【作者】 胡恭华；

【导师】 庄志雄；

【作者基本信息】 中南大学 ， 公共卫生与预防医学， 2013， 博士

【摘要】 1研究背景细胞常常受到外界环境及内源性代谢因素如辐射、化学物和氧化应激的攻击。而这些暴露则可能导致突变和DNA链的断裂,进而引发基因组不稳定性。为了克服环境有害因子的攻击并维持基因组稳定性,真核细胞通过长期进化,已形成了一种称为DNA损伤反应(DNA damage response, DDR)的复杂网络来感应DNA损伤、发送损伤信号并对之进行修复。DDR是由多种检查点蛋白和修复蛋白构成的,而这些相关蛋白能够协调复杂的DNA损伤信号级联反应,从而使得细胞发生细胞周期阻滞以使其有充分的时间来进行DNA修复或DNA损伤耐受,或者触发细胞凋亡。最近的研究证据表明,人类DNA聚合酶η(Polη)参与了DDR。 Polη是癌症易感性疾病—着色性干皮病变种(xeroderma pigmentosum variant, XPV)基因的编码产物。Polη属于Y族DNA聚合酶,它能够催化DNA合成反应以跨越因紫外线辐射所产生的能够阻碍复制性聚合酶进程的顺-反式环丁烷嘧啶二聚体(cyclobutane pyrimidine dimmers, CPDs)。研究表明,除了CPDs以外,Polη还能够跨越其它类型的DNA损伤,比如顺铂加合物、丁二烯所诱发生成的2’-脱氧尿苷以及一氧化氮所产生的加合物2’-脱氧肌苷等。此外,Polη参与了免疫球蛋白基因的体细胞超突变、同源重组和DNA脆性位点的复制。近年来的研究发现,Polη在人类细胞中参与了抑制8-羟基鸟嘌呤所诱发的突变,并可阻止基因组不稳定性以及与DNA双链断裂密切相关的γ-H2AX的形成。这些研究表明,除了参与跨损伤合成所介导的DNA损伤耐受以外,Polη在对各种内源性和外源性遗传毒性因子所致DNA损伤的反应当中还具有着其它重要的生物学功能。氢醌(hydroquinone, HQ)在自然界中天然地存在于细菌、植物和某些动物体内,也可因商业用途而被生产出来。目前,HQ主要用于化妆品中的皮肤增白剂、黑白摄影术的显影剂、橡胶抗氧化剂、不饱和单体如醋酸乙烯酯和丙烯酸单体的阻聚剂等。HQ也存在烟草、咖啡、麦类制品和红酒当中。人类可以通过环境、职业、饮食和吸烟等多种途径接触到HQ；也可通过对苯的暴露而接触到HQ,因为苯可在体内代谢为HQ。HQ可经皮肤、消化道和呼吸道等途径吸收,主要在肝脏内代谢。由于HQ应用的广泛性,国内外相关学者已对之进行了大量的毒性研究。啮齿类动物的慢性毒性研究表明,HQ可以引起肝肾肿瘤,并可引起白血病。许多研究表明HQ能够触发机体内的氧化应激过程,从而产生活性氧类,并可通过不同的机制引起明显的染色体异常和DNA损伤。因此,DNA损伤常被用作HQ的毒性终点。此外,HQ在人体细胞内能够诱发细胞凋亡,并干扰细胞周期的进展。虽然目前人们对于Polη在跨损伤合成中的作用已有了很好的了解,但是对于其在DDR过程当中的作用仍不太明确。为了更好地理解Polη在人类肝细胞内的功能性作用及其重要性,我们采用了RNA干扰(RNA interference, RNAi)技术来选择性敲低Polη在肝细胞内的表达水平,并通过测定其对细胞增生、细胞凋亡、细胞周期进展和DNA链断裂以及DNA损伤反应通路活化程度的影响,以确定肝细胞内Polη的缺失是如何影响HQ的细胞毒性及遗传毒性的。本研究的目的就是初步阐明Polη在肝细胞对HQ所致DNA损伤反应中的作用及其机制,从而为建立机体对HQ所致损伤的防御体系提供科学的理论依据。2研究方法2.1Polη缺陷肝细胞株的建立及鉴定根据POLH的目标序列,使用siRNA设计软件设计了3对编码shRNAs的寡核苷酸序列和1对编码非靶向shRNA的寡核苷酸序列,并送于上海生工生物技术服务有限公司合成。接着将所合成的shRNA寡核苷酸序列退火,并将之克隆到pLKO.1载体的Agel和EcoRI位点上以产生RNAi载体；接着将这些重组载体转化入感受态JM109细菌当中；收集各细菌克隆以备提取质粒之用。然后将阳性克隆进行测序鉴定。将经测序鉴定成功的阳性质粒分别命名为pLKO.1-POLH1、pLKO.1-POLH2、pLKO.1-POLH3和pLKO.1-C,接着采用脂质体2000试剂盒按照使用说明书将各阳性重组质粒转染293FT细胞,并进行慢病毒颗粒的包装及浓缩。慢病毒颗粒感染细胞,并采用嘌呤霉素筛选稳定表达POLH shRNA和阴性对照shRNA的细胞株,并将各细胞株分别命名为L02-POLH-sh1、L02-POLH-sh2、L02-POLH-sh3和L02-POLH-nsc(非特异性靶向siRNA对照)细胞。采用实时定量PCR和Western blot技术分别对POLH的,nRNA及蛋白质表达水平进行鉴定。此外,对上述各细胞株的生物学特征如细胞形态、细胞增生和细胞周期等进行比较分析。2.2Polη表达抑制对HQ作用条件下肝细胞的细胞生物学特征和DNA损伤的影响(1)将指数生长期的肝细胞用0、10、20、40、80、160和320gM的HQ作用24h,采用倒置显微镜观察各组细胞的形态学特征,并采用MTT法测定细胞存活率。(2)将L02-POLH-nsc和L02-POLH-sh肝细胞分别用0、10、20和40μM的HQ作用24h,采用流式细胞术检测各组细胞的凋亡情况,并采用DAPI荧光染色法观察各组凋亡细胞的细胞核形态。(3)将L02-POLH-nsc和L02-POLH-sh肝细胞分别用0、10、20和40μM的HQ作用24h,采用流式细胞术检测细胞周期的分布状况。每个样品至少收集10000个细胞,采用FACSAriaTM型流式细胞仪上所附带的CellQuest软件对数据进行分析,并采用ModFit LT软件对细胞周期各时相的构成比进行量化。(4)将L02-POLH-nsc和L02-POLH-sh肝细胞分别用0、10、20和40μM的HQ作用24h,采用彗星实验检测DNA损伤状况,采用CASP分析软件测算每个彗星的相关参数；采用激光扫描共聚焦荧光显微镜观察γ-H2AX焦点的生成情况。2.3Polη表达抑制对HQ作用条件下肝细胞内DNA损伤检查点蛋白的表达、磷酸化及其亚细胞定位的影响将L02-POLH-nsc和L02-POLH-sh肝细胞分别用0、10、20和40μM的HQ作用24h,采用实时定量PCR方法检测各DNA损伤检查点基因(ATM、ATR, Chk1、Chk2、p53、H2AX和RPA2)的mRNA表达水平,并采用RQ Manager1.2软件对各目的基因mRNA表达水平进行相对定量分析。