Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis

Although mutation of p53 tumor-suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis. Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3sigma, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in NPC.     

Identification Keratin 1 as a cDDP-resistant protein in nasopharyngeal carcinoma cell lines

Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.     

A novel Rhein derivative: Activation of Rac1/NADPH pathway enhances sensitivity of nasopharyngeal carcinoma cells to radiotherapy

Radiation resistance and recurrent have become the major factors resulting in poor prognosis in the clinical treatment of patients with nasopharyngeal carcinoma (NPC). New strategies to enhance the efficacy of radiotherapy have been focused on the development of radiosensitizers and searching for directly targets that modulated tumor radiosensitivity. A novel potential radiosensitizer 1,8-Dihydroxy −3-(2′-(4″-methylpiperazin-1″-yl) ethyl-9,10-anthraquinone −3-carboxylate (RP-4) was designed and synthesized based on molecular docking technology, which was expected to regulate the radiosensitivity of tumor cells through targeting Rac1. In order to assess the radiosensitization activity of RP-4 on NPC cells, the highly differentiated CNE1 and poorly differentiated CNE2 cells NPC lines were employed. According to the results, RP-4 showed higher binding affinity toward the interaction with Rac1 than lead compounds. We found that RP-4 could inhibit cell viability and proliferation in CNE1 and CNE2 cells and significantly induced apoptosis after non-toxic concentration of RP-4 combined with 2Gy irradiation. RP-4 could effectively modulated the radiosensitivity both CNE1 cells and CNE2 cells through activating Rac1/NADPH signaling pathway and its downstream JNK/AP-1 pathway. What's more, Rac1/NADPH signaling pathway were significantly activated in Rac1-overexpressed CNE1 and CNE2 cells after treated with RP-4. Taken together, Rac1 and its downstream pathway may probably be the direct targets of RP-4 in regulating radiosensitivity of NPC cells, our finding provided a novel strategy for the development of therapeutic agents in response to tumorous radiation resistance.     

Effect of Turkish propolis extracts on proteome of prostate cancer cell line

Background

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells.

Results

We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins.

Conclusion

The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.     

DNA-PK的活性与鼻咽癌细胞株CNE1/CNE2放射敏感性的关系

本文主要研究DNA依赖的蛋白激酶（DNA-dependent protein kinase,DNA-PK）与鼻咽癌细胞放射敏感性之间的关系。克隆形成实验分析鼻咽癌细胞CNEI／CNE2的剂量存活曲线,Signa TECT DNA-PK试剂盒检测DNA-PK活性,免疫荧光及激光显微共聚焦分析放疗前及放疗后15min、1h、6h、12h和24hCNE1／CNE2细胞中Kus及DNA-PKcs的亚细胞定位,Western blot分析两株细胞中Kus蛋白的表达。结果显示：CNE1细胞在每个剂量点的存活分数均高于CNE2细胞;同时发现放疗前后CNE1细胞中的DNA-PK活性也均高于CNE2细胞,但两株细胞中Ku70／Ku80蛋白表达无明显差异;放疗可使DNA-PK活性增加,且各个检测时间点CNE1细胞增加的幅度大于CNE2细胞;DNA-PK亚基可同时定位于胞浆和胞核,但主要位于胞核,细胞照射后Ku70、Ku80和DNA-PKcs从胞浆转运到胞核。结果表明：DNA-PK活性更高可能是CNE1细胞较CNE2细胞更能抵抗放射的原因之一;放疗所致DNA-PK活性增高可能与DNA-PK亚基从胞浆转运到胞核有关,而与Ku蛋白表达的总量无关。     

鼻咽癌细胞系与永生化的鼻咽上皮细胞系蛋白质表达差异分析

鼻咽癌对我国南部居民的健康造成严重的威胁.为了研究鼻咽癌的发病机理,本研究采用了蛋白质组学技术分析和比较了鼻咽癌细胞系（HNE1和CNE1）与永生化的鼻咽上皮细胞系的蛋白质表达谱.采用双向凝胶电泳分离提取的全细胞蛋白质,通过PDQuest软件分析找出在肿瘤中表达变化的蛋白质点,用基质辅助激光解析电离飞行时间串联质谱(MALDI- TOF/TOF-MS)进行鉴定.共得到了15个在肿瘤细胞系中表达上调和18个在肿瘤细胞系中表达下调的蛋白质,并对其中一些蛋白质的表达进行免疫印迹的验证.这些表达差异的蛋白质与细胞的增殖和调亡、癌症的转移,细胞骨架,信号传导等有关.本研究鉴定了一批可能作为鼻咽癌治疗的药物靶标的蛋白质,并对研究鼻咽癌发病机理提供了相关的线索.     

鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定

鼻咽癌中p53基因突变罕见,但绝大部分鼻咽癌中存在p53蛋白过表达/聚集且功能失活.然而,到目前为止p53蛋白失活的机制仍然不清楚.为揭示鼻咽癌中p53蛋白功能失活的机制,采用免疫共沉淀技术分别富集鼻咽癌细胞系HNE1和HNE2的p53结合蛋白,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）对免疫沉淀复合物进行分离,从胶中切取p53结合蛋白条带,胶内酶解后进行电喷雾串联质谱（LC-ESI-MS/MS）分析,得到相应的肽序列标签（peptide sequence tags, PST）,通过搜索数据库在鼻咽癌细胞系中鉴定了9个p53结合蛋白.分别是热休克蛋白70（HSP70）家族成员GRP-78和GRP-75、HSP90家族成员GRP-94、核纤层蛋白A/C （Lamin A/C）、α-actinin 4、Ezrin/Cytovillin、DNA复制准许因子/MCM3蛋白(DNA replication licensing factor/minichromosome maintenance 3 protein, MCM3)、CD98/4F2 heavy chain和蛋白激酶C（PKC）.并用免疫共沉淀和蛋白质印迹分析技术对HNE₁细胞蛋白条带3鉴定的p53相互作用蛋白之一HSP78进行了验证.首次在鼻咽癌细胞中鉴定了9个p53结合蛋白,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.     

鼻咽癌相关基因 BRD7 对鼻咽癌细胞CNE1的影响

为了研究BRD7基因对鼻咽癌细胞CNE1的影响,通过脂质体转染方法,将BRD7基因导入NPC细胞株CNE1细胞中.通过细胞生长曲线发现该基因能够抑制CNE1细胞的生长.为了探讨可能的作用机制,进而采用蛋白质组技术研究该基因对鼻咽癌蛋白质表达谱的影响,从而研究该基因在CNE1中的地位和作用.通过对过表达BRD7基因后鼻咽癌细胞系CNE1的蛋白质表达谱改变的研究,鉴定出19个差异表达蛋白,这些蛋白质包括:BCCIP (BRCA2 and CDKN1A(p21(Waf1/Cipl)),FHL2(four and a half LIM domains 2),Chloride channel regulatory protein;Hin-1(high-in-normal-1),WISP-1(connective tissue growth factor related protein),SREC-4(scavenger receptor expressed by endothelial cells-2),folate receptor.这些差异蛋白涉及到基因表达调控、细胞黏附等众多的事件.从另一个侧面研究了BRD7基因与鼻咽癌的关系,扩展了BRD7基因的研究范围,并进一步充实了该基因做为鼻咽癌候选抑瘤基因的证据.     

5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation

The radioresistance of tumor cells remains a major cause of treatment failure in nasopharyngeal carcinoma (NPC). Recently, several reports have highlighted the importance of epigenetic changes in radiation-induced responses. Here, we investigated whether the demethylating agent 5-azacytidine (5-azaC) enhances the radiosensitivity of NPC cells. The NPC cell lines CNE2 and SUNE1 were treated with 1 μmol/L 5-azaC for 24 h before irradiation (IR); clonogenic survival was then assessed. Tumor growth was investigated in a mouse xenograft model in vivo. The apoptosis, cell cycle progression and DNA damage repair were examined using flow cytometry, immunofluorescent staining and western blotting. Promoter methylation and the expression of four genes epigenetically silenced during the development of NPC were evaluated by pyrosequencing and real-time PCR. We found that pretreatment with 5-azaC significantly decreased clonogenic survival after IR compared to IR alone; the sensitivity-enhancement ratio of 5-azaC was 1.4 and 1.2 for CNE2 and SUNE1 cells, respectively. The combined administration of 5-azaC and IR significantly inhibited tumor growth in the mouse xenograft model, and enhanced radiation-induced apoptosis in vitro compared to 5-azaC alone or IR alone. 5-AzaC also decreased promoter methylation and upregulated the expression of genes which are epigenetically silenced both in vitro and in vivo in NPC. Thus, 5-azaC enhance the radiosensitivity of both the CNE2 and SUNE1 cell lines, possibly by altering DNA methylation levels and increasing the ability of irradiated cells to undergo apoptosis. The use of 5-azaC combined with IR maybe represent an attractive strategy for the treatment of NPC.     

