Ku蛋白与DNA修复

电离辐射易引起DNA双链断裂(DSB),Ku蛋白作为异二聚体,主要参与非同源末端联接修复DSB。Ku蛋白在维持端粒结构发挥至关重要的作用。在人的不同肿瘤中,Ku蛋白有着异常表达,且Ku蛋白的表达和功能与肿瘤放化疗抵抗有关。通过抑制Ku蛋白可增强放化疗敏感性。因此,Ku蛋白可作为放化疗增敏的靶点。     

神经上皮细胞转化基因1对细胞辐射敏感性的影响及其机制探讨

目的 探讨神经上皮细胞转化基因1(Net1)对细胞辐射敏感性的影响及相关的分子作用机制.方法 运用实时荧光定量PCR检测辐射后细胞中Net1基因表达水平的变化;采用RNAi干扰技术抑制细胞中Net1的表达,用克隆形成率分析细胞的辐射敏感性;利用免疫共沉淀技术发现Net1的结合蛋白.结果 电离辐射损伤后,细胞中的Net1 mRNA水平显著上升(t=-10.52,P＜0.05);与对照组相比,siRNA沉默细胞中的Net1表达后明显增加了细胞的辐射敏感性(t=15.31、11.65,P＜0.05);无论在正常状态下还是在细胞受到辐照后,Net1都能与非同源末端连接修复蛋白Ku70、Ku80和DNA-PKcs结合.结论 Net1对细胞的辐射防护作用可能是通过与非同源末端连接修复蛋白相互作用来调控辐射损伤修复实现的.     

Ku70对同源重组修复蛋白Rad51的调节作用

目的 研究非同源末端连接(NHEJ)蛋白Ku70对同源重组(HR)修复蛋白Rad51的调节作用,初步探讨HR与NHEJ间协同作用的机制。 方法 采用Western blot法观察特异性增高和降低Ku70蛋白水平后Rad51蛋白表达水平的变化情况。 结果 靶向Ku70基因小干扰RNA(siRNA)单纯转染组Rad51蛋白的表达水平较空白对照组明显下降;Ku70高表达载体PGCsi3.0-hKu70转染肿瘤细胞后,随着Ku70表达水平的升高,Rad51水平也随之升高。 结论 Ku70可能对Rad51有正调控作用,NHEJ可能通过Ku70对Rad51的调节来实现其与HR的协同作用。     

应用脉冲电场凝胶电泳分析X射线诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应

目的 研究电离辐射诱发人骨肉瘤肿瘤细胞DNA双链断裂与辐射损伤修复效应, 观察辐射损伤、损伤修复效应与肿瘤细胞辐射敏感性之间的关系。方法 选用强制均匀电场电泳, 分别测定经不同剂量X射线照射和相同剂量照射后培养不同时间, 人骨肉瘤Rho0和143.B肿瘤细胞株DNA双链断裂。结果 (1)X射线诱发人骨肉瘤肿瘤细胞的DNA双链断裂与辐射剂量呈线性正比关系; (2)培养后的人骨肉瘤肿瘤细胞对辐射诱发的DNA双链断裂具有一定修复能力; (3)Rho0比143.B细胞株具有更高的辐射敏感性; (4)脉冲电场凝胶电泳技术是分析人肿瘤细胞DNA双链断裂的敏感方法。结论 脉冲电场凝胶电泳是分析人肿瘤细胞DNA双链断裂的敏感方法; 电离辐射诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应和肿瘤细胞的辐射敏感性有密切关系。     

线粒体DNA4977bp缺失和彗星分析评价肿瘤细胞的辐射敏感性

目的 探讨mtDNA4977bp缺失和彗星分析用于肿瘤细胞辐射敏感性检测的可行性。方法 选取3种不同肿瘤细胞株:肝癌细胞(HepG₂)、食管癌细胞(EC-9706)和乳腺癌细胞(MCF-7)。采用MTT法检测肿瘤细胞经γ射线照射后的存活分数(SF);巢式PCR法测肿瘤细胞的mtDNA4977bp缺失率;单细胞凝胶电泳(SCGE)检测肿瘤细胞的DNA断裂水平。结果 用MTT法发现HepG₂ 和EC-9706细胞的辐射敏感性高于MCF-7细胞。8 Gy照射后HepG₂和EC-9706 mtDNA4977bp缺失率明显高于MCF-7细胞(P<0.05),证实HepG₂和EC-9706细胞的辐射敏感性高于MCF-7细胞。多种方法分析结果说明3种肿瘤细胞在8Gy照后表现出的辐射敏感性差异有统计学意义。结论 多种生物学指标的综合应用,可能更加客观准确地评价肿瘤细胞的辐射敏感性。     