采用Western blot检测DNA损伤检查点蛋白(ATM、ATR、Chk1、Chk2、p53、H2AX和RPA2)及其磷酸化蛋白(p-Chk1、p-Chk2、p-p53、y-H2AX和P-RPA2)进行检测,并采用ImageJ软件对各目的蛋白条带的灰度值进行分析。采用激光扫描共聚焦显微镜观察各DNA损伤检查点蛋白的亚细胞定位特征。2.4实验数据的统计学分析所有实验数据都以均数±标准差表示,采用SPSS14.0for windows软件对之进行统计分析。两组间的比较采用t检验；多组间比较采用单因素方差分析,当组间比较差异具有统计学意义时,采用Tukey法进行任两组之间的比较；规定P<0.05时,差异具有统计学意义。3研究结果3.1Polη缺陷肝细胞株的建立及鉴定实时荧光定量PCR实验结果显示,与未转染对照组细胞即正常L-02肝细胞相比较,L02-POLH-sh1、L02-POLH-sh2和L02-POLH-sh3细胞内POLH基因mRNA表达的抑制率分别为61%、60%和82%,差别具有统计学意义(P<0.05)；而L-02肝细胞、L02-POLH-nvc和L02-POLH-nsc细胞POLH基因mRNA表达水平之间的差别却无统计学意义(P>0.05)。Western blot实验结果显示,L02-POLH-sh1、 L02-POLH-sh2和L02-POLH-sh3细胞内Polη在蛋白质表达水平上的抑制率分别为57.46%、71.49%和85.84%,与L-02细胞相比较,差别也有统计学意义(P<0.05)；而L-02肝细胞、L02-POLH-nvc和L02-POLH-nsc细胞内Polη的蛋白质表达水平之间的差别则无统计学意义(P>0.05)。此外,L02-POLH-sh1、L02-POLH-sh2、 L02-POLH-sh3,L02-POLH-nsc、L02-POLH-nvc和L-02细胞在细胞生物学特征方面没有明显差别。由于PoIη在L02-POLH-sh3细胞内的抑制效率最好,因此将L02-POLH-sh3命名为L02-POLH-sh,并作为后续研究的工具细胞。3.2Polη表达抑制对HQ作用条件下肝细胞的细胞生物学特征和DNA损伤的影响(1)MTT实验结果显示,与未处理对照组相比较,其中10、20、40和80μM的HQ对L-02、L02-POLH-nsc肝细胞的相对存活率没有明显影响(P>0.05),而160和320μM的HQ则可明显降低肝细胞的存活水平,与未处理对照组比较差异有统计学意义(P<0.01)。而对于L02-POLH-sh细胞而言,10和20μM组与未处理对照组相比较,细胞相对存活率无明显差异(P>0.05)；当HQ作用剂量高于40μM就可导致细胞活力的明显降低(P<0.01)。L02-POLH-sh细胞对HQ的敏感性明显高于g-02和L02-POLH-nsc细胞。此外,细胞的形态学观察结果与MTT实验结果相符。(2)流式细胞术检测结果显示,在0～40μM浓度范围内,HQ可诱导L02-POLH-nsc和L02-POLH-sh细胞发生凋亡,并呈现一定的剂量依赖性。对L02-POLH-nsc细胞而言,0、10、20和40gM剂量组的细胞调亡率依次为：2.73%、3.77%、5.27%、6.63%；而对L02-POLH-sh细胞而言,0、10、20和40μM剂量组的细胞调亡率则依次为：3.24%、6.78%、10.70%、14.03%；并且各处理组与未处理对照组相比较,均明显地高于未处理对照组(P<0.05)。在相同的作用剂量条件下,HQ对L02-POLH-sh细胞所诱发的凋亡率明显地高于L02-POLH-nsc细胞(P<0.01)；而在0gM的HQ作用条件下,L02-POLH-nsc和L02-POLH-sh细胞之间的凋亡率差别无统计学意义(P>0.05)。此外,DAPI荧光染色法实验结果与上述流式细胞术的检测结果基本相符。(3)流式细胞术检测结果显示,HQ作用后,L02-POLH-nsc和L02-POLH-sh细胞的细胞周期各时相分布特征发生了明显地改变,表现为G1期细胞比例的减少、S期和G2期细胞比例的相应增加。对于L02-POLH-nsc细胞而言,10μM和20μM组的S期细胞比例与未处理对照组相比较其差异无统计学意义(P>0.05),而40μM组的S期细胞比例则明显地高于未处理组(P<0.05),其数值分别由未处理组的25.93%增加至27.54%、27.93%和35.02%。而对于L02-POLH-sh细胞来说,10、20和40μM的HQ与未处理对照组相比较均可引起S期细胞的明显增加(P<0.05),其具体数值分别由未处理组的27.80%增加至36.42%(10μM组)、40.72%(20μM组)和44.42%(40μM组)。此外,在相同的HQ作用剂量(10、20或40gM)下,与L02-POLH-nsc细胞相比较,L02-POLH-sh细胞的S期细胞比例均有明显地增加(P<0.05)。(4)彗星实验结果显示,HQ以剂量依赖性的方式诱导L02-POLH-nsc和L02-POLH-sh细胞DNA损伤水平增加。在相同的HQ作用剂量条件下, L02-POLH-sh细胞DNA损伤的程度明显地大于L02-POLH-nsc细胞,差别具有统计学意义(P<0.01)。细胞免疫荧光联合激光扫描共聚焦显微镜观察结果显示,HQ作用后,L02-POLH-nsc和L02-POLH-sh细胞内的y-H2AX焦点数均有随着HQ作用剂量的增加而增加的趋势,并呈现一定的剂量依赖性；在相同的HQ作用剂量条件下,L02-POLH-sh细胞内的y-H2AX焦点数明显地多于L02-POLH-nsc细胞(P<0.05)。3.3Polη表达抑制对HQ作用条件下肝细胞内DNA损伤检查点蛋白的表达、磷酸化及其亚细胞定位的影响(1)实时定量PCR实验结果显示,HQ能够诱导ATM、ATR、 CHK1、CHK2.P53和RPA2基因的mRNA表达水平在L02-POLH-nsc和L02-POLH-sh细胞内以剂量依赖性方式增加；在相同的HQ作用条件下,L02-POLH-sh细胞内CHK1、P53和RPA2基因的mRNA表达水平明显地高于L02-POLH-nsc细胞。(2) Western blot分析结果显示,HQ能够在L02-POLH-nsc和L02-POLH-sh细胞内诱导ATM、ATR、Chk2、p53、RPA2、p-chk1、 p-chk2、p-p53、p-RPA2和γ-H2AX的蛋白表达水平增加。