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

We examined apoptosis and expression of p53, E2F-1, bax, bclx_L and bcl2 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation.We found that there is no change in expression of E2F-1, bax, bclx_L or bcl2 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bcl2, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability. Received: 19 August 1999 / Accepted in revised form: 30 November 1999     

不同分化程度的鼻咽癌细胞系质膜差异蛋白质组分析

本研究以CNE1和CNE2为材料,采用亚细胞蛋白质组研究方法研究不同分化程度鼻咽癌细胞系的差异蛋白质．首先用Percoll密度梯度离心法获得高纯度质膜,通过双向凝胶电泳分离、PDQuest软件分析后找出在肿瘤细胞中表达变化的蛋白质点,再用基质辅助激光解析电离飞行时间串联质谱（MALDI-TOF/TOF-MS）进行鉴定,共鉴定到9个具有2倍或2倍以上差异的蛋白质.这些表达差异的蛋白质参与了细胞分化、代谢及细胞信号传导过程．我们对其中5个蛋白质进行了实时定量PCR分析,对其中4个蛋白质的表达进行了免疫印迹验证．本试验为研究不同分化程度的鼻咽癌提供了一种蛋白质组研究方法,并且找到了galectin-1、annexin Ⅱ等一些可能与分化相关的蛋白质.这些数据对于研究鼻咽癌的生物学特性具有非常重要的意义．     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

Association between TP53 codon 72 single-nucleotide polymorphism and radiation sensitivity of human fibroblasts

Inherent radiosensitivity varies widely between individuals. We hypothesized that amino acid substitution variants in two highly radiation-responsive proteins, TP53 (p53) and CDKN1A (p21, Waf1, Cip1), are associated with and could explain individual variations in radiosensitivity. The two non-synonymous single-nucleotide polymorphisms (SNPs) TP53 codon 72 Arg/Pro G>C and CDKN1A codon 31 Ser/Arg C>A were genotyped in 92 normal fibroblast cell strains of different radiosensitivity. The clonogenic surviving fraction at 2 Gy (SF2) ranged between 0.15 and 0.50 (mean = 0.34, SD = 0.08). The mean SF2 was used to divide the cell strains into radiosensitive (45) and normal groups (47). A significant association was observed between SF2 and the TP53 codon 72 haplotype (C compared to G, P = 0.01). No association was observed between CDKN1A codon 31 haplotype and radiosensitivity (P = 0.86). The variant TP53 Arg72 allele was associated with a decrease in radiosensitivity, presumably due to suboptimal function leading to less stringent control of cell division. We conclude that certain SNPs in susceptible genes can influence cellular radiation response. Such risk alleles could ultimately be used as predictive markers for radiosensitivity to help stratifying individuals during assessment of risk of radiation exposure.     

人NPCEDRG基因启动子的克隆及CCAAT/NFY结合位点初步分析

NPCEDRG基因是采用基因定位候选克隆策略获得的一个鼻咽癌候选抑瘤基因.NPCEDRG在鼻咽癌细胞和组织中表达下调,重新恢复NPCEDRG基因在CNE2细胞系的表达,可部分逆转CNE2的恶性表型.为揭示NPCEDRG基因在鼻咽癌细胞和组织中表达下调的分子机制,联合应用生物信息学和报告基因载体系统分析方法对NPCEDRG基因启动子区进行克隆及功能分析,系统发育进化足迹分析结果表明,NPCEDRG基因5′端调控区-180～+235 bp区间在脊椎动物中高度保守,该保守区域中存在包括CCAAT/NFY、STAT1和SP1等转录因子结合位点.构建Luc和/或EGFP报告基因表达载体并检测其启动子活性,-146～-8 bp区域有较强的启动子活性,电泳迁移阻滞分析实验(EMSA)提示,CCAAT/NFY转录因子结合位点是NPCEDRG基因的转录调控元件.因此,研究确定-146～-8 bp区域是NPCEDRG基因核心启动子区域且启动子核心元件CCAAT/NFY可能参与NPCEDRG基因的转录调控.     

Regulation of survivin and CDK4 by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cell lines

Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line- CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/ M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMPI. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.     

Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.     

The yeast nuclear pore complex functionally interacts with components of the spindle assembly checkpoint

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p-Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores. Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p. On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint. Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport.