mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis

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心脏特异性高表达hAPE1基因小鼠的构建与鉴定

本研究拟建立心脏特异性表达hAPE1转基因小鼠,为研究hAPE1基因功能及其突变与心脏发育和心血管疾病的关系提供工具动物。将人APE1(human APE1,hAPE1)基因插入到心脏特异性启动子α-肌球蛋白重链(α-MHC)下游,构建了心肌细胞特异性表达hAPE1的转基因表达载体,显微注射法导入C57BL/6J小鼠受精卵中,经胚胎移植获得转基因首建者小鼠,建立hAPE1转基因小鼠,PCR鉴定转基因小鼠基因型,Western blotting鉴定h APE1蛋白在心脏中的表达并筛选高表达的转基因品系。研究表明,将含有心肌细胞特异性α-MHC启动子和hAPE1基因的转基因载体进行显微注射于小鼠胚胎中,接着将胚胎移植入假孕母鼠的输卵管中发育,建立了心脏组织特异性高表达hAPE1转基因小鼠品系,获得子代小鼠40只。PCR检测发现有15只小鼠在其基因组上整合有hAPE1基因,Western blotting检测hAPE1在这些小鼠心脏中高度特异性表达。本研究成功获得了在小鼠心肌细胞中特异性表达hAPE1的转基因小鼠,为研究基因在心脏发育与相关疾病中的功能提供了有利的工具。     

蕈样肉芽肿和湿疹中Survivin 和Bcl-xL的表达分析

目的:探讨蕈样肉芽肿、湿疹、正常皮肤中Survivin、Bcl-xL蛋白的表达差异性及其临床意义。方法:运用免疫组织化学法检测30例蕈样肉芽肿、20例湿疹及15例正常人皮肤组织中Survivin、Bcl-xL蛋白的表达情况。结果:蕈样肉芽肿表皮中及真皮中Survivin、Bcl-xL的阳性表达率显著高于湿疹及正常皮肤,差异均有统计学意义(P0.05)。各时期蕈样肉芽肿表皮、真皮中Survivin、Bcl-xL的表达差异有统计学意义(P0.05)。结论:Survivin和Bcl-xL在蕈样肉芽肿中高表达其表达异常在蕈样肉芽肿的发生中起着重要作用,Survivin、Bcl-xL可能作为鉴别诊断蕈样肉芽肿、皮肤炎症性疾病有价值的参考指标。     

Low expression of Beclin 1, associated with high Bcl-xL,predicts a malignant phenotype and poor prognosis of gastric cancer

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.     

帕金森病相关蛋白Parkin 与PINK1的相互作用研究

帕金森病(Parkinson's disease,PD)是常见的神经系统变性疾病．分子遗传学研究发现,突变的Parkin蛋白及PINK1蛋白均参与了帕金森病的致病过程,但二者之间是否存在相互作用以及是否能够相互调节仍不十分清楚．为明确生理状态下Parkin蛋白与PINK1蛋白之间的相互作用,首先运用蛋白体外结合实验(GST pull-down)技术及免疫共沉淀技术证实了Parkin与PINK1在体外及体内均可相互结合．进一步构建PINK1的不同截短型,运用GST pull-down技术验证了PINK1与Parkin相互结合的区段为PINK1的蛋白激酶结构域．免疫细胞化学实验也证实Parkin与PINK1蛋白在细胞中存在共定位．进一步运用免疫共沉淀技术证实Parkin可减少PINK1通过泛素蛋白酶体系统(ubiquitin proteasome system,UPS)的降解,从而稳定PINK1．PINK1可增加Parkin通过UPS的降解,从而减少Parkin的水平,降低其稳定性．这些结果提示,帕金森病相关蛋白Parkin与PINK1能够直接结合,二者通过泛素蛋白酶体降解系统相互调节,可能协同作用参与了帕金森病的致病过程．     

PC12细胞APE/ref-1cDNA的克隆和表达

APE Ref 1是双功能核蛋白 ,它既能在碱基切除修复过程中切除脱嘌呤 脱嘌啶位点 ,又能促进包括AP 1、Myb和NF κB等受氧化还原调节的转录因子对DNA的结合 .从PC12细胞中抽提总RNA ,经逆转录PCR(RT PCR)扩增出APE ref 1cDNA并克隆到pQE3 1表达质粒上的BamHⅠ和PstⅠ位点间 .经测序表明 ,PC12细胞的APE ref 1cDNA以正确的阅读框架重组进入表达质粒 ,表达重组质粒pQE3 1 APE在宿主菌BL2 1中得到稳定表达 .SDS PAGE鉴定表明 ,带 6个组氨酸的融合蛋白分子量为 3 8kD ,并经Ni NTA琼脂糖亲和纯化得到电泳纯融合蛋白 .Western印迹证明重组蛋白为APE ref 1融合蛋白 .     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．     