HQ能够在L02-POLH-sh细胞诱导Chk1蛋白表达水平的升高,而在L02-POLH-nsc细胞内却无此现象。此外,在相同的HQ作用条件下,L02-POLH-sh细胞内Chk1、p53、RPA2、p-chk1、p-chk2、p-p53、 P-RPA2和γ-H2AX的蛋白表达水平明显地高于L02-POLH-nsc细胞。(3)细胞免疫荧光实验结果显示,HQ能够在L02-POLH-nsc和L02-POLH-sh细胞内诱导ATM、ATR、Chk1、Chk2、p53、RPA2、 p-chk1、p-chk2、p-p53、p-RPA2和γ-H2AX荧光强度的增加。在相同的HQ作用条件下,L02-POLH-sh细胞内Chk1、p53、RPA2、 p-chk1、p-chk2、p-p53、p-RPA2和γ-H2AX的荧光强度明显地高于L02-POLH-nsc细胞。各目的蛋白的亚细胞定位分析结果显示,HQ能够导致ATM和Chk2从细胞核移位到细胞浆；而Polη的缺陷则会导致Chk1和p53从细胞浆移位至细胞核内。此外,RPA2在L02-POLH-nsc和L02-POLH-sh细胞内能够与γ-H2AX共定位在一起,并且该种共定位会随着HQ作用剂量的增加而增强。此外,Polη的抑制会使RPA2与γ-H2AX的共定位效应得到明显的增强。4结论(1)本研究采用RNA干扰技术成功建立了Polη缺陷的肝细胞株,这为进一步的实验研究提供了基础。(2)Polη表达的抑制导致了HQ作用条件下肝细胞增生的减少,并使肝细胞对HQ所致细胞毒性的敏感性增强。(3) Polη的缺陷明显增加了HQ所致肝细胞DNA双链断裂的形成或积累程度。(4) Polη表达的抑制在肝细胞内增强了HQ所诱发的细胞凋亡效应,并增强了HQ在肝细胞内所引起的细胞周期的S期阻滞现象。(5)HQ作用条件下,Polη的缺陷明显增加了Chk1、p53和RPA2在肝细胞内的表达水平及其在细胞核内的定位。(6)HQ可在肝细胞内引起DNA损伤检查点蛋白Chk1、Chk2、 p53、H2AX和RPA2的强烈磷酸化；并且这些蛋白的磷酸化效应在Polη有缺陷的肝细胞内得到了明显的增强。(7)Polη可能通过调节细胞增生、凋亡、细胞周期进程、DNA双链断裂修复和DNA损伤检查点蛋白的活化而在肝细胞对HQ所引起的DNA损伤反应当中发挥着重要的作用。更多还原

【Abstract】 1BackgroundCells are constantly exposed to environmental and metabolic insults such as radiation, chemical agents and oxidative stress. Such exposure may generate DNA lesions that lead to mutations and DNA strand breaks and cause genomic instability. To overcome these attacks and maintain the integrity of the genome, eukaryotic cells have evolved a complex network to detect, signal the presence of and repair DNA damage, which is referred as DNA damage response(DDR) pathway. DDR is composed of numerous checkpoint and repair proteins that coordinate a complex signaling cascade to assess the damage, then either arrest cell cycle to provide time for DNA repair or DNA damage tolerance or trigger apoptosis.Recent evidences have suggested that human DNA polymerase eta (Polη) has been implicated in DDR. Polη is the product of the xeroderma pigmentosum variant (XPV) gene, which is mutated in the cancer-prone genetic disorder, xeroderma pigmentosum variant. Polη is a Y-family DNA polymerase with the ability to perform translesion synthesis across cys-syn pyrimidine dimmers (CPDs), the primary lesion induced by UV radiation, which blocks replicative DNA polymerases. In addition to CPDs, Polη has been shown to replicate across other DNA lesions, including cisplatin adducts, butadiene-derived2’-deoxyuridine adducts, and the nitric oxide-derived adduct2’-Deoxyinosine. Moreover, Polη is also involved in somatic hypermutation of immunoglobulin genes, homologous recombination, and replication of DNA fragile sites. Recent studies have found that Polη plays a suppressive role in the mutagenesis by8-hydroxyguanine in human cells and plays an important role in preventing genome instability and avoiding y-H2AX which is associated with