PI3K和MAPK信号通路对A549细胞FOXO1蛋白表达及其亚细胞定位的调节

目的探讨非小细胞肺癌中磷脂酰肌醇-3-激酶(PI3K)/AKT和丝裂原活化蛋白激酶MAPK/ERK信号通路对Forkhead转录因子(Fox蛋白家族)FOXO1活性的影响及分子机制。方法体外培养人非小细胞肺癌系A549,分别选用PI3K/AKT信号通路特异性抑制剂LY294002和MAPK/ERK信号通路特异性抑制剂UO126及两种抑制剂联合处理细胞之后,MTT比色法检测其对细胞增殖的影响;Western blot检测蛋白FOXO1、p-FOXO1及信号下游蛋白Bim表达的变化;免疫荧光法检测FOXO1蛋白在A549细胞中亚细胞的定位的变化。结果与对照组相比,LY294002和UO126都能明显地抑制A549细胞的增殖,且具有时间依赖性。Western blot结果显示,FOXO1的蛋白水平未见显著性变化,而FOXO1的磷酸化水平明显下降,Bim的表达相应增加。免疫荧光结果显示,FOXO1在A549细胞中的核转位增多。LY294002和UO126联合处理A549细胞时,较药物单独作用效果明显增加。结论 PI3K/AKT和MAPK/ERK信号通路能调节FOXO1磷酸化水平,且具有协同作用,抑制其转录活性。FOXO1通过激活Bim蛋白,促进细胞凋亡,抑制细胞的增殖。     

MNNG诱导TERC基因沉默的16HBE细胞恶性转化模型的建立

目的: 建立N-甲基em>N’-硝基-N-亚硝基胍(N-methyl-N-nitro-N-nitrosoguanidine, MNNG)诱导的人端粒酶RNA组分(telomerase RNA component, TERC)缺陷的人支气管上皮细胞株(16HBE)恶性转化细胞模型。方法:将靶向TERC基因的shRNA干扰质粒载体转染16HBE细胞,G418抗性克隆筛选得到稳定转染的16HBE-1细胞,RT-PCR检测16HBE-1细胞TERC mRNA的干扰效率;用1 mg/L MNNG对16HBE-1细胞进行隔代染毒,每次染毒1 h;直到染毒27次转化灶的出现。分离扩增转化灶细胞并命名为16HBE-T,用软琼脂克隆形成实验和裸鼠成瘤实验鉴定细胞的转化程度。结果:从转化灶分离培养的细胞能在软琼脂中生长,且转化细胞能在裸鼠体内成瘤,HE染色后光镜下显示为鳞癌。结论:成功建立MNNG诱导的TERC基因缺陷的16HBE细胞恶性转化模型。     

FOXO1质粒稳定转染人骨肉瘤细胞的筛选及意义

目的 建立稳定表达FOXO1-GFP的人骨肉瘤U2OS细胞系,并验证U2OS-FOXO1-GFP细胞模型对氧化应激损伤的指示作用.方法 将pcDNA3-GFP-FOXO1质粒转染到U2OS细胞中,利用G418和FCM筛选稳定细胞系.40mmol/LAAPH处理U2OS-FOXO1-GFP细胞4h,CCK-8检测细胞活力,荧光显微镜观察FOXO1-GFP的分布情况.结果 成功获得U2OS-FOXO1-GFP稳转细胞系.经G418和FCM筛选后,FOXO1-GFP阳性细胞占细胞总数的92％.CCK-8结果显示,AAPH处理后,U2OS-FOXO1-GFP细胞存活率下降至0.55 ±0.04明显低于对照组(P＜0.05).荧光显微镜观察发现,正常情况下,FOXO1-GFP位于细胞质;AAPH处理后,FOXO1-GFP易位到细胞核.结论 U2OS-FOXO1-GFP细胞系能很好的指示细胞氧化应激损伤,为筛选抗氧化应激药物提供了一种有利工具.     

