核孔蛋白与肿瘤

在真核生物中,细胞的物质交换在其增殖和凋亡中长期存在,而细胞核与质的交换只能通过核孔复合体（NPC）进行的穿梭运动实现。NPC位于细胞核膜,通过穿梭运动实现真核生物中核与质之间蛋白、RNA等物质的运输任务。特定的核孔蛋白会调节特定的运输路径,而正是由于此,通过NPC进行的运输和调节是高选择性和协调性,从而使NPC在基因表达、信号网络和保持细胞内环境中起到了重要作用。它们当中有的会改变核膜的结构性状,影响有丝分裂,有的可以通过与激酶结合导致异常的生化反应,或者与其他生物因子作用,在血管生成、细胞迁移或者细胞生成及凋亡的环节出现异常变化,从而导致肿瘤的发生。     

蛋白质的核质转位在细胞凋亡及癌变过程中的研究进展

0 引言 哺乳动物细胞中存在着数量繁多的可以通过核膜上的核孔复合体在胞核与胞质间进行交换的蛋白质,它们通过核孔复合体在胞核与胞质间进行交换的过程即称为蛋白质的核质转位.这些蛋白可以通过各种转位机制对细胞自身的生长、增殖、凋亡及癌变等各种生理和病理过程产生调控作用[1].正常的生理情况下,它们在胞核或胞质中发挥着调节细胞生长和增殖的作用,而当细胞在各种因素的作用下发生病理变化时,本应存在于胞核或胞质中的大量信号蛋白和分子便会通过核孔复合体发生异常核质转位.并以这种方式将胞质和胞核紧密联系起来,使导致细胞发生癌变或凋亡的信号蛋白和分子能够进入特定场所发挥作用[2],从而导致疾病的产生.     

XPO1 抑制剂在血液系统恶性肿瘤中的研究进展

核输出蛋白1（exportin 1，XPO1）是一种转运受体蛋白，能转运富含亮氨酸的蛋白质，从细胞核通过核孔复合体进入细胞质。在血液系统恶性肿瘤中，XPO1常过度表达而导致细胞生长凋亡调节异常或细胞周期异常。因此，XPO1抑制剂可作为靶向药物，阻碍过度表达的XPO1转运作用，从而达到治疗血液系统恶性肿瘤的效果。本文综述XPO1抑制剂在不同血液系统恶性肿瘤临床前和临床研究中的应用。     

胰岛素-胰高血糖素瘤克隆20在肿瘤细胞中的作用

胰岛素-胰高血糖素瘤克隆20( IG20)在人类细胞中至少可表达6种剪接异型体,这些蛋白主要通过细胞肿瘤坏死因子(TNF)与TNF相关死亡配体(TRAIL)等凋亡相关的信号通路来影响细胞的凋亡与增殖,此外其还作为GTP-GDP交换因子参与神经突触囊泡蛋白的运输.     

核质转运通路及其抑制剂在肿瘤靶向治疗中的研究进展

0 引言 恶性肿瘤是世界范围内死亡率较高的疾病,并有逐年上升的趋势.尽管近年来不断涌现出新的抗肿瘤药物,但肿瘤的高复发性和耐药性仍不可避免.肿瘤细胞的恶性增殖与一系列信号转导通路的异常激活或抑制密切相关.其中转录因子及转录辅助因子,通过核孔复合体(nuclear pore complexes,NPC)和出入核转运受体进出细胞核,诱导或阻滞下游基因的表达,可促使信号转导通路异常激活或抑制[1].已有许多报道证实通过出入核抑制剂阻断这些重要转录因子的作用可抑制肿瘤细胞的增殖,诱导细胞凋亡和逆转肿瘤的耐药性.本文对目前核质转运通路及其抑制剂在恶性肿瘤靶向治疗中的进展加以阐述.     

分子信号传导与肿瘤生物治疗

生长因子、细胞因子或激素均可与其相应受体结合,激活特定的信号通路,通过一系列分子间相互作用即分子信号传导,最终调节某些重要基因的转录,使细胞发生增殖、分化或凋亡.癌基因产物大都与细胞信号传导有关,其过度表达或突变使细胞增殖调节发生紊乱,进而导致肿瘤发生,基于大部分肿瘤由于信号通路活性过高导致细胞无限增殖,以分子信号通路为靶点,通过阻断信号传导抑制肿瘤生长是目前肿瘤治疗的重要研究领域,有的方案已进入临床试用,显示出良好的应用前景.     