double-strand breaks and single-stranded DNA in human cells. These studies suggest that in addition to its roles in DNA damage tolerance by translesion synthesis, Polη has other biological roles in response to DNA damage caused by a variety of endogenous and exogenous genotoxic agents.Hydroquinone (HQ) occurs naturally in bacteria, plants and some animals and is also manufactured for commercial use. It is used in cosmetics as a skin lightening agent, in photography as a black and white developer, in the production of antioxidants for rubber and as a polymerization inhibitor for vinyl acetate and acrylic monomers. HQ also occurs naturally in cigarette smoke, the leaves and bark of several plant species as a component of glucopyranoside, arbutin, and plant-derived foods such as coffee, wheat-based products, and red wine. Human exposure to HQ can occur following environmental, occupational, dietary and cigarette smoke exposure, and from exposure to benzene, which can be metabolized to HQ. HQ is absorbed dermally, orally or by inhalation, and is mainly metabolized in the liver.Due to the widespread use of HQ, numerous toxicity studies have been conducted. In chronic toxicity studies with rodents some evidence of carcinogenicity was demonstrated, including benign tumors of the kidney and liver and mononuclear cell leukemia. Many studies have indicated that HQ can trigger an oxidative stress process, giving rise to reactive oxygen species, and induce significant chromosome abnormities and DNA damage via a different mechanism. Thus, DNA lesions are usually used as toxic end points. In addition, HQ can induce apoptosis and interfere with cell cycle progression in human cells. Despite its well-established role in translesion synthesis, the role of Polη in DDR has not been well explored.In order to better understand the functional roles and importance of Polη in human hepatic cells, we used RNA interference (RNAi) to selectively knockdown Polη in hepatocytes and then determined how the loss of Polη affected the cytotoxicity and genotoxicity of HQ by measuring its effects on cell proliferation, apoptosis, cell cycle progression, DNA strand breaks, and the activation of the DNA damage response pathways in hepatocytes. The goal of this work was to characterize preliminarily the mechanism of polymerase Polη in DNA damage response induced by HQ in hepatocytes and provide scientific theoretical basis to establish the body defense system to the damage caused by hydroquinone.2Methods2.1Construction and identification of Polη-deficient hepatic cell lineAccording to the targeting sequences of POLH, three pairs of oligonucleotide encoding shRNAs and one pair of oligonucleotide encoding non-target shRNA were designed using siRNA