豨莶草调控sirt1/FOXO1通路对膝骨关节炎大鼠软骨损伤的影响

目的:探讨豨莶草调控sirt1/FOXO1通路对膝骨关节炎大鼠软骨损伤的影响。方法:SD大鼠随机分组为对照组、模型组、豨莶草(2 g/kg)组、尼克酰胺(NA,sirt1抑制剂,剂量:100 mg/kg)组、豨莶草+尼克酰胺组(豨莶草为2 g/kg,NA为100 mg/kg)。除对照组外,其余各组以改良伸直位固定法建立膝骨关节炎大鼠模型,按组别分别以药物处理后,测量各组大鼠膝关节宽度、被动活动度;检测各组大鼠压痛阈值、热痛阈值;以苏木精-伊红染色(HE)观察各组大鼠膝关节软骨病理损伤情况,进行Mankin′s评分;以酶联免疫吸附(ELISA)试剂盒检测各组大鼠血清中IL-1β、肿瘤坏死因子(TNF-α)、软骨寡聚基质蛋白(COMP)水平;以蛋白免疫印迹法检测各组大鼠膝关节软骨组织sirt1、FOXO1、acely-FOXO1蛋白表达。结果:与对照组相比,模型组大鼠膝关节宽度、Mankin′s评分、血清中IL-1β、TNF-α及COMP水平、膝关节软骨组织中acely-FOXO1升高(P<0.05),膝关节被动活动度、压痛阈值、热痛阈值、膝关节软骨组织中sirt1蛋白表达降低(P&...     

用高通量实时荧光PCR技术研究低浓度MNNG诱发的细胞基因应答反应

目的：研究低浓度 N-甲基-N'-硝基-N-甲基亚硝基胍对人羊膜FL细胞部分基因表达的影响，以助于阐明MNNG引起细胞应答反应的基因及其调控机制。方法：用ABI公司的高通量实时荧光定量PCR方法，检测FL细胞在0.2 μmol/L MNNG处理后基因表达发生的改变。数据用ABI公司的SDS 2.1软件分析。结果：MNNG处理后，在检测的95个基因中，29个基因表达发生改变，其中14个基因下调2倍以上，15个基因下调在1.5-2倍之间；其中有4个基因与细胞周期相关，6个基因与信号转导相关，6个基因与转录调节相关。结论：在低浓度 MNNG攻击后，FL细胞的基因表达发生了显著的变化。     

MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用

目的:研究MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用。方法:应用已构建的沉默MDC1的食管癌ECA109细胞株接受8 Gy电离辐射后1 h,检测相关因子核内斑点形成情况。构建沉默MCPH1的食管癌ECA109细胞株,检测此细胞株接受同样照射条件后相关因子核内斑点的形成情况。结果:成功构建沉默MCPH1的食管癌ECA109细胞;电离辐射使MDC1、MCPH1与γ-H2AX蛋白相互作用。沉默MDC1不影响γ-H2AX和MCPH1核内斑点的形成;沉默MCPH1使电离辐射导致的MDC1核内斑点减少,不影响γ-H2AX核内斑点的形成。结论:MCPH1在电离辐射诱导的DNA损伤通路中可能位于H2AX下游、MDC1上游,可以调控MDC1核内斑点形成。     

VDT低频脉冲磁场对MNNG诱致DNA损伤的影响

用视频显示终端（ＶＤＴ）产生的磁通密度峰值为４μＴ和４０μＴ，频率为１５．６ｋＨｚ低频脉冲磁场和甲基硝基亚硝酸胍（ＭＮＮＧ，已知可损伤ＤＮＡ的致癌剂）对培养的人羊膜细胞进行７２ｈ的辐射及接触，结果得出４０μＴ低频脉冲磁场辐射能增加ＭＮＮＧ引起的细胞３Ｈ／１４Ｃ放射活性比值；４μＴ低频脉冲磁场辐射则不能增加其放射活性比值，表明了ＶＤＴ低频脉冲磁场的磁通密度峰值在４０μＴ的情况下，能增强ＭＮＮＧ对细胞ＤＮＡ的损伤作用     

甲基硝基亚硝胍处理的vero细胞中磷酸化蛋白的差异显示

目的：初步了解哺乳类细胞非定标性突变形成是否与以蛋白质磷酸化级联反应为主要形式的细胞信号传导通路有关;方法：采用［３２Ｐ］体内预标记及凝胶双向电泳方法差异显示甲基硝基亚硝胍（ＭＮＮＧ）处理组和对照组磷酸化蛋白,蛋白质免疫印迹杂交试验初步鉴定差异显示蛋白斑点性质;结果：在处理组发现两个与对照组不同的蛋白斑点,分子量分别在６８×１０３和４３×１０３附近,与抗酪氨酸磷酸化蛋白抗体未发生阳性反应;结论：根据差异显示蛋白斑点的分子量和印迹杂交试验结果,初步认为所见蛋白斑点可能为丝／苏氨酸磷酸化蛋白,且可能为丝裂原激活的蛋白激酶（ＭＡＰＫ）成员     

