人细胞色素P4502E1基因在胚胎鼻咽组织、鼻咽癌细胞和组织中的表达

背景与目的有研究表明,亚硝胺类化学物质能诱导体内外鼻咽细胞癌变,但作为间接致癌物,其活化主要依赖细胞色素P4502E1(CYP2E1)的代谢.本研究旨在探讨人细胞色素P4502E1(hCYP2E1)基因在化学物质二亚硝基哌嗪(N,N-dinitrosopiperazine,DNP)致鼻咽癌变中的可能作用,为鼻咽癌病因和发病学机制提供新的证据.方法采用RTPCR方法检测8例胚胎鼻咽组织,10例鼻咽癌活检组织,5株鼻咽癌细胞系(CNE1,CNE2,HNE1,HNE2,HNE3)和1株体外恶性转化的鼻咽细胞系(7429)CYP2E1mRNA水平的表达.用不同浓度(200,250,300μg/ml)化学致癌物DNP作用于体外培养的正常鼻咽细胞后,也用RTPCR检测其CYP2E1mRNA表达.结果(1)100%胚胎鼻咽组织(8/8)和鼻咽癌细胞系(6/6,含7429细胞系)、80%(8/10)的鼻咽癌活检组织均存在CYP2E1的表达;(2)经DNP处理的胚胎鼻咽细胞CYP2E1表达增高.结论鼻咽部存在可诱导的CYP2E1基因表达,提示该基因在亚硝胺类间接致癌物(如DNP)致鼻咽癌变过程中可能发挥重要作用.     

鼻咽癌细胞中表达上调的新基因 NAG23 (FBXO 30) 的克隆与分析

目的：分离位于6号染色体长臂鼻咽癌高频等位基因不平衡位点D6S1581（6q25.3-27）附近与鼻咽癌相关的新基因。方法：运用差异RT－PCR检测定位于D6S1581附近的11个表达序列标签（expressed sequence tage,EST)在正常人鼻咽上皮细胞和鼻咽癌细胞系中的表达水平,并对其中一个表达上调的EST在鼻咽癌组织和正常鼻咽组织中的表达情况进行分析。用Northern杂交验证其表达差异并检测其在多脏器中的表达及所代表基因的转录本大小。利用生物信息学资源克隆并获得其全长cDNA ,对该序列进行初步分析。结果：获得了一个位于6q25．3－27的在鼻咽癌中表达上调的新基因,命名为NAG23（FBXO30）(GenBank收录编号：AF248640）。该基因在68．9％的鼻咽癌活检组织中表达上调,cDNA全长3301bp,预测其开放阅读框编码一个含390个氨基酸的胞浆蛋白,该蛋白含一个F－box基序。结论：NAG23基因是一个在鼻咽癌中表达上调的新基因,很可能编码一新的F－box蛋白。NAG23可能参与了鼻咽癌的发生发展。     

Activating enhancer-binding protein-2α induces cyclooxygenase-2 expression and promotes nasopharyngeal carcinoma growth

Activating enhancer-binding protein-2α (AP-2α) regulates the expression of many cancer-related genes. Here, we demonstrated a novel mechanism by which AP-2α up-regulated cyclooxygenase-2 (COX-2) expression to promote the growth of nasopharyngeal carcinomas (NPCs). High expression of AP-2α in NPC cell lines and tumor tissues from NPC patients was detected and significantly correlated with COX-2 expression. Overexpression of AP-2α and COX-2 in tumor tissues was associated with advanced tumor stage, clinical progression, and short survival of patients with NPCs. Knockdown of AP-2α by siRNA markedly inhibited COX-2 expression and PGE2 production in NPC cells. Exogenous expression of AP-2α up-regulated the COX-2 and PGE2. Knockdown of AP-2α also significantly suppressed cell proliferation in NPC cells in vitro and tumor growth in a NPC xenograft mouse model. Moreover, we found that p300 played an important role in the AP-2α/COX-2 pathway. AP-2α could co-localize and interact with p300 in NPC cells. Overexpression of the p300, but not its histone acetyltransferase (HAT) domain deletion mutant, promoted the acetylation of AP-2α and its binding on the COX-2 promoter, thereby up-regulated COX-2 expression. Our results indicate that AP-2α activates COX-2 expression to promote NPC growth and suggest that the AP-2α/COX-2 signaling is a potential therapeutic target for NPC treatment.     

Reduced expression of intercellular adhesion molecule-1 in ovarian adenocarcinomas.

Ovarian adenocarcinomas develop as the result of multiple genetic and epigenetic changes in the precursor ovarian surface epithelial (OSE) cells which result in a malignant phenotype. We investigated changes in gene expression in ovarian adenocarcinoma using a cDNA array containing 588 known human genes. We found that intercellular adhesion molecule-1 (ICAM-1) was expressed at lower levels in the ovarian tumour cell lines OAW42, PEO1 and JAM than in the immortalised human ovarian surface epithelial cell line HOSE 17.1. Further investigation revealed ICAM-1 was expressed in the surface epithelium of normal ovaries and both mRNA and protein expression levels were reduced in the majority of ovarian adenocarcinoma cell lines and primary tumours. ICAM-1 expression was increased in 8/8 cell lines treated with the de novo methyltransferase inhibitor 5-aza-2'-deoxycytidine, indicating that methylation of CpG islands may play a role in the down-regulation of its expression in primary tumours. There was a significant association between patients whose tumours expressed ICAM-1 and survival (P = 0.03), suggesting that expression levels of ICAM-1 may have clinical relevance.     

组织芯片技术检测人鼻咽癌组织及细胞株KIAA1173基因的表达

背景与目的:鼻咽癌(nasopharyngealcarcinoma,NPC)致病的分子机制至今仍不清楚,已有研究表明,染色体3p21～22区域存在与鼻咽癌发生密切相关的抑癌基因。KIAA1173基因是定位于3p22.1的一个新的肿瘤相关基因,其与NPC发病的关系尚未见报道。本研究采用KIAA1173基因特异性原位杂交探针,检测其在NPC组织及细胞株中的表达,探讨KIAA1173基因与NPC发病的关系。方法:克隆KIAA1173基因片段(354bp),并制备cDNA探针;采用组织芯片技术,通过原位杂交检测73例鼻咽部不同组织标本(41例NPC、18例鼻咽非典型增生上皮、14例正常鼻咽粘膜上皮)和6种NPC细胞株(CNE1、CNE2、HNE1、HNE2、6-10B、5-8F)中KIAA1173基因mRNA的表达情况。结果:KIAA1173基因在NPC细胞、非典型增生上皮和正常鼻咽粘膜上皮的阳性率分别为21.9%(9/41)、83.3%(15/18)、92.8%(13/14),而6种NPC细胞株均未见表达;在正常鼻咽粘膜上皮和非典型增生上皮中强阳性率分别是64.3%(9/14)和38.9%(7/18),而NPC中无强阳性,在鼻咽部不同上皮组织中的表达差异有显著性(P<0.001);在38例伴有淋巴细胞浸润的NPC组织中,癌细胞与浸润淋巴细胞之间KIAA1173基因表达有显著性差异(P=0.026),并且呈明显负相关(κ=-0.337,P=0.020)。结论:KIAA1173基因在鼻咽部不同组织中表达不同,正常鼻咽上皮强表达,而NPC细胞中低表达甚至不表达,提示该基因可能参与NPC演变的过程。     

Identification of interleukin-13 receptor alpha2 chain overexpression in situ in high-grade diffusely infiltrative pediatric brainstem glioma