design software, and synthesized by Sangon. The oligonucleotides were annealed and cloned into the AgeI and EcoRI sites of the pLKO.1vector to generate RNAi vectors, and these recombination vectors were then transformed into competent JM109cells. Bacterial colonies were pooled and used for plasmid preparation. The positive clones were confirmed by sequencing. The resulting plasmids were designated as pLKO.1-POLH1, pLKO.1-POLH2, pLKO.1-POLH3and pLKO.1-C.293FT cells were then transfected with pLKO.1-POLHs or pLKO.1-C using Lipofectamine2000according to the manufacturer’s instructions. The lentivirus expression vectors were established and replication incompetent lentiviruses carrying POLH shRNA were produced by transfected293FT cells. L-02cells were stably infected with lentivirus carrying POLH shRNA or negative control shRNA. After infection, stable cell lines were generated by selection with puromycin. The puromycin-resistant clones selected were considered to be stably infected cells and designated as L02-POLH-sh1, L02-POLH-sh2, L02-POLH-sh3and L02-POLH-nsc (non-specific siRNA control) cells, respectively. POLH mRNA and protein expression levels were confirmed by Real-time PCR and Western blot. In addition, the biological characteristics(cell morphology, cell proliferation and cell cycle) between the above cell lines were compared.2.2Exploration on effects of inhibition of Polη expression on cell biological characteristics and DNA damage in hepatocytes exposed by HQ(1) Exponentially growing hepatic cells were treated with HQ of different final concentrations(0,10,20,40,80,160, and320μM) for24h, then morphological features of hepatic cells were observed by inverted microscope and cellular viability was determined by the MTT assay.(2) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, apoptosis was determined by flow cytometry, and the nuclei changes induced by HQ were observed under fluorescence microscope after DAPI staining.(3) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, and cell cycle distribution was determined using flow cytometry. At least10,000cells per sample were collected and the data were then analyzed by a FACSAriaTM flow cytometer using CellQuest software. The percentage of cell cycle phases was quantified using ModFit LT software.(4) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, DNA damage was determined by the Comet assay and the parameters of each comet was calculated using CASP analysis software; y-H2AX foci were visualized by confocal laser scanning fluorescence microscopy.2.3Exploration on effects of inhibition of Polη expression on the expression, phosphorylation and subcellular localization of DNA checkpoint proteins in hepatocytes exposed by