DNA,保持遗传信息的完整性,从而抑制癌症的发生.随着对DSBR与肿瘤关系的深入研究,DSBR中相关基因在卵巢癌的发生发展及治疗等方面产生了重要影响.

Abstract：

DNA double-strand break repair (DSBR) pathways are important repair pathways in human. DSBR pathways repair damaged DNA, maintain the integrity of the genetic information and therefore suppress cancer. More and more researches have indicated important roles of DSBR pathway genes in the development and treatment of ovarian cancer.     

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells

The induction of collateral radioresistance after the development of cisplatin resistance is a well-documented phenomenon; however, the exact processes that are responsible for the cisplatin-induced radioresistance remain to be elucidated. There was no obvious difference in the level of radiation-induced DNA double strand breaks (DSBs), in DSB rejoining rates, or the level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in the cisplatin- and radiation-sensitive 2780/WT and cisplatin-resistant 2780/CP cell lines. However, there was a significantly (P < 0.01) lower level of DSB misrejoining activity within nuclear protein extracts derived from the cisplatin- and radiation-sensitive 2780/WT and OAW42/WT tumour cell lines than in similar extracts from their cisplatin- (and radiation-) resistant 2780/CP and OAW42/CP counterparts. All of the DSB misrejoining events involved deletions of between 134 and 444 bp that arose through illegitimate recombination at short repetitive sequences, such as those that arise through non-homologous repair (NHR). These data further support the notion that the radiosensitivity of DSB repair proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. Furthermore, our data suggest that the induction of acquired cisplatin resistance is associated with a two- to threefold decrease in the activity of a non-conservative DSB rejoining mechanism that appears to be a manifestation of NHR.     

The BLM helicase is necessary for normal DNA double-strand break repair

Experiments with the supF20 mutagenesis system demonstrate that extracts from Bloom's syndrome (BS) cells are unable to use microhomology elements within the supF20 gene to restore supF function after the induction of a double-strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable with those of normal extracts when ligatable ends are present, a significant 5-fold increase in mutation rate with BS extracts is observed when terminal phosphates are removed from the DNA substrate that needs repair. Mutant plasmids recovered after DSB repair by BS extracts contain smaller deletions within the lacZalpha gene not commonly recovered from normal extracts. This work demonstrates that BS cells, lacking the BLM helicase, process DSBs differently than normal cells and strongly suggests a role for the BLM helicase in aligning microhomology elements during recombinational events in DSB repair.     

Liquid-liquid phase separation in DNA double-strand break repair

<正>DNA repair factors function through spatiotemporal condensation and dissolution at DNA double-strand breaks(DSBs). Recent advances have indicated that some DSB repair factors undergo liquid-liquid phase separation(LLPS) and show droplet-like properties, as well as dynamic material exchange. Importantly, LLPS regulates various biological processes, and aberrant LLPS is involved in the pathologic progression of various diseases.     

Radiation-induced DNA double-strand break rejoining in human tumour cells.

Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell.     

Long-term XPC silencing reduces DNA double-strand break repair

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.     

小鼠DNA双链断裂修复缺陷细胞的γ射线剂量率效应

目的：探讨γ射线照射后小鼠DNA双链断理解修复缺陷细胞（SCID）的剂量率效应和潜在致死性损伤的修复。方法：采用低剂量率和高剂量率以及间隔24h的2次γ射线照射正常细胞（CB．17＋／＋）和SCID细胞，通过成克隆分析法观察被照射细胞的存活分数。结果：应用间隔24h的2次γ射线照射CB．17＋／＋细胞时，其存活分数明显高于相同剂量的单次照射，而SCID细胞二者无明显差异。在高剂量率单次和2次γ射线照射时，SCID细胞均比CB．17＋／＋细胞更敏感。在低剂量率γ射线照射时，SCID细胞亦显示比CB．17＋／＋细胞更敏感。低剂量率γ射线照射 CB．17＋／＋细胞和SCID细胞后，二者的存活分数均明显高于剂量率照射。结论：SCID细胞不具有DNA双链断裂的修复能力。SCID细胞和CB．17＋／＋细胞均具有剂量率效应。     

DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells

Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs. Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay. The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay. Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line. The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells. Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma. These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs. Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.     

DNA double-strand break repair capacity and risk of breast cancer

A tumorigenic role of the non-homologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks (DSBs) has been suggested by our finding of a significant association between increased breast cancer risk and a cooperative effect of single-nucleotide polymorphisms in NHEJ genes. To confirm this finding, this case-control study detected both in vivo and in vitro DNA end-joining (EJ) capacities in Epstein-Barr virus-immortalized peripheral blood mononuclear cells (PBMCs) of 112 breast cancer patients and 108 healthy controls to identify individual differences in EJ capacity to repair DSB as a risk factor predisposing women to breast cancer. PBMCs from breast cancer patients consistently showed lower values of in vivo and in vitro EJ capacities than those from healthy women (P < 0.05). Logistic regression, simultaneously considering the effect of known risk factors of breast cancer, shows that the in vitro EJ capacity above the median of control subjects was associated with nearly 3-fold increased risks for breast cancer (adjusted odds ratio, 2.98; 95% confidence interval, 1.64-5.43). Furthermore, a dose-response relationship was evident between risk for breast cancer and EJ capacity, which was analyzed as a continuous variable (every unit decrease of EJ capacity being associated with an 1.09-fold increase of breast cancer risk) and was divided into tertiles based on the EJ capacity values of the controls (P for trend < 0.01). The findings support the conclusion that NHEJ may play a role in susceptibility to breast cancer.     