转录因子FOXO1对胰岛β细胞致炎因子产生的影响

目的观察转录因子FOXO1(forkhead box O1)对胰岛β细胞炎症因子产生的影响,探讨转录因子FOXO1在糖尿病发病机制中的作用。方法运用脂质体转染方法,将pcDNA-FOXO1真核表达载体瞬时转染至NIT-1细胞,通过Western blot检测细胞中FOXO1的过表达情况;以LPS分别处理重组质粒(pcDNA-FOXO1)、空质粒(pcDNA3.1)转染以及未转染的NIT-1细胞24 h后,应用ELISA法检测3组细胞致炎因子IL-1β、TNF-α表达情况。以LPS和PBS分别处理NIT-1细胞24 h后,ELISA测定两组细胞致炎因子IL-1β、TNF-α表达情况,Western blot比较两组细胞FOXO1蛋白表达及磷酸化水平的差异。结果 FOXO1在瞬时转染48 h的NIT-1细胞中实现了过表达;经LPS刺激后,重组质粒转染的NIT-1细胞产生的IL-1β和TNF-α显著高于空质粒转染和未转染组(P<0.01);经LPS处理24 h后的NIT-1细胞产生了致炎因子IL-1β和TNF-α,而PBS处理组未见表达。LPS处理组中FOXO1蛋白表达高于PBS处理组,但磷酸化水平显著低于PBS处理组(P<0.01)。结论炎症状态下,过表达转录因子FOXO1可增加胰岛β细胞致炎因子的产生,这主要与非磷酸化FOXO1的相对增多有关,它有望成为糖尿病抗炎治疗的新靶点。     

Functional FEN1 polymorphisms are associated with DNA damage levels and lung cancer risk

Flap endonuclease 1 (FEN1) is a key enzyme in maintaining genomic stability and protecting against carcinogenesis. This study investigated whether functional variations in FEN1 gene are associated with DNA damage and lung cancer risk. Thirty DNA samples were sequenced to identify variants and function of the variants was examined by a set of biochemical assays. DNA damage levels were detected by comet assays in a cohort of 303 coke‐oven workers and 297 controls. The association with lung cancer risk was examined in two independent case–control panels consisted of a total 1,840 lung cancer patients and 1,958 controls. We identified two single nucleotide polymorphisms (SNPs) located in the FEN1 promoter c.?69G>A (rs174538:G>A) and 3′‐untranslational region c.4150G>T (rs4246215:G>T) that were associated with reduced FEN1 expression. Among coke‐oven workers, DNA damage levels were significantly higher in the ?69GG or GA carriers compared with the ?69AA carriers. The ?69GG or 4150GG carriers had a significantly increased risk for developing lung cancer compared with the ?69AA or 4150TT carriers. These results highlight FEN1 as an important gene in human carcinogenesis and genetic polymorphisms in FEN1 confer susceptibility to lung cancer. Hum Mutat 30:1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells

Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs-induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs-induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs-induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549-R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549-R and A549 cell lines treated with COF condensate (COFC). RT-PCR and Western blot results showed that the expression of hOGG1 in A549-R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549-R cell line was much lower than that in the A549 cell line, and more COFC-induced DNA damage was detected in the A549-R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549-R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF-induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1-mediated repair pathways, and oxidative DNA damage may be involved in COF-induced carcinogenesis.     