Human malignant glioma cell lines and adult brain tumors overexpress high levels of interleukin-13 receptor alpha2 chain (IL-13Ralpha2). Because the IL-13Ralpha2 chain is an important target for cancer therapy and prognosis for patients with brainstem glioma (BSG) remains dismal, we investigated the expression of this receptor in specimens of diffusely infiltrative pediatric BSG relative to normal brain tissue. Twenty-eight BSG specimens and 15 normal brain specimens were investigated for IL-13Ralpha2 protein expression by immunohistochemical analysis (IHC) using two different antibodies in two different laboratories. Highly sensitive Q-dot-based IHC and in situ hybridization (ISH) assays were also developed to identify IL-13Ralpha2 protein and RNA in these specimens. The results were evaluated independently in two laboratories in a blinded fashion. By Q-dot IHC or a standard IHC assay, 17 of 28 (61%) tumor specimens showed modest to strong staining for IL-13Ralpha2, while 15 normal brain tissue samples showed weak expression for IL-13Ralpha2 protein. Significant interrater agreement between the two laboratories was seen in the assessment of IL-13Ralpha2 intensity. High-level IL-13Ralpha2 RNA expression was detected in tumor samples by Q-dot ISH, but only weak RNA expression was observed in normal brain. Significant agreement between ISH and IHC assays was observed (simple kappa [kappa] estimate=0.358, weighted kappa=0.89, p=0.001). IL-13Ralpha2 protein and mRNA are expressed to significantly higher levels in BSG than in normal brain tissue. Both IHC and ISH represent robust methods to detect expression of the IL-13Ralpha2 receptor in BSG that could represent an important new drug target for treatment of this disease.     

Significance of Expression of Human METCAM/MUC18 in Nasopharyngeal Carcinomas and Metastatic Lesions

Human METCAM/MUC18, a cell adhesion molecule (CAM) in the immunoglobulin-like gene super family,plays a dual role in the progression of several epithelium cancers; however, its role in the nasopharyngealcarcinoma (NPC) remains unclear. To initiate the study we determined human METCAM/MUC18 expression intissue samples of normal nasopharynx (NP), NPCs, and metastatic lesions, and in two established NPC cell lines.Immunoblotting analysis was used for the determination in lysates of frozen tissues, and immunohistochemistry(IHC) for expression in formalin-fixed, paraffin-embedded tissue sections of 7 normal nasopharynx specimens, 94NPC tissue specimens, and 3 metastatic lesions. Human METCAM/MUC18 was expressed in 100% of the normalNP, not expressed in 73% of NPC specimens (or expressed at very low levels in only about 27% of NPC specimens),and expressed again in all of the metastatic lesions. The level of human METCAM/MUC18 expression in NPCtissues was about one fifth of that in the normal NP and metastatic lesions. The low level of human METCAM/MUC18 expression in NPC specimens was confirmed by a weak signal of RT-PCR amplification of the mRNA.Low expression levels of human METCAM/MUC18 in NPC tissues were also reflected in the seven establishedNPC cell lines. These findings provided the first evidence that diminished expression of human METCAM/MUC18is an indicator for the emergence of NPC, but increased expression then occurs with metastatic progression,suggesting that huMETCAM/MUC18, perhaps similar to TGF-β, may be a tumor suppressor, but a metastasispromoter for NPC.     

pRb2/p130、cyclin D1和MUC1在食管鳞癌中的表达

 目的探讨pRb2/p130、cyclinD1和MUC1在食管鳞癌中的表达及意义。方法应用免疫组化法检测了pRb2/p130、cyclinD1和MUC1在16例正常食管粘膜上皮及60例食管鳞癌中的表达水平,分析它们与临床病理指标及其三者之间相关关系。结果食管鳞癌中pRb2/p130的低表达与癌组织分化程度、TNM分期、淋巴结转移、浸润深度有关;cyclinD1的高表达与癌组织的分化程度、淋巴结转移相关;MUC1的高表达与淋巴结转移相关;Kaplan-Meier生存分析表明pRb2/p130高表达组生存率高于低表达组(P=0.0032,LogRank检验);Cox比例风险模型分析显示pRb2/p130、淋巴结转移、癌组织的分化程度是食管鳞癌的独立预后指标。结论食管鳞状细胞癌中存在pRb2/p130的低表达以及cyclinD1和MUC1的高表达,促进了细胞的生长和肿瘤的发展,是食管癌发生发展中的重要事件。     

鼻咽癌表达下调基因NASG3′UTR可变剪接分析及其在多种肿瘤中的表达

背景与目的：采用抑制性消减杂交技术已分离获得了人鼻咽组织特异性基因NASG。本研究对人鼻咽组织特异性表达且在鼻咽癌表达下调的NASG基因3′非编码区(untranslated region,UTR)的可变剪接进行分析,并考察NASG基因在多种肿瘤组织中的表达。方法：在NASG基因3′UTR存在可变剪接部位的两端设计引物进行RT—PCR扩增,分离扩增产物并测序。用RT—PCR检测NASG基因在鼻咽癌中的表达,采用了肿瘤表达谱阵列(cancer profiling array)杂交分析NASG基因在多种肿瘤组织的表达状况。结果：NASG基因3′UTR存在3种剪接产物,NASG基因在71％的鼻咽癌活检组织中表达下调,25％的肺癌组织中表达上调．而在其他肿瘤及其配对的正常组织末见明显表达。结论：NASG基因3′UTR存在3种剪接产物,NASG基因的表达异常是鼻咽癌和肺癌发生、发展过程中重要的分子事件。     

Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β

Transforming growth factor-β (TGF-β) promotes cancer metastasis via the epithelial-mesenchymal transition (EMT) but the underlying mechanisms in nasopharyngeal carcinoma (NPC) remain unclear. Flotillin-2 (Flot2), a specialized lipid raft domain in cellular membrane, was reported to promote cancer metastasis. Recently, in neuropathy, it was also suggested that Flot2 was involved in Src activation, which is known as the downstream signal of TGF-β. Therefore, we intended to find out the relationship between Flot2 and TGF-β in the process of nasopharyngeal carcinoma (NPC) metastasis. In this study, we found that Flot2 expression level positively correlated with the cancer stage in NPC tissues. Elevated Flot2 in tumor tissue was an independent prognostic marker, and higher Flot2 expression level showed shorter overall survival time in 181 NPC patients. In NPC cells, silencing Flot2 reversed the metastatic effect induced by TGF-β. Moreover, TGF-β-induced Src phosphorylation was significantly inhibited by Flot2 knocking down. As the consequence of Flot2 inhibition, the expression of the epithelial biomarker E-cadherin was upregulated, while the mesenchymal marker vimentin and signaling transducer β-catenin was suppressed. In conclusions, Flot2 is an indispensable member for TGF-β signaling, which is essential for the EMT process in NPC metastasis. Suppressing Flot2 may be a novel way against TGF-β-induced EMT.     

Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma

Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation-specific PCR (MSP). We have detected dense methylation of TIG1 5'CpG island in the 5 TIG1-negative NPC cell lines and xenograft (C666-1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1-expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5-aza-2'-deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis.     

Renal cell carcinoma induces interleukin 10 and prostaglandin E2 production by monocytes.

Immunotherapy with interleukin 2 (IL-2) is not an effective anti-cancer treatment in the majority of patients with renal cell carcinoma (RCC), suggesting that the activation of cytotoxic T cells or NK cells may be impaired in vivo in these patients. The production of immunosuppressive factors by RCC was investigated. Using immunohistochemistry, IL-10 was detectable in 10 of 21 tumour samples tested. IL-10 was undetectable in the supernatant of cell lines derived from these RCCs. However, these cell lines or their conditioned medium (RCC CM), but not normal renal epithelial cells adjacent to the RCC or breast carcinoma cell lines, were found to induce IL-10, as well as prostaglandin E2 (PGE2) and tumour necrosis factor (TNF)alpha production by autologous or allogeneic peripheral blood mononuclear cells (PBMCs) and monocytes. IL-10 production induced by RCC CM was found to be dependent on TNF-alpha and PGE2 since an anti-TNF-alpha antibody (Ab) inhibited 40-70% of IL-10 production by monocytes, and the combination of anti-TNF-alpha Ab and indomethacin, an inhibitor of PGE2 production, inhibited 80-94% of RCC CM-induced IL-10 production by monocytes. The RCC CM of the five cell lines tested were found to induce a down-regulation of the expression of HLA-DR and CD86, as well as a strong inhibition of mannose receptor-dependent endocytosis by monocytes. The blockade of HLA-DR and CD86 expression was partially abrogated by indomethacin and anti-IL-10 Ab respectively, and completely abrogated by an anti-TNF-alpha Ab. The inhibition of mannose receptor-dependent endocytosis was partially abrogated by an anti-IL-10 Ab and completely abrogated by an anti-TNF-alpha Ab. These results indicate that RCCs induce IL-10, PGE2 and TNF-alpha production by monocytes, which down-regulate the expression of cell-surface molecules involved in antigen presentation as well as their endocytic capacity.     

