Role of CAGE, a novel cancer/testis antigen, in various cellular processes, including tumorigenesis, cytolytic T lymphocyte induction, and cell motility

A cancer-associated antigen gene (CAGE) was identified by serological analysis of a recombinant cDNA expression library (SEREX). The gene was identified by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera from patients with gastric cancer. CAGE was found to contain a D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. The CAGE gene is widely expressed in various cancer tissues and cancer cell lines. Demethylation plays a role in the activation of CAGE in certain cancer cell lines where the gene is not expressed. The functional roles of CAGE in tumorigenesis, the molecular mechanisms of CAGE expression, and cell motility are also discussed.     

Identification of differentially expressed genes in normal and tumor human gastric tissue

The search for differentially expressed genes in gastric cancer may help define molecular alterations and molecular diagnosis of gastric cancer. Using the differential display PCR technique, we identified 18 genes that are differentially expressed between normal and tumor human gastric tissues. Their expressions were verified with reverse Northern blot analysis and Northern blot analysis. Oxidative phosphorylation-related genes, antizyme inhibitor of ornithine decarboxylase, protein phosphatase-1beta, 35-kDa peroxisomal membrane protein, and cystic fibrosis transmembrane conductance receptor were highly expressed in tumor tissue, whereas pepsinogen A, Na-K ATPase alpha subunit, nerve growth factor receptor, and alpha-tropomyosin were highly expressed in normal tissue. In addition, 3 unknown genes were found to be differentially expressed in paired gastric tissues. These differentially expressed genes may provide significant opportunities for further understanding of gastric carcinogenesis and the molecular diagnosis of gastric cancer.     

Identification and characterization of a novel cancer/testis antigen gene CAGE

We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human x hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.     

Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA⁺ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.     

Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

应用二维电泳和质谱技术筛选喉癌及癌旁组织中的差异表达蛋白质

目的分离并鉴定喉癌和癌旁正常粘膜组织的差异表达蛋白质,为喉癌早期临床诊断、治疗提供新的有关的肿瘤生物学标记和靶标。方法收集5对人喉癌组织和对应的癌旁正常粘膜组织,提取组织总蛋白质,采用二维凝胶电泳技术分离蛋白并进行比较。选择在喉癌中明显差异表达的蛋白质点,进行质谱分析。结果获得了分辨率和重复性均较好的凝胶蛋白图谱。筛选出的在喉癌及癌旁正常粘膜组织中明显差异表达的10个蛋白质点,并成功鉴定。其中在喉癌组织中高表达的7个,低表达的3个。结论喉癌组织与癌旁正常粘膜组织蛋白存在明显的差异,筛选并鉴定出的这些蛋白质可能成为喉癌早期临床诊断、治疗的标志物和靶标。     

幽门螺杆菌毒素蛋白CagA诱导的蛋白的差异表达分析及其基因在胃癌组织中的表达

为了研究胃癌细胞中幽门螺杆菌(Hp)毒素蛋白CagA诱导的蛋白差异表达及其基因在人胃癌组织中的表达,用Hp感染胃癌细胞系SGC 7901和AGS及用含CagA基因的表达载体稳定转染SGC 7901细胞, 构建3组实验模型.提取各组细胞的总蛋白进行双向凝胶电泳,筛选3组重叠的差异表达蛋白质斑点进行质谱鉴定.共获得135个差异表达的蛋白质,其中上调蛋白质73个,下调蛋白质62个. 鉴定出10个差异表达蛋白质, 其中有6个差异表达蛋白是首次发现,它们主要参与细胞的能量代谢和信号转导等.最后定量检测了这10个差异表达蛋白基因在人胃癌组织中的表达, 发现有4个基因高表达和1个基因低表达. 本结果将为研究幽门螺杆菌感染引起胃癌的分子机制提供新的线索.     

Quantitative proteomics of primary tumors with varying metastatic capabilities using stable isotope-labeled proteins of multiple histogenic origins

The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins were identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors versus corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.     

Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.     