Polymorphisms in DNA double-strand break repair genes and skin cancer risk

UV can cause a wide range of DNA lesions. UVA-induced oxidative DNA damage and blocked DNA replication by UVB-induced photoproducts can lead to double-strand breaks (DSBs). We selected 11 haplotype-tagging single nucleotide polymorphisms in three DSB repair genes XRCC2, XRCC3, and LigaseIV and evaluated their associations with skin cancer risk in a nested case-control study within the Nurses' Health Study [219 melanoma, 286 squamous cell carcinoma (SCC), 300 basal cell carcinoma (BCC), and 873 controls]. We observed that the XRCC3 18085T (241Met) allele and its associated haplotype were significantly inversely associated with the risks of SCC and BCC, whereas the XRCC3 4552C allele along with its associated haplotype and the XRCC2 30833A allele were significantly associated with increased BCC risk. The LigaseIV 4044T and 4062T alleles were associated with decreased BCC risk; two of four haplotypes were significantly associated with altered BCC risk. A trend toward decreased risk of nonmelanoma skin cancer was found in those harboring a greater number of putative low risk alleles (P for trend, 0.05 for SCC, <0.0001 for BCC). The main effects of these genotypes were essentially null for melanoma risk. This study provides evidence to suggest the role of the DSB repair pathway in skin cancer development, especially for BCC.     

Synthetic lethal targeting of DNA double-strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double-strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant [V-C8(Rev1)]. ii) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested SL in CH ovary cells expressing a dominant-negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. SL was also demonstrated in CH cells expressing a dominant-negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising SL target in cancer.     

DNA双链断裂修复通路和卵巢癌的关系

卵巢癌发生发展与DNA损伤累积引起的基因不稳定性和细胞行为异常密切相关。DNA双链断裂修复通路中最为重要的是同源重组和非同源末端连接机制以及最近发现的微同源介导的末端修复形式,能够快速准确地修复DNA双链断裂,对维持基因组稳定性起着至关重要的作用,对卵巢癌的基因诊断、基因治疗以及卵巢癌分子水平研究均有重要意义。     

DNA双链断裂修复蛋白表达水平与肿瘤细胞放射敏感性的关系研究

目的：检测肿瘤细胞株中DNA双链断裂修复蛋白（Ku80、DNA-PKcs和ATM）的表达水平和放射敏感性参数,探讨3个蛋白预示肿瘤细胞放射敏感性的价值。方法：培养4株人宫颈癌细胞HeLa、SiHa、C33A和Caski,3株人乳腺癌细胞MCF-7、MDA-MB-231和MDA-MB-453,及1株人肺癌细胞A549,Western blot检测这8株细胞中Ku80、DNA-PKcs和ATM蛋白的表达水平;流式细胞仪检测X线（10 Gy,6 MV）照射48 h后的凋亡率;克隆形成实验检测SF2（surviving fraction at 2 Gy）值和α、β值;Pearson线性相关分析蛋白表达水平与照射后凋亡率、SF2值和α/β比值的相关性。结果：3种蛋白在同一株细胞中的表达及同一蛋白在不同细胞株的表达均存在明显差异;DNA-PKcs的表达水平与SF2之间存在正相关关系（r=0.723,P=0.043）;Ku80和ATM的表达与SF2值间无明显相关关系（P〉0.05）。3种蛋白与凋亡率和α/β比值均无相关性（均P〉0.05）。结论：DNA-PKcs蛋白表达越高,细胞对放射线越抵抗,其表达水平可能成为指示肿瘤细胞放射敏感性的指标。     

Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells

DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ～10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability.     

Faster repair of DNA double-strand breaks in radioresistant human tumor cells

To investigate the molecular basis of radiation resistance in human tumor cells, the induction and repair of radiation-induced DNA single- and double-strand breaks was determined by DNA elution analysis in two normal human cell lines and 12 early-passage human tumor cell lines of varying radiosensitivities. The radiosensitivities (D0) of the cell lines ranged from 1 to 2.9 Gy. Inherent cellular radiosensitivity was found to directly correlate with the rate at which the DNA double-strand breaks were repaired. Radioresistant cell lines repaired approximately 90% of their radiation-induced DNA double-strand breaks within 1 hr of irradiation while more radiosensitive cell lines required 2-4 hr to repair the same fraction of damage. Radioresistant cell lines also had lower initial DNA double-strand break frequencies. DNA single-strand break induction and repair was not found to be an important factor in the radiation response of human tumor and normal cell lines. Therefore, the rate at which DNA double strand breaks are repaired is a critical factor underlying radioresistance in human tumor cell lines.     

A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of gamma-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.     

DNA double-strand break repair,DNA-PK,and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation.Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5′-³³P] Poly (dA)·(oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting.Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e^-αD-βD2, the two cell lines roughly have the same α value (∼0.40 Gy^-1) whereas the β value was considerably higher in UM-SCC-14A (0.067 Gy^-2 ± 0.007 Gy^-2 [SEM]) as compared to UM-SCC-1 (0.013 Gy^-2 ± 0.004 Gy^-2 [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A (p < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines.Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.