DNA损伤反应中细胞周期检查点蛋白p27~(Kip1)表达及其调控机制

为了研究DNA损伤反应中p2 7Kip1的表达及其调控机制 ,应用免疫印迹的实验结果表明 :10Gy 60 Coγ射线照射后 3h ,HeLa细胞中p2 7Kip1蛋白水平开始下降并持续到 2 4h ,进而失去它对CDKs的抑制功能 .Northern印迹结果显示 ,电离辐射 (IR)对p2 7Kip1mRNA表达水平无明显影响 ,说明电离辐射诱导p2 7Kip1表达水平的降低主要与蛋白质降解相关 ,但其具体的调控机制还不清楚 .已知在G1—S期p2 7Kip1蛋白的降低主要依赖细胞周期蛋白E Cdk2激酶将其磷酸化后的泛素化蛋白酶体途径 (ubiquitin proteasomepathway) .酶动力学研究结果揭示 :电离辐射后细胞周期蛋白E Cdk2激酶活性增高 ,12h细胞周期蛋白E Cdk2激酶活性达到最大 .当在照前用细胞周期蛋白E Cdk2抑制剂olomoucine (10 μmol L)抑制细胞周期蛋白E Cdk2激酶活性时 ,p2 7Kip1蛋白表达水平增加 .此外 ,还观察到电离辐射可诱导p2 7Kip1泛素化水平的增高 ,而在使用蛋白酶体抑制剂MG 132 (5 μmol L)处理HeLa细胞后 ,可抑制辐射诱导p2 7Kip1蛋白水平的下调 .研究结果提示 :泛素化蛋白酶体途径参与了辐射诱导P2 7Kip1蛋白表达下调的降解机制 .     

Saposin C下调LNCaP细胞雄激素受体的多泛素化降解（英）

本研究旨在证实鞘脂活化蛋白C(saposin C)对雄激素受体（AR）多泛素化降解的影响及其机制. 通过将真核表达载体saposin C转染LNCaP细胞,发现saposin C上调AR的蛋白水平和转录激活活性. 进一步将野生型和突变型泛素质粒Ubwt和UbK48R分别与saposin C 共转染LNCaP细胞发现,saposin C能够促进AR蛋白的单泛素化形式的稳定性,抑制AR的多泛素化修饰及其在蛋白酶体中的降解. 其分子机制是saposin C、Ub和AR三者形成复合体,抑制了AR的进一步多泛素化过程. 同时还发现,在这一机制中,细胞内低浓度的雄激素（0.1 nmol/L）与saposin C具有协同作用.     

Saposin C抑制雄激素受体的多泛素化以及在蛋白酶体中的降解(英文)

本研究旨在证实鞘脂活化蛋白C(saposin C)对雄激素受体(AR)多泛素化降解的影响及其机制.通过将真核表达载体saposin C转染LNCaP细胞,发现saposin C上调AR的蛋白水平和转录激活活性.进一步将野生型和突变型泛素质粒Ubwt和UbK48R分别与saposin C共转染LNCaP细胞发现,saposin C能够促进AR蛋白的单泛素化形式的稳定性,抑制AR的多泛素化修饰及其在蛋白酶体中的降解.其分子机制是saposin C、Ub和AR三者形成复合体,抑制了AR的进一步多泛素化过程.同时还发现,在这一机制中,细胞内低浓度的雄激素(0.1 nmol/L)与saposin C具有协同作用.     

去泛素化酶OTUD3通过稳定PTEN抑制癌症发生发展

PTEN(phosphatase and tension homology,deleted in chromosome 10)是重要的抑癌分子,其蛋白剂量水平的精细变化与肿瘤的发生发展密切相关。PTEN的蛋白水平调控研究一直是该领域的研究热点,虽然已经发现了数个PTEN的泛素连接酶,但能调控PTEN蛋白稳定性的去泛素化酶却一直不明确。该文介绍了PTEN泛素化修饰的最新进展,去泛素化酶OTUD3(OTU domaincontaining protein 3)通过去除PTEN的多聚泛素化维持其蛋白水平,并发挥抑癌功能。这一研究进展弥补了通过拮抗泛素化降解从而稳定PTEN这一长期未被阐释的调控机制。     