缺氧条件下骨肉瘤MG-63细胞中TG2通过线粒体途径调节细胞凋亡的机制

目的探讨缺氧条件下TG2在骨肉瘤MG-63细胞凋亡中的作用以及TG2通过阻止细胞色素C的释放和调节Caspase-3的表达及活性抑制细胞凋亡的机制。方法建立骨肉瘤细胞体外缺氧培养模型,设立四组:(1)常氧组;(2)单纯缺氧组;(3)对照si RNA缺氧组;(4)TG2 si RNA缺氧组。观察各组在缺氧培养不同时相(6、12、24、48、72 h)骨肉瘤MG-63细胞TG2、Caspase-3表达、胞核和胞质细胞色素C的变化以及细胞凋亡率。结果与常氧组比较,单纯缺氧组及对照si RNA缺氧组的TG2活性、m RNA及蛋白表达水平明显增强(P<0.01),且随缺氧时间延长明显升高;Caspase-3活性未见明显增加,而Caspase-3及细胞质内细胞色素C蛋白的表达水平和细胞凋亡率轻度增加。与前三组比较,在TG2 si RNA缺氧组,当通过转染TG2 si RNA抑制TG2的表达时,Caspase-3的活性及胞质内细胞色素C蛋白表达水平明显增强(P<0.01);细胞凋亡率也显著增加(P<0.01)。结论缺氧条件下,MG-63骨肉瘤细胞TG2表达增强,并且可以阻止细胞核内细胞色素C向胞质释放,从而降低Caspase-3的表达及活性,抑制肿瘤细胞的凋亡。     

γ-干扰素对TRAIL诱导人甲状腺癌细胞凋亡影响的研究

目的:探讨γ-干扰素(IFN-γ)对TRAIL诱导人甲状腺癌细胞凋亡作用的影响.方法:流式细胞术、蛋白质印迹法、蛋白质羰基测定法和共聚焦显微镜分别用于检测细胞凋亡、GAPDH蛋白表达、氧化损伤程度和细胞核内GAPDH免疫染色表达.运用微小RNA干扰技术下调GAPDH表达.结果:与耐药细胞系相比,TRAIL敏感的FRO和KTC2细胞核提取液中可见不同程度的GAPDH蛋白表达、细胞内羰基蛋白显著集聚.IFN-γ可增加耐药ARO细胞核中GAPDH水平.IFN-γ或TRAIL单药组细胞凋亡率均＜5%,而IFN-γ/TRAIL联合组细胞凋亡率达28.5%.siGAPDH组与siRNA对照组间细胞凋亡率差异有统计学意义(t=4.909,P=0.08),使对照组细胞凋亡率下降11%.结论:IFN-γ加强TRAIL诱导人甲状腺癌细胞凋亡可能是通过上调细胞核内GAPDH表达实现的.     

格尔德霉素增强子宫颈癌HeLa细胞热疗效的影响

背景与目的：苯醌安莎霉素类如格尔德霉素（geldanamycin,GA）通过抑制热休克蛋白（heat shock protain 90,HSP90）从而导致细胞内信号调节通路网络的多点阻断,如导致涉及细胞增殖、细胞周期调节及幸存调节等众多效应蛋白的降解.前期研究证明HSP90可作为抗肿瘤治疗的一个分子靶位.本研究以子宫颈癌HeLa细胞为研究对象,观察HSP90分子伴侣复合物阻止剂格尔德霉素能否增强加热对HeLa细胞的杀伤效应.方法：采用四唑盐比色实验（MTT）、流式细胞术（flow cytometry,FCM）、吖啶橙荧光染色等方法观察比较应用单纯热疗组、GA组、热疗联合GA组对HeLa细胞活力、凋亡率及细胞形态学的影响.结果：四唑盐比色实验观察到加热与GA化疗间有交互作用,加热联合GA组显著增强单独加热对HeLa细胞杀伤效应（P＜0.01）;Annexin,V-FITC染色流式细胞术结果显示加热联合GA组、单纯加热组、单纯GA治疗组所致细胞凋亡率分别为92.1%、52.2%及26.8%.用吖啶橙荧光染色法观察到加热联合GA组明显见HeLa细胞固缩着色不均而且较深,细胞核浓缩、裂解等凋亡特征.结论：HSP90分子伴侣复合物阻止剂GA能增强热诱导的HeLa细胞的杀伤作用.     

