ATM在人胃癌细胞系的表达及其在细胞DNA损伤中的作用

目的:研究DNA损伤剂对ATM基因缺陷和正常的胃癌细胞系的作用机制.方法:Western-blot方法检测6株胃癌细胞系ATM蛋白的表达情况,并用DNA损伤剂顺铂作用于ATM基因缺陷的MKN28细胞系和ATM基因正常的BGC823细胞系,采用Western-blot、DNAladder、Hoechest 33258 /PI双荧光染色等方法观察凋亡相关激酶的表达和细胞的应答反应.结果:Western-blot显示,ATM蛋白在胃癌细胞系MGC803、MKN28、MKN45、SGC7901的表达水平显著低于BGC823和AGS细胞系,BGC823细胞在CDDP作用后,ATM蛋白表达水平升高,磷酸化chk1和chk2活性24小时后增强,而Cdc25C表达减少;MKN28细胞中G2期检测点蛋白ATM、p-chk1、p-chk2表达较低,Cdc25C表达较高,但在CDDP作用前后无明显变化.MKN28细胞在DNA损伤剂作用后出现显著凋亡,而BGC823细胞在DNA损伤剂作用48小时后未见显著凋亡.结论:ATM在细胞周期的多个环节均有调控作用,特别是在细胞周期检测点和DNA损伤的修复中有重要的监视和启动作用.     

钬元素对小鼠肝脏细胞DNA损伤的影响

背景与目的: 通过研究钬离子溶液对小鼠肝脏细胞DNA的损伤,探讨钬元素对诱导动物细胞凋亡的影响 。材料与方法: 处理组1： 小鼠定时灌胃氯化钬溶液50 mg/(kg•d),连续5 d; 组 2～3： 小鼠分别定时腹腔注射氯化钬溶液60 mg/(kg•d)和120 mg/(kg•d),连续2 d; 组4： 小鼠一次性腹腔注射氯化钬溶液320 mg/kg; 每次染毒相间24 h,组1～4： 小鼠均在末次染毒24 h后处死,提取肝脏DNA。 组5： 小鼠一次性腹腔注射等体积生理盐水; 组6～9： 小鼠一次性腹腔注射氯化钬溶液80 mg/kg,分别于注射后12、24、48和96 h处死小鼠提取肝脏DNA。通过琼脂糖凝胶电泳研究各组DNA带型变化。 结果：处理组7 DNA电泳中出现连续的弥散带型,其它各组均未观察到明显的拖尾现象。也未观察到“DNA ladder"。 结论： 钬离子对小鼠肝脏细胞DNA的断裂作用可能与其剂量大小、处理时间及DNA修复作用有关,而且无特异性。本实验结果表明,钬元素未诱导小鼠肝脏细胞凋亡。     

DNA损伤检控点ATM与胶质瘤放化疗耐受关系

胶质瘤放化疗耐受现象的分子机制是一个多信号通路构成的复杂网络,近年来的研究表明细胞DNA损伤检控点ATM、ATR、Chk1、Chk2、Rad17、Rad1、Rad9、Hus1等及其调节通路在细胞增殖、基因组的稳定以及肿瘤的发生和肿瘤放化疗耐受机制中发挥重要的作用.干预细胞DNA损伤检控点能够增加放化疗耐受肿瘤细胞对放化...     

CREB1和CREB3在胃癌及癌前病变组织中的表达及其临床意义

目的 探究环磷腺苷反应元件结合蛋白1(CREB1)和环磷腺苷反应元件结合蛋白3(CREB3)在胃癌及癌前病变组织中的表达及临床意义。方法 收集慢性浅表性胃炎40例,慢性萎缩性胃炎伴肠化生40例,异型增生40例,胃癌50例,采用免疫组化法检测CREB1和CREB3在四种组织中的表达,并分析其与临床病理参数的关系。另收集同期新鲜组织慢性浅表性胃炎、慢性萎缩性胃炎伴肠化生、胃癌共30例,应用蛋白印迹法(Western blot)检测CREB1和CREB3在不同胃组织中的表达。利用Kaplan-Meier plotter分析CREB1和CREB3的表达与胃癌患者总生存时间(OS)和无进展生存时间(FP)的相关性。利用STRING数据库分析CREB1和CREB3在信号通路中的位置以及与之相关的上下游基因。结果 免疫组化显示：胃癌组、异型增生组、萎缩性胃炎伴肠化生组CREB1、CREB3阳性表达率明显高于慢性浅表性胃炎组(P<0.05);并且CREB1、CREB3在胃癌组的阳性表达率明显高于慢性萎缩性胃炎伴肠化生组(P<0.05),但与异型增生组无统计学差异(P>0.05)。临床...     