ANTP-SmacN7对肿瘤细胞的辐射增敏作用及其机制

目的 探讨ANTP-SmacN7融合蛋白对肿瘤细胞的辐射增敏作用及其机制。方法 合成ANTP-SmacN7融合蛋白并观察其细胞渗透能力,Western blot法检测辐射对XIAP表达量的影响,克隆形成实验观察ANTP-SmacN7融合蛋白的辐射增敏作用。Annexin V-FITC/PI双染法测定药物及射线对肿瘤细胞凋亡的影响,Western blot法检测ANTP-SmacN7阻断XIAP前后Caspase-8、Caspase-9和Caspase-3的变化。结果 ANTP-SmacN7融合蛋白可以进入细胞内,并在细胞内蓄积;辐射诱导肿瘤细胞体外XIAP表达量与肿瘤辐射敏感性呈负相关（r=0.82）;ANTP-SmacN7对肿瘤细胞有辐射增敏作用,增敏比为1.41;ANTP-SmacN7融合蛋白可促进4、8和10 Gy γ射线诱导的XIAP高表达肿瘤细胞的凋亡（t=-14.924、-7.294和-15.866,P<0.05）。辐射可诱导Caspase-3表达水平的升高,ANTP-SmacN7对Caspase-3蛋白表达水平不产生影响,但可增加活化Caspase-3的裂解片段的数量。结论 ANTP-SmacN7融合蛋白可通过诱导Caspase-3的活化降低肿瘤细胞的辐射抗性。     

UHRF1对人乳腺癌细胞MDA-MB-231辐射敏感性的影响及作用机制

目的 了解基因UHRF1的不同表达水平对乳腺癌细胞MDA-MB-231辐射敏感性的影响及潜在的作用机制。方法 利用克隆形成实验观察细胞存活;流式细胞术测定细胞周期;利用DNA片段分析和Annexin V试剂盒测定细胞凋亡;Western blot 测定蛋白表达变化;利用经典的染色体分析,观察染色体畸变(着丝粒环和双着丝粒)。 结果 与对照相比较,UHRF1转染可将D₀值由2.08 Gy提高至3.17 Gy,即降低MDA-MB-231细胞对X射线的辐射敏感性。利用siRNA抑制UHRF1的表达,可将X射线照射后细胞的生存率由41%降低至17%,即增强了细胞的辐射敏感性。UHRF1的高表达,可诱导G₂/M期阻滞,抑制细胞凋亡,下调促凋亡蛋白Bax,上调DNA损伤修复蛋白Ku70和 Ku80的表达水平,而且,抑制X射线诱导的染色体畸变。结论 UHRF1的高表达可以抑制细胞凋亡,促进DNA损伤修复。因此,通过抑制UHRF1的表达,可作为临床提高乳腺癌放疗疗效的新靶标。     

组蛋白去乙酰酶阻滞剂的放射增敏作用

放射治疗是肿瘤的重要治疗手段之一,辐射可以导致细胞DNA双链断裂。细胞主要通过同源重组修复和非同源末端连接修复方式修复DNA双链断裂。随着对双链DNA损伤修复机制认识的深化,组蛋白去乙酰酶（HDAC）阻滞剂成为提高放射敏感性的一种新策略。HDAC可分为4类。HDAC阻滞剂可非特异性地或特异性地阻滞这4类HDAC,使组蛋白乙酰化水平提高,染色体解螺旋,核小体结构改变。一方面使DNA更易受到辐射的影响;另一方面通过降低E2F1转录因子活性抑制损伤修复蛋白Ku80、Rad51等的表达,使其不能募集DNA损伤修复蛋白,且不能形成相应的蛋白复合物,使同源重组修复和非同源末端连接修复作用延缓,在伴有或不伴有肿瘤细胞凋亡增加的情况下,提高放射敏感性。现已有一些临床试验在进行中,并取得了初步的结果。     