KIF14在恶性肿瘤中的研究进展

KIF14作为驱动蛋白超家族(KIFS)中的成员,通过调节有丝分裂纺锤体的形成、染色体的分离和胞质分裂的完成,从而促进肿瘤的发生和发展.KIF14的异常已经在人类多种恶性肿瘤中得到证实,其作用的增强可影响有丝分裂的进程而导致肿瘤的发生;其作用降低可导致Akt激酶失活,从而降低细胞的迁移,促进细胞的凋亡.此外,KIF14的缺失也可增强肿瘤对化疗的敏感性.综上所述,KIF14在肿瘤的发生发展过程中起到了重要作用,本文就KIF14与恶性肿瘤的关系做一综述.     

Functional analysis of nuclear pore complex protein Nup62/p62 using monoclonal antibodies

The nuclear pore complex (NPC) is an enormous structure embedded in the double membrane of the nuclear envelope that acts as a passageway for nucleocytoplasmic transport. The vertebrate NPC is comprised of about 30 unique proteins. Nup62/p62, a major component of the NPC, has been reported to interact directly with several nuclear transport factors, including importin-beta and NTF2. However, it has not been shown how the interaction of Nup62/p62 with transport factors is involved in nucleocytoplasmic transport. The present study reports on the preparation of monoclonal antibodies (MAbs) directed against human Nup62/p62 and a functional analysis of Nup62/p62 using antibodies in living cells. Hybridomas producing the antibodies were produced by the hybridization of mouse myeloma cells with medial iliac lymph node cells from an immunized rat. These MAbs specifically recognized Nup62/p62 as evidenced by immunoblotting analysis using a nuclear membrane fraction. In the immunostaining using MAbs, a punctuate nuclear rim staining pattern was observed. Moreover, cytoplasmic injected-anti-Nup62/p62 MAbs were rapidly targeted to the nuclear pore of cultured cells and some of them inhibited normal cell division, causing the formation of abnormal nuclei. The antibodies described in this study provide the means for immunochemical analyses of the NPC protein Nup62/p62 in mammalian cells, and represent useful molecular tools that should permit a better understanding of the biological roles and cellular dynamics of this protein in nucleocytoplasmic transport, cell division, and nuclear organization.     

The role of nuclear pore complex in tumor microenvironment and metastasis

One of the main reasons for cancer mortality is caused by the highly invasive behavior of cancer cells, which often due to aggressive metastasis. Metastasis is mediated by various growth factors and cytokines, operating through numerous signaling pathways. Remarkably, all these metastatic signaling pathways must enter the nucleus through a single gatekeeper, the nuclear pore complex (NPC). NPCs are the only gateway between the cytoplasm and the nucleus. NPCs are among the largest proteinaceous assemblies in the cell and are composed of multiple copies of around 30 different proteins called nucleoporins. Here, we review what is currently known about the NPC, and its role in the mechanisms of tumor progression. We will also explore potential strategies to target metastatic pathways by manipulating the karyopherins (importins/exportins) of nucleocytoplasmic traffic through NPCs.     

Liposome DNA delivery and uptake by cells (review)

Human gene therapy is a rapidly emerging field; therapeutic genes are engineered into adenovirus, retrovirus, or into plasmid-liposome complexes for their delivery into cells in culture or in vivo. Steps to improve for successful liposome-mediated gene delivery to somatic cells include persistence of the plasmid in blood circulation, port of entry and transport across the cell membrane, release from endosomal compartments into the cytoplasm, nuclear import by docking through the pore complexes of the nuclear envelope, expression driven by the appropriate promoter/enhancer control elements, and persistence of the plasmid in the nucleus for long periods. A number of strategies for enhancing the efficiency of uptake by the cells and release from endosomal compartments of liposome-plasmid or liposome-oligonucleotide complexes are reviewed here; emphasis is given to the direction of liposomes to caveolae vesicles.     