HB—EGF蛋白在胃癌组织和人胃癌细胞系中的表达

目的：研究HB—EGF蛋白在胃癌组织和多个胃癌细胞系中的表达及其与胃癌发生的相关性。方法：免疫印迹和免疫荧光法检测新鲜胃癌组织和不同胃癌细胞系中HB—EGF蛋白的表达。结果：在癌旁正常胃黏膜中,HB—EGF蛋白表达较低,而在胃癌组织中,蛋白表达明显升高（P〈0．05）;在正常胃黏膜上皮细胞系GES-1中,未检出HB—EGF蛋白的表达,而在4种人胃癌细胞系中,HB—EGF蛋白表达均为阳性（P〈0．01）;其中SGC7901和MKN45中的表达强于BGC823和MKN28中的表达。结论：HB—EGF蛋白在胃癌组织和多种胃癌细胞系高表达,其表达水平升高可能与胃癌的发生和发展相关。     

PIG3在细胞凋亡及DNA损伤修复中的作用

PIG3作为p53下游调控细胞凋亡的靶基因,能够通过参与合成活性氧簇及对氧化应激的调控,而在细胞凋亡过程中发挥着重要的作用。近年来,其在DNA损伤应答通路中的作用也被大家广泛地研究。鉴于PIG3在细胞中扮演着两种不同的角色,而且这两种角色分别对应着不同的预后,因而了解PIG3在不同情形下发挥的作用尤为重要。本文针对PIG3在细胞凋亡和DNA损伤修复中的作用进行综述,并探讨了在不同细胞状态下PIG3表达的调控机制。     

Caspase-3及其底物DFF45在人肺癌组织中的表达及意义

目的:检测Caspase-3、DNA裂解因子(DFF45)在肺癌组织中的表达及与临床病理特征的关系.方法:在57例肺癌组织中应用免疫组织化学SP法、Western blot检测Caspase-3、DFF45蛋白的表达.结果:Western blot实验结果与免疫组化基本相符.57例肺癌组织中Caspase-3、DFF45蛋白表达阳性率分别为66.67%,29.82%.Caspase-3和DFF45蛋白的阳性表达率与组织学类型无关(P＞0.05),与肺癌的分化程度和淋巴结转移显著相关(P＜0.05).在肺癌组织中,Caspase-3与DFF45的表达具有显著正相关性(P=0.024).结论:Caspase-3和DFF45蛋白的低表达,促进了肺癌细胞的生长和淋巴结的转移.     

STIP1调控EGR1表达并促进胃癌细胞DNA损伤修复

目的 探讨应激诱导磷酸化蛋白1 (STIP1)表达对于胃癌化疗抵抗的影响,并进一步探讨其潜在机制。方法 构建STIP1过表达及敲低胃癌细胞系后,应用CCK-8、单克隆形成等实验检测胃癌细胞对5-氟尿嘧啶及顺铂、奥沙利铂的敏感性;并对过表达STIP1的胃癌细胞系进行RNA测序并验证获得STIP1所调控的下游分子及信号通路;最后应用免疫荧光、免疫印迹等方法检测STIP1对胃癌细胞化疗抵抗的影响是否依赖于其下游分子。结果 STIP1过表达可显著增强胃癌细胞对于5-氟尿嘧啶及顺铂、奥沙利铂等细胞毒性药物的抵抗作用;并可促进化疗抵抗相关基因的表达;RNA表达谱测序发现STIP1可能促进重组修复相关信号通路的活化,而免疫印迹及qPCR等检测证实STIP1过表达可促进该通路相关基因的表达;免疫荧光检测进一步证实STIP1表达可影响5-氟尿嘧啶诱导的DNA损伤的重组修复能力;而应用重组修复抑制剂干预胃癌细胞后。免疫荧光检测获得STIP1表达对于5-氟尿嘧啶所诱导的胃癌细胞DNA损伤的影响主要是通过影响其同源重组修复途径。对于其下游机制的探索,通过生物信息学分析、免疫印迹、qPCR及免疫荧光,证实STI...     