慧星电泳用于肿瘤细胞辐射敏感性检测的研究

目的：探讨彗星电泳方法检测肿瘤细胞对γ射线的辐射敏感性的可行性。方法：以自行设计的图像分析系统，用彗星电泳方法检测4种人肿瘤细胞受γ射线照射后细胞初始DNA损伤，以及细胞径30min修复时DNA损伤残余率；用细胞集落存活法检测2Gy γ射线照射后细胞存活率。结果：4种肿瘤细胞初始DNA损伤与辐射敏感性无关，2Gyγ射线照射后4种细胞存活率（SF2）与细胞经30min修复后的DNA损伤残余率相关明显（r=-0.87)，结论：本实验方法有可能成为一种快速、准确检测肿瘤细胞内在辐射敏感性的方法。     

FHIT基因转染增强人肝癌细胞HepG2辐射敏感性

目的探讨脆性组胺酸三联体基因转染对肿瘤细胞辐射敏感性的影响。方法首先构建了含有FHIT基因编码序列的真核表达质粒pcDNA／FHIT，并转染人肝癌细胞株Hep G2得到表达正常FHIT蛋白的细胞株Hep G2，FHIT。然后利用CCK-8和流式细胞仪等方法研究了Hep G2／FHIT细胞接受^60Coγ射线电离辐射后细胞周期、增殖和凋亡的改变。结果研究表明人肝癌细胞Hep G2重新表达FHIT后其对电离辐射的敏感性增加，表现为接受电离辐射后细胞存活率下降，凋亡细胞增加。细胞周期检测发现Hep G2重新表达FHIT后接受电离辐射后发生G2期阻滞的程度减轻。结论FHIT基因缺失的肿瘤细胞重新表达正常的FHIT蛋白可以提高其辐射敏感性，并且辐射敏感性提高可能是和FHIT基因抑制了ATR／CHK1通路活性有关。FHIT基因．电离辐射联合治疗肿瘤，具有一定的协同作用，可以作为一个较好的肿瘤基因治疗的靶点。     

Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells

Purpose:?To investigate the radiosensitising effect of Ku autoantigen 70 (Ku70) and Ku autoantigen 80 (Ku80) knockdown by lentivirus-mediated RNA interference (RNAi) in the MCF10A immortalised human mammary epithelial cell line.

Materials and methods:?MCF10A cells were infected with lentiviral vectors for RNAi of Ku70. The Ku70-knockdown cell line (Ku70i) and a mock-infected control cell line (LVTHM) were used to perform radiation experiments. For the in?vitro Micronucleus (MN) assay, both cell lines were irradiated with doses of 2 and 4 Gy ⁶⁰Co γ-rays. For cell survival experiments, doses ranging between 0 and 8 Gy were used.

Results:?Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. A significantly higher radiation-induced MN yield was obtained in the Ku70i cell line compared to the control LVTHM cell line. RNAi of Ku70 also resulted in a lower survival yield after irradiation compared to the control cell line. Analysis of cell death mechanisms showed that MCF10A cells (Ku70i and LVTHM) do not undergo apoptosis, but undergo post-irradiation cellular senescence.

Conclusion:?RNAi of Ku70 resulted in increased chromosomal and cellular radiosensitivity in the MCF10A human mammary cell line after irradiation with ⁶⁰Co γ-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.     

低剂量电离辐射对7721细胞细胞周期及p53、Ku70和Ku80表达的影响

目的　观察电离辐射对 772 1细胞 (人肝癌细胞 )细胞周期和p53、Ku70和Ku80基因表达的影响。方法　以人肝癌细胞株 772 1为研究对象 ,通过克隆形成实验拟合出 772 1细胞的剂量存活曲线 ;用流式细胞技术检测 75mGyX射线照射后 772 1细胞周期变化 ;用原位杂交方法检测 772 1细胞p53、Ku70和Ku80基因在 75mGyX射线照射前、后的表达情况。结果　与对照组相比 ,75mGyX射线照射后 0 5～ 6h ,772 1细胞S期延长 (P <0 0 5)。p53、Ku70和Ku80基因的表达照射前与照射后比较差异无显著性。结论　 772 1细胞在 75mGyX射线照射后 ,未出现G1期阻滞 ,p53、Ku70和Ku80基因在照射前、后表达无明显变化 ,是由于这些基因自身存在缺陷或激活机制存在缺陷所致     