Cytoplasmic APE1 promotes resistance response in osteosarcoma patients with cisplatin treatment

Chemotherapy resistance has become a hold back and major clinical challenge in osteosarcoma cancer. The alteration and subcellular distribution of apurinic/apyrimidinic endonuclease 1 (APE1) has been reported to be involved in chemotherapy resistance in many cancers. Here, we report that the cytoplasmic distribution of APE1 plays a key role in the sensitivity of combination platinum chemotherapy in osteosarcoma. Interestingly, the prevalence of cisplatin-induced DNA damage and apoptosis in low cytoplasmic APE1 osteosarcoma cell lines was higher than in high expression of cytoplasmic APE1 cell lines. Overexpression of cytoplasmic APE1 protected the osteosarcoma cells from CDDP-induced apoptosis. In addition, clinical data also show that the level of cytoplasmic APE1 was negatively associated with sensitivity to combination chemotherapy of cisplatin in osteosarcoma patients. Our findings suggest that cytoplasmic APE1 plays a significant role in chemotherapy resistance. This role is a supplement to the extranuclear function of APE1, and cytoplasmic APE1 expression level could be a promising predictor of platinum treatment prognosis for osteosarcoma patients.     

Pro-apoptotic meiogynin A derivatives that target Bcl-xL and Mcl-1

The biological evaluation of a natural sesquiterpene dimer meiogynin A 1, is described as well as that of five non-natural analogues. Although active on a micromolar range on the inhibition of Bcl-xL/Bak and Mcl-1/Bid interaction, meiogynin A 1 is not cytotoxic on three cell lines that overexpress Bcl-xL and Mcl-1. Contrarily, one of its analogues 6 with an inverted configuration on the side chain and an aromatic moiety replacing the cyclohexane ring was active on both target proteins, cytotoxic on a micromolar range and was found to induce apoptosis through a classical pathway.     

~(131)I-Herceptin对HER2过表达乳腺癌细胞Bcl-xL表达的影响

目的:探讨131I-Herceptin对HER2过表达乳腺癌细胞Bcl-x L表达的影响。方法:采用Iodogen法制备131I-Herceptin,超滤法纯化后测定其标记率、放射化学纯度和免疫结合率。通过免疫荧光法检测乳腺癌细胞表面HER2表达水平。131I(4.625 MBq/m L)、Herceptin(125μg/m L)及131I-Herceptin(4.625 MBq/m L)干预乳腺癌BT474细胞后,Western blot检测细胞中Bcl-x L的表达。结果:131I-Herceptin的标记率、放射化学纯度和免疫结合率分别为(89.71±2.93)%、(91.80±1.43)%和(58.84±3.35)%。BT474细胞膜表面HER2表达水平明显高于MDA-MB-231细胞。Herceptin、131I-Herceptin组BT474细胞内Bcl-x L表达水平明显低于对照组及131I组(均P0.01),而Herceptin与131I-Herceptin组之间细胞内Bcl-x L含量差异无统计学意义(P0.05)。结论:131I-Herceptin保留Herceptin对HER2过表达乳腺癌细胞Bcl-x L表达的抑制作用并促进细胞凋亡,进而与131I产生协同作用,较Herceptin更有效地杀伤HER2过表达乳腺癌细胞。     

p-MAPK、CyclinD1和CDK4蛋白在大肠癌中的表达及相关性

目的 检测p-MAPK、CyclinD1和CDK4蛋白在大肠癌中的表达,并探讨其相关性.方法 选取78例大肠癌组织,距癌灶3 cm以外的癌旁组织,进行组织切片,HE染色进行病理分型,应用S-P免疫组化法对组织中的p-MAPK、Cyclin D1和CDK4进行检测.结果 病理分型后,78例大肠癌中p-MAPK、CyclinD1和CDK4蛋白的阳性表达率分别为67.9 %（53/78）、57.7 %（45/78）和39.7 %（31/78）;78例相应癌旁组织中阳性表达率分别为6.4 %（5/78）、10.3 %（8/78）和19.2 %（15/78）.三者阳性表达率和阳性强度均以大肠癌中为高（P＜0.01）;大肠癌中p-MAPK与CyclinD1表达呈明显正相关（P＜0.01）,而与CDK4蛋白表达无明显相关性（P＞0.05）,CyclinD1和CDK4表达呈正相关（P＜0.05）,RT-PCR检测结果与免疫组化相似,p-MAPK、CyclinD1和CDK4蛋白的阳性表达率分别为8.2 %（61/78）、67.9 %（53/78）和53.8 %（42/78）;相应癌旁组织中阳性表达率分别为3.8 %（3/78）、2.6 %（2/78）和6.4 %（5/78）.结论 MAPK活化可能是cylinD1激活的原因之一,并在大肠癌发生过程中可能起重要作用;而Cyclin D1 在细胞周期中可与CDK4 结合,从而促进细胞周期进程.     

Unconventional initiation of PINK1/Parkin mitophagy by Optineurin

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RNA-cleaving properties of human apurinic/apyrimidinic endonuclease 1 (APE1)

We have recently identified apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease that cleaves c-myc mRNA in vitro and regulates c-myc mRNA levels and half-life in cells. This study was undertaken to further unravel the RNA-cleaving properties of APE1. Here, we show that APE1 cleaves RNA in the absence of divalent metal ions and, at 2 mM, Zn²⁺, Ni²⁺, Cu²⁺, or Co²⁺ inhibited the endoribonuclease activity of APE1. APE1 is able to cleave CD44 mRNA, microRNAs (miR-21, miR-10b), and three RNA components of SARS-corona virus (orf1b, orf3, spike) suggesting that, when challenged, it can cleave any RNAs in vitro. APE1 does not cleave strong doublestranded regions of RNA and it has a strong preference for 3’ of pyrimidine, especially towards UA, CA, and UG sites at single-stranded or weakly paired regions. It also cleaves RNA weakly at UC, CU, AC, and AU sites in single-stranded or weakly paired regions. Finally, we found that APE1 can reduce the ability of the Dicer enzyme to process premiRNAs in vitro. Overall, this study has revealed some previously unknown biochemical properties of APE1 which has implications for its role in vivo.     

急性肝损伤时肝脏内Pink1/Parkin的表达变化及意义

目的:观察C57小鼠急性肝损伤(Acute liver injury, ALI)中线粒体自噬相关蛋白Pink1/Parkin表达的变化及意义。方法:领取28只小鼠,随机分为对照组、ALI 1 d组、ALI 4 d组、ALI 7 d组。分别检测血清谷氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)的变化;肝组织病理变化用HE染色观察;Real Time quality PCR检测Pink1/Parkin在m RNA水平的表达变化;进一步利用Western Blot方法分别检测肝组织中Pink1/Parkin蛋白水平的表达变化。结果:ALI组1 d、4 d、7 d组与对照组相比,ALT、AST表达显著升高,具有统计学意义(P0.05);肝组织病理学检查ALI组1 d和4 d组中,释放出大量的炎性细胞,肝细胞出现大面积坏死,7 d组肝细胞坏死与1 d和4 d比较明显缓解;m RNA水平检测Pink1/Parkin在ALI 1 d(4.79/1.82倍)、4 d(1.74/4.17倍)和7 d(2.91/1.25倍)与对照组相比表达水平明显升高(P0.05);Pink1/Parkin在蛋白水平检测时ALI 1 d(2.03/1.82倍)、4 d(1.78/4.17倍)和7 d(1.25/1.25倍)时肝组织内表达也显著增加,差异具有统计学意义(P0.05)。结论:急性肝损伤状态下Pink1/Parkin在m RNA和蛋白水平表达升高,且其表达量随着肝脏的损伤缓解而表达水平降低,提示Pink1/Parkin可能对于治疗急性肝损伤具有重要意义。     

E-Cadherin and the Cytoskeletal Network in Colorectal Cancer Development and Metastasis

Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers (CRC). Cell–cell adhesion molecule E-cadherin regulates cell polarity, differentiation, proliferation and migration through its intimate association to the actin cytoskeletal network. During colorectal carcinogenesis changes in intercellular adhesion and dynamic rearrangements in the actin cytoskeleton result in altered signalling and migration with loss of contact inhibition. The adenomatous polyposis coli (APC) protein, besides its established role in the β catenin/Wnt signalling pathway, can coordinate microtubule and actin organization during cell migration. The actin-bundling protein Fascin promotes cell motility and is overexpressed in CRC. Based on recent molecular and pathological studies, this review focusses on the role of these molecules sharing the common feature of being associated with the cytoskeletal network during colorectal carcinogenesis and metastasis. The potential use of these molecules as prognostic markers and/or therapeutic targets will also be discussed.     

Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication

Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase beta with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase beta was accomplished by DNA containing photoreactive group at the 3 -end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase beta depending on the nature of the 5 -group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5 -phosphate group in the nick/gap) or long-patch (DNA containing 5 -sugar phosphate or 5 -flap) base excision repair (BER) intermediates. Other DNA duplexes (3 -recessed DNA and DNA with the 5 -hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5 -sugar phosphate group in the nick/gap, whereas DNA polymerase beta binds to DNA duplex with a mononucleotide gap flanked by the 5 -p group. When APE1 and DNA polymerase beta are both present, a ternary complex APE1-DNA polymerase beta-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5 -flap or mononucleotide-gapped DNA with 5 -p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase beta, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3 -5 exonuclease activity on 3 -recessed DNA duplex.