HQL02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, the mRNA expression levels of DNA damage checkpoint genes(ATM, ATR, Chk1, Chk2, p53, H2AX and RPA2) were determined by real-time PCR and the data were analyzed by RQ Manager1.2software; the protein expression levels of DNA damage checkpoint proteins(ATM, ATR, Chk1, Chk2, p53, H2AX and RPA2) and its phosphorylation(p-Chkl, p-Chk2, p-p53, y-H2AX and p-RPA2) were determined by Western blot, the relative amounts of target proteins were calculated from the scanning profiles and analyzed by ImageJ software; subcellular localization of DNA damage checkpoint proteins were visualized by confocal laser scanning fluorescence microscopy.2.4Statistical analysisAll data are presented as mean±SD. Statistical evaluation of data analysis was carried out using SPSS14.0for Windows. Differences between the mean values of two groups were analyzed by t test. Differences between the mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. P<0.05was considered statistically significant.3Results3.1Polη-deficient hepatic cell line was constructed by RNAiThe results of real-time PCR analysis showed that the expression levels of Polη mRNA were down-regulated by61%in L02-POLH-sh1cells, by60%in L02-POLH-sh2cells and by82%in L02-POLH-sh3cells compared with normal L-02cells. Different degrees of POLH knockdown were also observed at the protein level in these cells, which were down-regulated by58%in L02-POLH-shl cells, by72%in L02-POLH-sh2cells and by86%in L02-POLH-sh3cells compared with normal L-02cells. The expression levels of both Polη, mRNA and protein were not obviously changed in L02-POLH-nsc and L02-POLH-nvc cells. In addition, there were no significant differences in cell biology characteristics between L02-POLH-sh1, L02-POLH-sh2, L02-POLH-sh3, L02-POLH-nsc, L02-POLH-nvc and L-02cells, however, the levels of Polη mRNA and protein were significantly reduced in L02-POLH-sh1, L02-POLH-sh2and L02-POLH-sh3cells, compared to L-02cells(P<0.05). Because Polη was down-regulated most effectively in L02-POLH-sh3cells, this clone was used in all subsequent studies, and is hereafter referred to as L02-POLH-sh.3.2Effects of inhibition of Polη expression on cell biological characteristics and DNA damage in hepatocytes exposed by HQ(1) Cell viability of L-02and L02-POLH-nsc cells in the treated groups (160and320μM) was significantly less than that in the untreated group (0μM)(P<0.01). However, cell viability in the treated groups (40,80,160and320μM) of L02-POLH-sh cells was significantly less than that in the untreated group (P<0.01). In particular, the viability of L02-POLH-sh cells was significantly lower than that of L-02and L02-POLH-nsc cells treated with40,80,160or320μM HQ, respectively (P<0.01). Whereas the cytotoxicity of HQ in parental L-02cells was similar to that observed in L02-POLH-nsc cells (P>0.05). The results also showed that L02-POLH-sh cells were2-fold more sensitive to HQ compared with L-02and L02-POLH-nsc cells. In addition, the results of morphological observations were consistent with the MTT assay.(2) The results showed that HQ induced apoptosis in L02-POLH-nsc and L02-POLH-sh cells in a dose-dependent manner, and suppression of Polη expression had little effect on the percentage of apoptotic cells in untreated controls. However, treatment with increasing concentrations of HQ resulted in significant dose-dependent increases in the levels of apoptosis in both cell lines. The rate of apoptosis increased from2.73%to3.77%, to5.27%, to6.63%in L02-POLH-nsc cells and from3.24%to6.78%, to10.70%, to14.03%in L02-POLH-sh cells at0,10,20, and40μM HQ treatment, respectively, and a significantly higher percentage of apoptosis in all treated L02-POLH-sh cells was observed compared with L02-POLH-nsc cells(P<0.01). In addition, the results of DAPI staining assay were consistent with above data.(3) Exposure of cells to HQ induced a significant alteration in cell cycle distribution with a decrease in the fraction of cells in G1phase and a corresponding increase in the fraction of cells in S and G2phase in both cell lines. No significant alteration was found in S phase cell populations after incubation with10and20μM HQ as compared with untreated cells (P>0.05), whereas treatment with40μM HQ significantly increased the percentage of L02-POLH-nsc cells in S phase (P<0.05), the proportion of cells in S phase increased from25.93%in the control to27.54,27.93and35.02%, respectively. In contrast, treatment with10,20and40μM HQ significantly increased the percentage of L02-POLH-sh cells in S phase (P<0.05):27.80,36.42,40.72and44.42%, respectively. In addition, our data revealed that the fraction of L02-POLH-sh cells in S phase was significantly higher than that in L02-POLH-nsc cells treated with10,20, and40μM HQ (P<0.05).(4) Comet assay showed that HQ induced a marked dose-dependent increase in DNA damage in L02-POLH-nsc and L02-POLH-sh cells, as measured by comet parameters. However, DNA damage due to HQ treatment was significantly greater in L02-POLH-sh cells as compared with L02-POLH-nsc cells after24h of exposure(P<0.01). Further more, immunofluorescence assay showed that treatment of L02-POLH-nsc and L02-POLH-sh cells with HQ could induce the accumulation of y-H2AX foci in the nucleus in a dose-dependent manner, y-H2AX foci were more abundant in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment(P<0.05).3.3Effects of inhibition of Polη expression on the expression, phosphorylation and subcellular localization of DNA checkpoint proteins in hepatocytes exposed by HQ(1) Results of real-time PCR showed that HQ induced a dose-dependent increase in mRNA expression levels of ATM, ATR, CHK1, CHK2, P53, and RPA2in L02-POLH-nsc and L02-POLH-sh cells, and the mRNA expression levels of CHK1, P53and RPA2were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment.(2) Results of Western blot analysis showed that HQ induced an increase in protein expression levels of ATM, ATR, Chk2, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX in L02-POLH-nsc and L02-POLH-sh cells; the protein expression levels of Chkl was increased in L02-POLH-sh, but not in L02-POLH-nsc cells following HQ treatment. In addition, the protein expression levels of Chk1, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment.(3) Results of immunofluorescence assay showed that HQ induced an increase in the fluorescence intensity of ATM, ATR, Chk1, Chk2, p53, RPA2, p-chkl, p-chk2, p-p53, p-RPA2and y-H2AX in L02-POLH-nsc and L02-POLH-sh cells, and the fluorescence intensities of Chkl, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment. Subcellular localization analysis showed that ATM and Chk2translocated from the nucleus to the cytoplasm in L02-POLH-nsc and L02-POLH-sh cells following HQ treatment. Polη deficiency resulted in translocation of Chkl and p53from the cytoplasm to the nucleus. In addition, the majority of RPA2colocalized with γ-H2AX in the nucleus of L02-POLH-nsc and L02-POLH-sh cells, and this co-localization could be enhanced after HQ treatment in dose-dependent manner; furthermore, inhibition of Polη enhanced co-localication of RPA2and γ-H2AX.4Conclusion4.1Polη-deficient L02-POLH-sh cell line was successfully constructed by RNAi technology mediated by lentiviral vector, which provided basis for later studies.4.2Inhibition of Polη expression led to a decrease in cell proliferation and an enhanced susceptibility to HQ cytotoxicity in hepatocytes.4.3Polη deficiency significantly increased the formation or accumulation of DSBs induced by HQ in hepatocytes.4.4Inhibition of Polη expression resulted in an enhanced apoptosis and more pronounced S phase arrest upon HQ treatment in hepatocytes.4.5Polη deficiency significantly increased the expression levels and nuclear localization of Chkl, P53and RPA2in hepatocytes induced by HQ.4.6Phosphorylation of Chkl, Chk2, p53, H2AX and RPA2was strongly activated by HQ, and this activation is enhanced in hepatic cells lacking Polη.4.7The results demonstrate that Polη may play an important role in the DNA damage response induced by HQ in human hepatic cells, through regulating cell proliferation, apoptosis, cell cycle progression, DNA double-strand break repair and activation of DNA damage checkpoint proteins.更多还原