In vitro studies of cellular response to DNA damage induced by boron neutron capture therapy

The aim of these studies was to evaluate the mechanisms of cellular response to DNA damage induced by BNCT. Thyroid carcinoma cells were incubated with ¹⁰BPA or ¹⁰BOPP and irradiated with thermal neutrons. The surviving fraction, the cell cycle distribution and the expression of p53 and Ku70 were analyzed. Different cellular responses were observed for each irradiated group. The decrease of Ku70 in the neutrons +BOPP group could play a role in the increase of sensitization to radiation.     

Radioresistance in a tumour cell line correlates with radiation inducible Ku 70/80 end-binding activity

PURPOSE: The aims of the present study were to better understand the role of Ku 80, which is involved in double-strand break repair in mammalian cells in the mechanism of radiation resistance and to verify the possibility of increasing cell radiosensitivity by targeted inhibition of Ku autoantigen 80 (Ku 80). MATERIALS AND METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were performed on the human bladder carcinoma cell line RT112 (radioresistant) and on the human colorectal carcinoma cell line SW48 (radiosensitive) to assess the expression levels of DNA-dependent protein kinase (DNA-PK) components and the DNA-binding activity of the Ku 70/80 heterodimer after exposure to radiation, respectively. Ku 80 silencing was carried out with the use of small interfering RNA (siRNA). RESULTS: Greater differences in the DNA-binding activity of Ku 70/80 and Ku 80 phosphorylation level were observed in RT112 as compared to SW48 after X-ray treatment. There is no correlation between Ku expression and DNA-binding activity at lower doses. A significant increase in nuclear Ku 80 expression was observed one hour after the exposure, only at the higher doses, while the DNA-PK catalytic subunits (DNA-PKcs) and Ku 70 levels did not change significantly. Inhibition of Ku 80 expression by siRNA induced radiosensitivity in the RT112 cell line. CONCLUSIONS: Our data demonstrate that in a bladder tumour cell line up-regulation of Ku end-binding activity without any marked change in Ku expression underlie radiation resistance.     

Radioresistance in a tumour cell line correlates with radiation inducible Ku 70/80 end-binding activity

Purpose: The aims of the present study were to better understand the role of Ku 80, which is involved in double-strand break repair in mammalian cells in the mechanism of radiation resistance and to verify the possibility of increasing cell radiosensitivity by targeted inhibition of Ku autoantigen 80 (Ku 80).

Materials and methods: Western blot and electrophoretic mobility shift assay (EMSA) were performed on the human bladder carcinoma cell line RT112 (radioresistant) and on the human colorectal carcinoma cell line SW48 (radiosensitive) to assess the expression levels of DNA-dependent protein kinase (DNA-PK) components and the DNA-binding activity of the Ku 70/80 heterodimer after exposure to radiation, respectively. Ku 80 silencing was carried out with the use of small interfering RNA (siRNA).

Results: Greater differences in the DNA-binding activity of Ku 70/80 and Ku 80 phosphorylation level were observed in RT112 as compared to SW48 after X-ray treatment. There is no correlation between Ku expression and DNA-binding activity at lower doses. A significant increase in nuclear Ku 80 expression was observed one hour after the exposure, only at the higher doses, while the DNA-PK catalytic subunits (DNA-PKcs) and Ku 70 levels did not change significantly. Inhibition of Ku 80 expression by siRNA induced radiosensitivity in the RT112 cell line.

Conclusions: Our data demonstrate that in a bladder tumour cell line up-regulation of Ku end-binding activity without any marked change in Ku expression underlie radiation resistance.     