DNA damage induced in human peripheral blood lymphocytes by industrial solid waste and municipal sludge leachates

Exposure of humans to toxic compounds occurs mostly in the form of complex mixtures. Leachates, consisting of mixtures of many chemicals, are a potential risk to human health. In the present study, leachates of solid wastes from a polyfiber factory (PFL), an aeronautical plant (AEL), and a municipal sludge leachate (MSL) were assessed for their ability to induce DNA damage in human peripheral blood lymphocytes using the alkaline Comet assay. The leachates also were examined for their physical and chemical properties. Lymphocytes were incubated with 0.5-15.0% concentrations (pH range 7.1-7.4) of the test leachates for 3 hr at 37 degrees C, and treatment with 1 mM ethyl methanesulfonate served as a positive control. All three leachates induced significant (P < 0.05), concentration-dependent increases in DNA damage compared with the negative control, as measured by increases in Olive tail moment (arbitrary units), tail DNA (%), and tail length (mum). A comparison of these variables among the treatment groups indicated that the MSL induced the most DNA damage. Inductively coupled plasma emission spectrometry analysis of the leachates indicated that they contained high concentrations of heavy metals, viz. iron, manganese, nickel, zinc, cadmium, chromium, and lead. The individual, synergistic, or antagonistic effects of these chemicals in the leachates may be responsible for the DNA damage. Our data indicate that the ever-increasing amounts of leachates from waste landfill sites have the potential to induce DNA damage and suggest that the exposure of human populations to these leachates may lead to adverse health effects.     

Dna damage in human mononuclear cells induced by bacterial endotoxin

Damage to nuclear DNA in human peripheral blood mononuclear cells was studied after in vitro treatment with bacterial endotoxin by alkaline comet assay. It was found that LPS induced DNA damage as soon as over the first 30 min of incubation, while by the 4th hour of incubation DNA damage was found in more than 95% cells. Exogenous superoxide dismutase completely protected DNA, which suggests that superoxide radical is the primary extracellular damaging agent. Polyphenol antioxidant (water-soluble lignin) and specific NADPH oxidase inhibitor (diphenyleneiodonium chloride) also produced a protective effect. Our results show that LPS-activated mononuclear cells can be used ex vivo as a convenient and adequate experimental system for evaluation of the efficiency of various substances in protection of lymphocyte DNA from the damaging effect of reactive oxygen species of LPS-stimulated monocytes. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 9, pp. 275–277, September, 2008     

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has potential tumour-suppressive activity in human lung cancer

The expression of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is decreased in various tumours, but the role of IGFBP-rP1 in lung cancer is not yet clear. In this study, IGFBP-rP1 expression in lung cancer cell lines was evaluated and reduced expression of IGFBP-rP1 was found. In tissue microarrays containing 138 primary tumours and 20 normal lung tissues analysed by immunohistochemistry, 58 tumours (42%) exhibited no expression of IGFBP-rP1, while all 20 normal lung tissues showed high expression. In squamous cell lung cancer, low expression of IGFBP-rP1 was significantly linked to high-grade tumours. Treatment with 5-aza-2'-deoxycytidine restored the expression of IGFBP-rP1 in three of four lung cancer cell lines. Sequencing of PCR products of sodium bisulphite-treated genomic DNA from the three lung cancer cell lines revealed a heterogeneous methylation pattern in the region of exon 1 and intron 1. Stable transfection of IGFBP-rP1 full-length cDNA into the H2170 lung cancer cell line led to increased expression of IGFBP-rP1 protein. IGFBP-rP1-positive transfectants exhibited remarkably reduced colony-forming ability in soft agar, suppression of tumour growth rate in nude mice, and increased apoptotic cell number as well as activated caspase-3 expression level. The data suggest that IGFBP-rP1 is a tumour suppressor inactivated by DNA methylation in human lung cancer.     

非小细胞肺癌中Bmi-1和WWOX蛋白的表达及其临床意义

目的探讨非小细胞肺癌中BMi-1与WWOX蛋白的表达及其临床意义。方法应用免疫组化和Western blot检测86例非小细胞肺癌中Bmi-1蛋白和WWOX蛋白的表达水平,分析BMi-1与WWOX蛋白与临床患者特征的关系,并对患者生存影响因素进行分析。结果 Bmi-1蛋白在非小细胞肺癌中的表达水平显著高于癌旁正常组织中表达水平(0.01),而WWOX蛋白在非小细胞肺癌中的表达水平(0.52±0.05)则显著低于癌旁正常组织中表达水平(0.01)。Bmi-1和WWOX蛋白表达水平均与淋巴结转移有关(0.05)。结论 Bmi-1的高表达和WWOX蛋白的低表达均与淋巴结转移有关。