RNF8在前列腺癌细胞中的表达与定位

目的:构建Flag-RNF8真核表达质粒,证实融合蛋白在前列腺癌细胞内的表达与定位。方法:提取人前列腺癌细胞CWR22RV1总RNA,反转录为cDNA。PCR扩增RNF8全长编码区cDNA序列,亚克隆至含有Flag标签的真核表达载体中。将构建的重组质粒测序并转染到前列腺癌细胞CWR22RV1中,提取细胞蛋白进行Western blot检测,同时利用共聚焦激光扫描显微镜观察Flag-RNF8在CWR22RV1细胞内定位。使用免疫沉淀的方法纯化RNF8蛋白。结果:RNF8蛋白全长编码区cDNA克隆到了真核表达载体pcDNA3-Flag中,酶切鉴定片段约为1500bp。Western blot检测到了融合蛋白Flag-RNF8的表达,分子量约为57kDa。Flag-RNF8在CWR22RV1细胞中表达并定位在细胞核中。成功纯化RNF8蛋白。结论:成功构建了Flag-RNF8全长编码区cDNA的真核表达质粒;鉴定了Flag-RNF8融合蛋白的表达并纯化RNF8蛋白;在CWR22RV1细胞中,Flag-RNF8蛋白主要定位在细胞核中。为进一步研究RNF8的生物学特性及其功能奠定了基础。     

PARP and CHK inhibitors interact to cause DNA damage and cell death in mammary carcinoma cells

The present studies examined viability and DNA damage levels in mammary carcinoma cells following PARP1 and CHK1 inhibitor drug combination exposure. PARP1 inhibitors [AZD2281 ; ABT888 ; NU1025 ; {"type":"entrez-nucleotide","attrs":{"text":"AG014699","term_id":"3649917","term_text":"AG014699"}}AG014699] interacted with CHK1 inhibitors [UCN-01 ; AZD7762 ; LY2603618] to kill mammary carcinoma cells. PARP1 and CHK1 inhibitors interacted to increase both single strand and double strand DNA breaks that correlated with increased γH2AX phosphorylation. Treatment of cells with CHK1 inhibitors increased the phosphorylation of CHK1 and ERK1/2. Knock down of ATM suppressed the drug-induced increases in CHK1 and ERK1/2 phosphorylation and enhanced tumor cell killing by PARP1 and CHK1 inhibitors. Expression of dominant negative MEK1 enhanced drug-induced DNA damage whereas expression of activated MEK1 suppressed both the DNA damage response and tumor cell killing. Collectively our data demonstrate that PARP1 and CHK1 inhibitors interact to kill mammary carcinoma cells and that increased DNA damage is a surrogate marker for the response of cells to this drug combination.     

胃癌组织MMP-9基因及蛋白表达与肿瘤临床病理关系研究

目的：研究胃癌患者基质金属蛋白酶-9表达与肿瘤临床病理参数之间相关性。方法：应用酶联免疫吸附（ELISA）方法检测术前外周血清及胃液中MMP-9蛋白水平。结果：胃癌组、对照组血清中金属蛋白酶平均水平分别为406．03±257．18ng／ml、167．35±54．04ng／ml,（P〈0．01）;TNM晚期、淋巴结转移阳性者术前外周血清蛋白含量显著高于TNM早期、淋巴结转移阴性者（P〈0．01）。胃癌组术前胃液中MMP-9蛋白平均水平为77．59±191．97ng／ml,对照组胃液中MMP-9蛋白平均水平为0．81±2．39ng／ml,（P〈0．01）。肿瘤侵犯至浆膜层、TNM晚期、淋巴结转移阳性者蛋白含量显著高于肿瘤未侵犯至浆膜层、TNM早期、淋巴结转移阴性者（P〈0．01）。结论：检测胃癌患者术前胃液中血清中蛋白酶水平能反映肿瘤进展。     

ATM蛋白在不同放射敏感性鼻咽癌细胞株中的表达

背景与目的:ATM(Ataxia-telangiectasiamutant)蛋白与细胞放射敏感性密切相关。本研究探讨ATM蛋白在具有不同放射敏感性的鼻咽癌细胞系CNE1,CNE2中的表达。方法:采用免疫荧光标记技术,对CNE1、CNE2中的ATM蛋白进行激光扫描共聚焦显微镜(LSCM)分析。结果:ATM蛋白定位于细胞核及胞浆中,并以细胞核为主,且CNE1细胞核中ATM蛋白阳性细胞的荧光强度较CNE2强;半定量分析,CNE1中ATM蛋白表达的积分吸光度值为9×106,CNE2中为3×106。结论:ATM蛋白的表达在CNE1、CNE2中有差别,这种差别可能与它们所具有的不同放射敏感性有关。     