BRD7交互作用蛋白基因BRD2、BRD3在鼻咽癌组织中的表达

背景与目的：BRD7基因是通过cDNA代表性差异分析获得的一个鼻咽癌（nasopharyngeal carcinoma,NPC）密切相关基因。采用酵母双杂交技术,已从人胎脑的cDNA文库中筛选到了两个与BRD7蛋白存在交互作用且包含溴区结构域（bromodomain)的蛋白BRD2、BRD3。本研究的目的在于进一步证实BRD7蛋白与BRD2、BRD3蛋白之间的交互作用,探讨BRD2、BRD3基因在鼻咽癌中的表达和作用模式。方法：将BRD2、BRD3基因分别与BRD7基因共转化酵母Y187后将菌落影印到尼龙膜,X－Gal检测酵母中报告基因LacZ的表达,推断BRD2、BRD3与BRD7蛋白之间的交互作用。RT－PCR方法检测BRD2、BRD3基因在正常鼻咽上皮组织和鼻咽癌活检组织中的差异表达以及在BRD7基因稳定转染的鼻咽癌细胞株（HNE1）中,BRD7基因的重表达对BRD2、BRD3基因表达水平的影响。结果：通过特异性酵母双杂交技术,发现酵母转化子的颜色呈蓝色,进一步证实BRD2、BRD3蛋白能分别与BRD7蛋白发生交互作用。RT－PCR结果显示,在鼻咽癌组织中,BRD2和BRD3基因表达下调或缺失;在BRD7基因稳定转染的HNE1细胞株中,随着BRD7基因表达的增强,BRD2、BRD3基因的表达存在明显的上调。结论：BRD7蛋白能分别与BRD2、BRD3蛋白发生交互作用,且在mRNA水平存在一定程度的协同表达作用,彼此可能形成二聚体或三聚体,共同参与鼻咽癌的发病过程。     

Liver X Receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization

Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity.Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.     

间隙连接蛋白Cx43、Cx45在鼻咽癌组织中的表达

背景与目的：间隙连接在细胞间的营养物质,离子和细胞调节因子的交换中起重要作用。间隙连接异常,细胞间的物质交换障碍,往往导致细胞分裂失控,在有些癌组织和癌细胞中存在间隙连接的功能异常。恢复这些癌细胞的间隙连接功能,它们表现出正常细胞的生物学表型,因此,探讨细胞间隙连接蛋白在鼻咽癌中的表达,将有可能为临床诊断提供方法。为鼻咽癌的发病机理提供新思路。方法;采用免疫组化技术检测间障连接蛋白Cx43,Cx45在鼻咽组织中的表达。结果：（1）Cx43,Cx45在鼻咽慢性炎症组织和鼻咽癌组织中有表达差异,在鼻咽癌中,Cx43,Cx45阳性率为44．8％,46．6％,与鼻咽慢性炎症柱状上皮细胞阳性率为85％和100％比较,显著性下降（P＜0．01）,（2）鼻咽慢性炎症组织比鼻咽癌组织含鳞状细胞的百分率低（P＜0．01）,分别为29．7％和56．9％,（3）Cx43,Cx45在鼻咽癌旁柱状上皮细胞中的表达低于癌旁鳞状上皮细胞（P＜0．001）,高于癌细胞（P＜0．01）,结论：Cx43,Cx45在鼻咽组织中的表达异常,可能与鼻咽组织鳞化和癌变有关。