Regulation of cell death by the Abl tyrosine kinase

The c-abl proto-oncogene encodes a protein tyrosine kinase that is distributed in the nucleus and the cytoplasm of proliferating cells. In the nucleus, c-Abl activity is negatively regulated by the retinoblastoma protein (RB) and positively regulated by DNA damage signals. Activation of the c-Abl kinase by DNA damage requires the function of ATM, which regulates cell cycle checkpoint, DNA repair and apoptosis in response to DNA damage. Cells lacking c-Abl can activate cell cycle checkpoints and DNA repair, but show defects in apoptosis. The apoptosis defect of c-Abl deficient cells is correlated with a defect in the induction and activation of p73, which is a functional homologue of the p53 tumor suppressor protein and has pro-apoptotic activity. The inhibition of c-Abl by RB is consistent with RB's ability to block apoptosis; while the activation of c-Abl by ATM is consistent with ATM's ability to activate cell death. The oncogenic Bcr-Abl tyrosine kinase is a potent inhibitor of apoptosis, and it is retained exclusively in the cytoplasm of transformed cells. Interestingly, when Bcr-Abl is trapped inside of the nucleus through a combined disruption of its cytoplasmic retention and its nuclear export, this oncogenic Abl kinase induces apoptosis. Taken together, the current results support a role for the nuclear c-Abl tyrosine kinase in the regulation of apoptosis. Whether the cytoplasmic c-Abl kinase can actively inhibit apoptosis remains to be determined; however, a deliberate retention of c-Abl in the cytoplasm could potentially contribute to the attenuation of apoptosis response.     

倍半萜烯内酯对鼻咽癌细胞内核因子-кB活化的影响

背景与目的:探讨倍半萜烯内酯化合物对人鼻咽癌(nasopharyngeal carcinoma,NPC)细胞内核因子-κB(NF-κB)信号转导活化的影响。材料与方法:采用小白菊内酯(parthenolide,PN)作为受试物,以对PN诱导转归敏感的CNE1细胞给予TNF-α预诱导建立模型,PN处理后提取胞质和胞核蛋白,分别检测IκBα降解、NF-κB p65亚单位活化后核内迁移,电泳迁移率改变试验(EMSA)检测核内活化NF-κB的DNA结合活性。进行PN剂量和作用时间依赖关系分析。结果:阴性对照组IκBα蛋白存在于胞质中,PN处理组使TNF-α诱导的胞质IκBα蛋白降解被抑制、胞核内蛋白含量减少;相应地,阴性对照组p65亚单位在胞质中含量高于胞核内,PN处理组抑制TNF-α诱导的胞质p65核转位;同步进行的EMSA可见,PN处理组NF-κB核结合活性比TNF-α诱导组明显降低。随PN处理时间(0.5~4h)和剂量(5~25μmol/L)增加,胞质中IκBα蛋白降解的抑制作用增强(其蛋白含量增加),胞核内p65亚单位蛋白减少,EMSA结合活性降低,呈明显的剂量和时间依赖性(P均<0.05)。结论:PN可影响NPC细胞内NF-κB因子的活化,提示PN对TNF-α诱导NF-κB信号的抑制作用可能是PN诱导NPC细胞凋亡敏感性的分子机制之一。     

Reactive nitrogen species-dependent DNA damage in EBV-associated nasopharyngeal carcinoma: the relation to STAT3 activation and EGFR expression

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Recently, reactive nitrogen and oxygen species are considered to participate in inflammation-related carcinogenesis through DNA damage. In our study, we obtained biopsy and surgical specimens of nasopharyngeal tissues from NPC patients in southern China, and performed double immunofluorescent staining to examine the formation of 8-nitroguanine, a nitrative DNA lesion and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative DNA lesion, in these specimens. Strong DNA lesions were observed in cancer cells and inflammatory cells in stroma of NPC patients. Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8-nitroguanine-positive cancer cells. DNA lesions and iNOS expression were also observed in epithelial cells of EBV-positive patients with chronic nasopharyngitis, although their intensities were significantly weaker than those in NPC patients. In EBV-negative subjects, no or little DNA lesions and iNOS expression were observed. EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, but NF-kappaB was not expressed, suggesting that STAT3-dependent mechanism is important for NPC carcinogenesis. IL-6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of EBV-infected patients. EBV-encoded RNAs (EBERs) and latent membrane protein 1 (LMP1) were detected in cancer cells from all EBV-infected patients. In vitro cell system, nuclear accumulation of EGFR was observed in LMP1-expressing cells, and IL-6 induced phosphorylated STAT3 and iNOS. These data suggest that nuclear accumulation of EGFR and STAT3 activation by IL-6 play the key role in iNOS expression and resultant DNA damage, leading to EBV-mediated NPC.     

Nuclear topography and expression of the BCR/ABL fusion gene and its protein level influenced by cell differentiation and RNA interference

Nuclear topography, expression of the BCR/ABL fusion gene and its protein level/cellular pattern were studied in CML cell line K562 stimulated to differentiation, apoptosis and influenced by ABL-RNA interference (ABL-RNAi). Phorbol ester-induced maturation of K562 cells was accompanied by repositioning of down-regulated BCR/ABL genes closer to the nuclear membrane. This nuclear rearrangement could be connected with differentiation-related heterochromatinization of the amplified BCR-ABL locus, as demonstrated by increased histone H3(K9) dimethylation and decreased H3(K9) acetylation of B3A2 breakpoint. Topography of BCR/ABL in differentiated K562 cells was compared with other leukemic cell types: PMA-maturation of HL60 cells did not influence the nuclear positioning of individual BCR and ABL genes. Moreover, BCR and ABL genes in non-stimulated HL60 as well as in the bone marrow cells of CML patients, i.e. also BCR/ABL fusion genes, were positioned more interiorly in comparison with BCR/ABL multiple loci of K562 cells. Decreased expression of BCR/ABL gene was also found after cell stimulation by selectively pro-apoptotic agent etoposide and by ABL-RNAi leading to apoptosis. In order to compare the efficiency of selected experimental strategies, levels of Bcr/Abl and c-Abl proteins were determined and in all cases tested were reduced. In K562 cells the Bcr/Abl and c-Abl proteins were distributed homogeneously in both the cell nucleus and cytoplasm, while differentiation of K562 cells was characterized by a distinct pattern of Bcr/Abl and c-Abl proteins that were focally distributed rather in the cytoplasm while apoptotic population was completely absent of Bcr/Abl and c-Abl signals.     

p16 Gene transfer increases cell killing with abnormal nucleation after ionising radiation in glioma cells

It is well established that cells synchronised at the G1-S phase are highly radiosensitive. In this study, p16-null human glioma cell lines were induced into G1 cell cycle arrest by adenovirus-mediated p16 gene transfer, and examined for radiation-induced cell killing. Clonogenic analysis and trypan blue extraction test showed that the p16 gene transfer enhanced radiation-induced cell killing in p16-null glioma cell lines. TUNEL assays and pulse-field gel electrophoresis confirmed that the radiation-induced cell killing of p16-transfected cells could be caused by a nonapoptotic mechanism. Gimsa staining demonstrated that irradiation alone or Ax-mock infection plus irradiation results in a slight increase in the frequency of cells with abnormal nucleus, compared to unirradiated uninfected or Ax-mock infected cells. However, Ax-hp16 or Ax-hp21 infection alone modestly increased the frequency of cells with abnormal nucleus (especially bi- and multinucleation), and 4-Gy irradiation of Ax-hp16 or Ax-hp21 infected cells substantially enhanced this frequency. These results suggest that there exists some unknown interaction between radiation and p16 in cytoplasm/membranes, which decreases cytokinesis and promotes abnormal nucleation. Thus, p16 expression prevented radiation-induced apoptosis by promoting abnormal nucleation, thereby leading to another mode of cell death.     

Dual specificity kinases — a new family of signal transducers

Phosphorylation/dephosphorylation reactions are one of the dynamic mechanisms through which cells modulate protein activity in response to environmental stimuli. The eukaryotic molecules which are responsible for the phosphorylation of serine, threonine and tyrosine residues appear to have co-ordinately evolved from simple prokaryotic enzymes which primarily respond to nutritional cues. In multicellular eukaryotes the complexity of data transfer greatly exceeds that of simple bacteria. The eukaryotic cell needs to exchange information with neighouring and distant sister cells. Positional, nutritional and hormonal data are transmitted from the extracellular milieu across the plasma membrane and into the cytoplasm. In certain cases the signal must pass into the nucleus or other subcellular organelles where it is decoded and the proper cellular response initiated. All of these events have been shown to have a protein kinase component and it seems likely that in mammalian cells over 1,000 different kinase molecules have evolved to form the requisite signal transducing networks. In this review we describe a previously unappreciated family of protein kinases, the dual specificity or DSK kinases, which play important roles in the regulation of normal cellular growth and differentiation.