泛素-蛋白酶体降解途径在细胞周期调控中的作用

细胞周期的进程由一系列细胞周期蛋白依赖性激酶(CDK)和CDK活性调节因子驱动。泛素-蛋白酶体对细胞周期调节因子的降解是细胞调控分裂进程的重要手段。CDK活性抑制因子的降解是细胞分裂所必需的,而细胞周期正调控因子的降解则对维持细胞稳态至关重要。本从参与调控的2类泛素连接酶SCF复合物、APC／C复合物的结构和功能的角度阐述了泛素-蛋白酶体降解途径在整个细胞周期调控中的作用和意义。     

Skp2RNA干扰对人结肠癌HCT116细胞p27表达及对5-氟尿嘧啶敏感性影响的研究

目的:细胞S期激酶相关蛋白2(S-phase kinase associated protein,Skp2)作为泛素化降解过程中的一种F-box蛋白,在泛素化降解中起着特异性的底物识别和关键性的速率限制作用.依赖Skp2的泛素化降解途径参与p27、p21等众多细胞周期调控因子的降解,进而通过影响G1-S检测点而调控细胞周期.本研究探讨靶向干扰人结肠癌HCT116细胞中Skp2基因对细胞p27表达的影响及细胞对5-氟尿嘧啶(5-FU)敏感性的变化.方法:将已合成好的特异性的Skp2RNA干扰载体转染人结肠癌HCT116细胞,G418稳定筛选后,通过RT-PCR和Western印迹法分别检测细胞内p27mRNA和蛋白的表达情况,MTT法检测细胞对5-Fu敏感性的变化.结果:在转染干扰载体后,HCT116细胞内的p27mRNA表达无明显变化,蛋白水平却明显上升.MTT显示HCT116细胞对5-FU敏感性较其他组显著提高,IC50值明显下降(P＜0.05).结论:Skp2基因的RNA干扰能够上调结肠癌细胞内源性p27蛋白的表达,并提高5-FU对癌细胞的杀伤作用,为大肠癌的基因治疗提供了新的思路.     

抑制eIF3I蛋白的泛素化降解促进人宫颈癌细胞增殖

目的:研究eIF3I蛋白在细胞中的泛素化修饰,阐明其对人宫颈癌细胞系Hela增殖的影响.方法:通过点突变技术获得突变体K282R,与野生型eIF3I比较泛素化的水平,研究细胞内的泛素化修饰调控.经流式细胞仪分析细胞周期,研究野生型蛋白eIF3I和突变体K282R对Hela细胞的细胞周期影响.再从周期蛋白水平研究eIF3I对细胞增殖的调控作用.结果:突变体K282R比野生型eIF3I蛋白的外源表达量大.在Hela细胞中K282R突变体的泛素化水平低,抑制了该蛋白的泛素-蛋白酶体途径降解.过表达eIF3I能上调周期蛋白Cyclin D1的表达量,促进细胞进入由G1期进入S期.同时,泛素化程度低的突变体K282R具有较强的促进细胞增殖的作用.结论:抑制eIF3I的泛素-蛋白酶体途径降解能上调周期蛋白CyclinD1的表达,促进肿瘤细胞增殖,提示eIF3I在细胞增殖和肿瘤发生发展中发挥作用.     

SAHA抑制间充质干细胞C3H10T1/2的增殖与成脂分化

本文研究组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid,SAHA）对间充质干细胞(mesenchymal stem cells, MSCs) C3H10T1/2增殖和成脂分化的影响及其可能的作用机制.用Western印迹验证SAHA对细胞内蛋白乙酰化的影响;用MTT和流式细胞术检测细胞活性和细胞周期;利用油红O染色检测细胞成脂分化,实时定量PCR检测PPARγ2和成脂分化标志物Fabp4、perilipin以及adipoq mRNA的转录.Western印迹结果显示,SAHA可促进细胞内蛋白的乙酰化.MTT和流式细胞术结果显示,SAHA对C3H10T1/2细胞活性的抑制呈浓度依赖性,随着SAHA浓度增加,细胞形态趋向于展平,并将细胞周期抑制在G0/G1期;SAHA可呈浓度依赖性抑制C3H10T1/2 细胞的成脂分化作用.同时实时定量PCR结果显示,SAHA抑制成脂关键转录因子PPARγ2,脂肪因子Fabp4、perilipin和adipoq mRNA的转录.综上所述,SAHA可影响间充质干细胞C3H10T1/2细胞形态,并呈剂量依赖性地抑制其增殖和成脂分化.     