Ku80基因沉默联合γ射线对人食管癌细胞移植瘤生长的影响

目的 探讨shRNA抑制Ku80蛋白表达联合照射对裸鼠移植瘤食管癌生长的抑制作用。 方法 构建干扰Ku80载体shRNA-Ku80,采用Western blotting方法观察shRNA干扰Ku80蛋白的表达。将20只裸鼠随机分为对照组、单纯照射组、shRNA-H₂组和联合治疗组,观察裸鼠移植瘤的生长情况。免疫组织化学法检测移植瘤内Ku80表达。结果 成功构建了shRNA-Ku80载体,并挑选出有效的靶序列。Ku80干扰载体和放射对移植瘤生长均有抑制作用,抑瘤率分别为32.0%和39.9%。二者联合作用抑制更显著,抑瘤率为68.9%。shRNA-H2降低移植瘤内Ku80表达58%(t=3.77,P<0.05)。结论 shRNA干扰Ku80联合照射能显著提高食管癌细胞移植瘤的放射效应。     

Clinical studies of immunohistochemical staining of DNA-dependent protein kinase in oropharyngeal and hypopharyngeal carcinomas

PURPOSE: DNA-dependent protein kinase (DNA-PK), a serine/threonine kinase composed of p470 catalytic subunit (DNA-PKcs) and p85/p70 heterodimer (Ku antigen), is considered a critical enzyme in the repair of the DNA double-strand breaks (DSB) that are the major lethal lesions induced by ionizing radiation. We investigated the expression of DNA-PK subunits in human tumors. MATERIALS AND METHODS: We examined immunohistochemically the biopsy specimens of 44 patients with oropharyngeal carcinoma and 32 patients with hypopharyngeal carcinoma who had been treated with radiotherapy. RESULTS: Immunopositivity to Ku85 and DNA-PKcs was found in all patients. The staining of Ku85 and DNA-PKcs was nuclear, with none of the normal epithelial cells or malignant cells exhibiting cytoplasmic or membrane immunoreactivity. Normal epithelial cells were all stained intensely. In tumors, intense nuclear staining of DNA-PKcs was seen in 75 of 76 tumors, while that of Ku85 was seen in all 76 patients. The radiation responses of a primary tumor that was stained weakly with DNA-PKcs were excellent. CONCLUSION: Our results suggest the possibility of predicting the intrinsic radiosensitivity of human tumors in clinics able to perform immunohistochemical analysis of DNA-PK.     

Identification of proteins in the hamster DNA end‐binding complex

Purpose: To identify the protein components of the DNA end‐binding complex in hamster cells.

Materials and methods: DNA end‐binding complexes were identified as follows. Nuclear extracts from Chinese hamster ovary cells (0.5–1.0?µg protein/lane) were incubated with 0.5?ng ³²P‐labelled probe (144?bp) for 20?min at room temperature in the presence of 1?µg closed circular pUC18 plasmid, a non‐specific competitor in a final volume of 20?µl. The electrophoretic mobility of the protein–DNA complexes was analysed by electrophoresis in 5% polyacrylamide gels subjected to autoradiography. Antibodies to various DNA repair‐associated proteins were added to the DNA end‐binding complex reaction and a supershift identified DNA end‐binding complex components. These were confirmed by Western analysis of purified DNA end‐binding complex contents.

Results: Using both supershift and Western analysis, the following proteins were identified in the DNA end‐binding complex: Ku70, Ku80, DNA‐dependent protein kinase catalytic subunit, DNA ligase IV, X‐ray cross complementing protein 4, meiotic recombination protein 11 (Mre11), Werner's syndrome protein, Bloom's syndrome protein, p53, poly(ADP‐ribose) polymerase, replication protein A (RPA) 14, and RPA32, ataxia telangiectasia mutant, c‐Abl, Rad50, Nijmegen breakage syndrome protein 1 (NBS1), and DNA ligase III were not detected in the binding complex by any assay. Using a combination of electro‐elution and autoradiography, it was estimated that the single DNA end‐binding complex contains at least 15 proteins whose molecular weights of the DNA end‐binding proteins ranged from 620 to 12?kDa.

Conclusions: A combination of both a supershift assay and Western analysis of the DNA end‐binding complexes has identified 12 of at least 15 proteins present in the DNA end‐binding complex of Chinese hamster ovary cells. This protein complex contains Mre11, but not Rad50 or NBS1, suggesting that under some conditions, Mre11 might function independently of Rad50 and NBS1.