Oxidative DNA damage in gastric cancer: CagA status and OGG1 gene polymorphism

Oxidative DNA damage is thought to play an important part in the pathogenesis of H. pylori-induced mucosal damage. 8-OHdG is a sensitive marker of DNA oxidation and is repaired by a polymorphic glycosylase (OGG1) more effectively than by OGG1-Cys(326). The aims of this study were to ascertain the respective roles of H. pylori, cagA status and OGG1 polymorphism in determining 8-OHdG levels in benign and premalignant stomach diseases and in gastric cancer (GC). The study involved 50 GC patients (for whom both neoplastic tissue and surrounding mucosa were available), 35 with intestinal metaplasia and atrophy (IMA) and 43 controls. H. pylori and cagA status were determined by histology and polymerase chain reaction for urease and cagA. 8-OHdG was assayed using HPLC with an electrochemical detector (HPLC-ED). The OGG1 1245C-->G transversion was identified using RFLP analyses. 8-OHdG levels were significantly higher in GC, with no differences in relation to H. pylori or cagA status. OGG1 polymorphism was documented in 34% of GC (15 Ser/Cys, 2 Cys/Cys). OGG1 1245C-->G polymorphism was detected in 54% of IMA patients, but only 16% of controls (p = 0.0004) and coincided with significantly higher 8-OHdG levels. In the multivariate analysis, 8-OHdG levels were predicted by histotype and OGG1 status. OGG1 1245C-->G polymorphism was common in both GC and IMA, but very rare in controls, and correlated more closely with 8-OHdG levels than do H. pylori infection or cagA status.     

Antisense oligodeoxynucleotides targeting ATM strengthen apoptosis of laryngeal squamous cell carcinoma grown in nude mice

Background

To conserve laryngeal function and elevate living quality of laryngeal squamous cell carcinoma (LSCC) patients, we designed antisense oligodeoxynucleotides (AS-ODNs) to reduce expression of ATM and to enhance the apoptosis of hep-2 (Human epidermoid laryngeal carcinoma) cells to radiation in vitro and in vivo.

Methods

The expression of ATM mRNA and protein in hep-2 cells were examined by real-time quantitative PCR and western blotting respectively. Clonogenic survival assay was carried out to detect the survival ability of hep-2 cells after irradiation, and analyzed the cell apoptosis by flow cytometry. The volume of solid tumors was measured, while TUNEL assay and western blotting used to analyze cell apoptosis and protein expression after irradiation.

Results

The relative ATM mRNA and protein expression in hep-2 cells treated with ATM AS-ODNs were decreased to 11.03 ± 2.51% and 48.14 ± 5.53% of that in untreated cells respectively (P <0.05). After irradiation, the survival fraction (SF) of cells treated with ATM AS-ODNs was lower than that of other groups at the same dose of radiation (P < 0.05). The inhibition rate in hep-2 cells solid tumor exposed to X-ray alone was 5.95 ± 4.52%, while it was 34.28 ± 2.43% in the group which irradiated in combination with the treatment of ATM AS-ODNs (P < 0.05). The apoptotic index for the group irradiated in combination with ATM AS-ODNs injection was 17.12 ± 4.2%, which was significantly higher than that of others (P < 0.05).

Conclusion

AS-ODNs of ATM reduce ATM expression and enhance hep-2 cells apoptosis to radiation in vitro and in vivo.     

Gene-specific oxidative DNA damage in Helicobacter pylori-infected human gastric mucosa

To study the status of oxidative DNA damage in Helicobacter pylori infection in more detail, we examined oxidative DNA damage to individual genes by determining the loss of PCR product of a targeted gene before and after gastric mucosal DNA was treated with 8-hydroxyguanine glycosylase, which cleaves DNA at the 8-hydroxyguanine residues. The results showed that, of the 5 genes tested, p53, insulin-like growth factor II receptor and transforming growth factor-beta receptor type II showed significant oxidative DNA damage in H. pylori-positive tissues and that the BAX and beta-ACTIN genes were relatively undamaged. These results suggest that in H. pylori infection, oxidative DNA damage does not occur homogeneously throughout the genomic DNA but, rather, in a gene-specific manner. We conclude that the progressive accumulation of preferential oxidative DNA damage in certain genes, such as p53, likely contributes to gastric carcinogenesis.     

胃癌相关基因的研究进展

胃癌的发生发展是一个复杂的多基因作用过程,近年来人们已经认识到涉及胃癌发生、发展的基因包括原癌基因（如ｃ－ｍｙｃ、ＥＧＦＲ）、抑部基因（如Ｐ１６）和凋亡基因（如ｂｃｌ－２）。以上３类基因参与胃癌发生不同时期的分子病理过程,且呈现出一定规律性。通过对某些胃癌发生的相关基因进行检测。对于肿瘤早期诊断、预后评估都具有重要意义。