抑癌基因Pdcd4的表达调控及产物泛素化研究进展

程序性细胞死亡因子4(programmed cell death4,Pdcd4)基因是近年新发现的一种抑癌基因,它的表达产物通过抑制相关基因的转录和翻译抑制肿瘤生成.Pdcd4在多种人肿瘤组织细胞中表达缺失或低表达.有研究发现,5'CpG岛的甲基化可能是脑胶质瘤中Pdcd4基因沉默的主要原因之一.另外,在多种肿瘤细胞中发现microRNA-21的表达水平与Pdcd4蛋白的表达呈负相关性,microRNA-21在转录后水平调控Pdcd4,其作用位点位于Pdcd4mRNA的3′-UTR区.肿瘤细胞中Pdcd4的表达水平与细胞对抗肿瘤药物的敏感性有关,上调Pdcd4表达会增加细胞对某些抗肿瘤药物的敏感性,反之,下调Pdcd4表达则会引起某些药物细胞毒活性的减弱.有些药物本身也可以影响细胞中Pdcd4的表达水平.Pdcd4可以抑制p53的乙酰基化.Pdcd4蛋白能被蛋白激酶Akt/PKB磷酸化,引起Pdcd4的核易位,并减弱Pdcd4对转录激活因子AP-1的抑制作用.Pdcd4可以被蛋白激酶S6K1磷酸化并在泛素连接酶SCFβTRCP介导下经泛素化途径降解.     

NIRF泛素化p53的作用过程中NIRF与p53结合域的测定

NIRF(Np95/ICBP90-like RING finger protein)是2002年发现的一种核蛋白,其功能涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制等领域．已有研究报道,NIRF能与p53相互作用, NIRF本身也是一个高度调节蛋白,在细胞正常的生理状态下发挥泛素化E3连接酶的作用,结合p53并将其降解,但NIRF与p53结合的蛋白结合域目前尚不清楚．本文研究证明,NIRF能与p53结合成复合体参与泛素化蛋白降解途径,并测定出NIRF与p53结合的区域．为了检测NIRF的蛋白结合域,将空载体和NIRF缺失突变体质粒分别转染于HEK293细胞,蛋白表达水平通过Western印迹用两种抗体分别检测. 结果显示,所有的突变体都能在细胞中表达,并且两种抗体检测结果完全一致. 同时,免疫共沉淀技术用于进一步分析实验结果. 由于泛素化蛋白通常伴随蛋白酶体通路介导的降解,免疫共沉淀的蛋白纯化过程中用蛋白酶体抑制剂MG-132以抑制蛋白降解. 本研究结果显示,NIRF 通过PHD区域与p53形成复合体. 该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动,从而形成与细胞增殖相关的新的信号通路,在肿瘤的发生发展中可能发挥某种程度的作用．     

Study of a novel brain relatively specific gene LRRC4 involved in glioma tumorigenesis suppression using the Tet-on system

LRRC4 is a novel relatively specific gene,which displays significant down-regulation inprimary brain tumor biopsies and has the potential to suppress brain tumor growth.In this study,we inves-tigated the growth inhibitory effect of LRRC4 on tumorigencity in vivo and on cell proliferation in vitro by atetracycline-inducible expression system.Results showed that LRRC4 significantly reduced the growth andmalignant grade of xenografts arising from glioblastoma U251MG cells.Cell proliferation was markedlyinhibited after U251MG Tet-on-LRRC4 cell induction with doxycycline.Flow cytometry and Western blotanalysis demonstrated that LRRC4 mediated a delay of the cell cycle in late G_1,possibly through up-regulat-ing the expressions of p21Wafl/cip 1 and p27Kip 1 and down-regulating the expressions of cyclin-dependentkinase 2,retinoblastoma protein and epidermal growth factor receptors.Together,these findings provideclues to the function of LRRC4 as a negative regulator of cell growth and underscore a link between theabove-mentioned cyclins,cyclin-associated molecules and tumorigenicity.     