Coordinated expression of ncRNAs and HOX mRNAs in the human HOXA locus

In the human HOXA locus a number of ncRNAs are transcribed from the intergenic regions in the opposite direction to HOXA mRNAs. We observed that the genomic organization of genes for the ncRNAs and HOXA proteins is highly conserved between human and mouse. We examined the expression profiles of these ncRNAs and HOXA mRNAs in various human tissues. The expression patterns of ncRNAs in human tissues coincide with those of the adjacent HOXA mRNAs that are collinearly expressed along the anteroposterior axis. This coordinated expression was observed even in transformed tumors and cancer cell lines, suggesting that the expression of ncRNAs is prerequisite for the regulated expression of HOXA genes. HIT18844 ncRNA transcribed from the most upstream position of the HOXA cluster possesses an ultra-conserved short stretch which potentially forms an evolutionarily conserved secondary structure. Our data suggest a critical role for ncRNAs in the regulation of HOXA gene expression.     

胃粘膜非典型增生中微环境蛋白质的相互作用

微环境在胃癌发病过程中发挥重要作用。了解胃粘膜早期癌变的分子机制,对防治胃癌具有十分重要的意义。为了解胃粘膜非典型增生过程中,微环境中蛋白质的相互作用及调节机制,采用激光捕获显微切割（laser capture microdissection, LCM）技术,纯化正常胃粘膜组织（normal gastric mucosa tissue, NGM）和胃粘膜非典型增生（gastric mucosal atypical hyperplasia, GMAH）间质,通过同位素标记定量蛋白质组学技术分析,鉴定NGM和GMAH间质的差异表达蛋白质。利用生物信息学软件,分析NGM和GMAH间质差异表达蛋白质的相互作用及其联系。共鉴定出165个GMAH间质差异表达蛋白质,其中GMAH组织中表达上调者99个,下调者66个。它们涉及一些与肿瘤相关的信号通路,如p53信号通路、MAPK信号通路、细胞周期与凋亡等信号通路,且与细胞生长、增殖、凋亡和体液免疫应答等生物学过程有关。这些差异表达蛋白质,在STRING网络中呈现相互作用,两两间相互联系。 本文的研究提示,胃粘膜非典型增生微环境中存在S100A6和SOD3等蛋白质间的相互作用,它们通过影响p53信号通路、MAPK信号通路、细胞周期与凋亡等信号通路,在胃癌发病过程中发挥作用。     

Analysis of gene expression profiles of gastric normal and cancer tissues by SAGE

In an attempt to understand the molecular bases of gastric cancer progression, we have analyzed the differentially expressed genes in gastric cancer by SAGE. Four SAGE cDNA tag libraries were constructed from two sets of gastric cancer and normal tissues and 241,127 tags were obtained. By comparing the tags from cancer and normal tissues, 414 differentially expressed tags, representing 383 genes, were identified in cancer tissues (p 相似文献   

RTVP-1, a novel human gene with sequence similarity to genes of diverse species, is expressed in tumor cell lines of glial but not neuronal origin

A novel gene, RTVP-1, which shows significant sequence identity to the mammalian testis-specific proteins, a family of plant pathogenesis-related proteins and the vespid venom allergen, antigen-5, has been isolated from a cDNA library of the human glioblastoma brain tumor cell line, U-251 MG. The highest degree of sequence identity was with the human testis-specific protein, TPX1 (38.7% over 119 amino acids). Northern hybridization analysis revealed that in fetal tissue RTVP-1 RNA was detected only in the kidney, but its expression was ubiquitous in adult tissues including brain. Multiple mRNAs encoded by RTVP-1 were highly expressed in a panel of cell lines from nervous system tumors arising from glia, although expression was low or absent in non-glial-derived nervous system tumour cell lines. The GenBank DNA database accession number for this sequence is X91911.     

转化相关蛋白p86的鉴定

以转化细胞Rat3-3免疫大鼠,获得单克隆抗体E5(MeAb E5)。其对应抗原耐热,是分子量为86kD的蛋白质(P86)。它在c-Ha-ras癌基因转化的细胞上均有较高的阳性表达,而在其相应的非转化细胞上含量甚微。以人工合成的反义c-Ha-ras寡聚核苷酸As-Ⅱ处理Rat3-3细胞,该细胞生长受抑(44.2％),,此对p86的表达亦降低(39.1％)。P86在五种人胃癌细胞系中均有较高表达,在原发胃癌及其转移灶中阳性率达82.6％。p86不仅是一种与C-Ha-ras转化相关的蛋白,而且为胃癌相关抗原。