基于网络药理学分析瑞香素抗多种肿瘤的作用机制及体外实验验证

基于网络药理学预测瑞香素抗恶性胶质瘤、肝癌和三阴性乳腺癌的共同靶点及可能机制,并对其进行体外实验验证。利用Swiss Target Prediction和GeneCards等数据库检索瑞香素与恶性胶质瘤、肝癌和三阴性乳腺癌的共同靶点。使用Cytoscape构建瑞香素-三种肿瘤蛋白质相互作用网络图(PPI)并筛选出核心靶点,并对核心靶点进行GO及KEGG富集分析;通过AutoDock Tools对瑞香素与核心靶点进行分子对接。体外实验验证:采用CCK-8和Western blot法行体外实验验证不同浓度瑞香素对U-251 MG、HepG-2和MDA-MB231细胞系细胞抑制率和P53、RRM2蛋白的表达水平的影响。共筛选出瑞香素抗三种肿瘤核心靶点56个,富集分析显示靶点富集在P53通路和癌症通路,参与细胞周期调节、细胞凋亡、DNA生物合成和修复等生物过程;分子对接结果显示瑞香素与P53、RRM2有较好的结合作用。体外验证实验显示,与对照组比较,瑞香素能显著抑制U-251 MG、HepG-2、MDA-MB231的增殖(P<0.01),并显著上调其P53蛋白及下调RRM2蛋白的表达,且呈剂量依赖性(P<0.05)。我们的结果提示瑞香素能抑制U-251 MG、HepG-2和MDA-MB231细胞增殖,其机制可能与调控P53/RRM2通路有关。     

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.     

Adenovirus-mediated overexpression of p15INK4B inhibits human glioma cell growth, induces replicative senescence, and inhibits telomerase activity similarly to p16INK4A.

The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40-50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated beta-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway.     

Expression and functional characterization of LRRC4, a novel brain-specific member of the LRR superfamily

LRRC4, a novel member of LRR superfamily thought to be involved in development and tumorigenesis of the nervous tissue, has the potential to suppress tumorigenesis and cell proliferation of U251MG cells. This study aimed at revealing the correlation between expression of LRRC4 and the maintenance of normal function and tumorigenesis suppression within the central nervous system. We systematically analyzed the expression and tissue distributions of the gene in tissues. Results showed that LRRC4 expression was limited to normal adult brain, both in human and in mouse, and exhibited a development-regulated pattern, but was down-regulated in brain tumor tissues and U251MG cell line. Furthermore, dynamic alterations in gene expression associated with cell cycle progression were investigated by using Tet-on system. Results showed that LRRC4 induced a cell cycle delay at the late G1 phase, probably through the alteration of the expression of different cell cycle regulating proteins responsible for mediating G1-S progression, such as p21(Waf1/Cip1) and p27(Kip1), Cdk2 and PCNA, p-ERK1/2. These findings suggest that LRRC4 may play an important role in maintaining normal function and suppressing tumorigenesis in the central nervous system.     

Brazilein,a compound isolated from Caesalpinia sappan Linn., induced growth inhibition in breast cancer cells via involvement of GSK-3β/β-Catenin/cyclin D1 pathway

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC₅₀ value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 μmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3β and following reduced β-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3β/β-Catenin pathway was involved in its action mechanism.     

Effect of Bone Morphogenetic Protein 4 in the Human Brain Glioma Cell Line U251

The role of bone morphogenetic protein 4 (BMP4) in gliomas is not clear. We hypothesized that BMP4 inhibits proliferation in the brain glioma cell line U251 through a signaling pathway involving BMP4 and the mothers against decapentaplegic homolog 4 (SMAD4) protein. We exposed U251 cells to Adriamycin (1 g) for 48 h; cell proliferation (MTT assay), expression of BMP4 and SMAD4 (mRNA: qPCR; protein: Western blot) were studied. We further altered expression of BMP4 by overexpression or siRNA silencing, and documented cell responses to Adriamycin. Proliferation of U251 cells was significantly inhibited upon exposure to Adriamycin. This inhibition was associated with increased expression of BMP4. Further, proliferation of U251 cells was inhibited when BMP4 was overexpressed. BMP4 expression negatively correlated with expression of SMAD4, such that elevated levels of BMP4 were associated with decreased expression of SMAD4 and vice versa. The Adriamycin-induced inhibition of proliferation of U251 cells was attenuated when BMP4 was knocked down by siRNA. To conclude, BMP4 is associated with inhibition of proliferation of U251 cells; the effects of BMP4 involve the BMP4-Smad signaling pathway. BMP4 has a potential as a target for glioma therapy.