Cloning and characterization of F-LANa, upregulated in human liver cancer

Differentially expressed genes between normal liver and hepatocellular carcinomas were investigated using differential display. Consequently, we identified a fragment cDNA upregulated in tumor tissues. We screened the liver library and cloned the full-length cDNA, named F-LANa. Increased expression of F-LANa was confirmed by Northern blot analysis in 10 of 14 (71%) cases of hepatocellular carcinomas. Human F-LANa gene maps to chromosome 17p at D17S1828-D17S786, spans at least 11.8 kb, and contains 7 exons. This gene encodes a 239 aa protein exhibiting 97.9% similarity to the mouse ortholog gene, identified later by in silico cloning. Homology analysis was carried out in various species and showed that F-LANa was evolutionarily conserved from yeast to human. In addition, F-LANa antisense oligonucleotide suppressed F-LANa expression in human hepatocellular carcinoma BEL-7404 cells and significantly inhibited cell growth. Together, our data demonstrate that overexpression of evolutionarily conserved F-LANa occurs frequently and may play an important role in proliferation.     

Haymaker gene expression in malignant and normal gynecologic tissues.

We previously reported that cell lines established from human carcinomas and leukemias/lymphomas expressed high levels of an intracellular membrane-bound protein, Haymaker, whereas cell lines derived from non-malignant connective tissue cells and lymphoid cells expressed low levels of this gene product. To determine whether these findings reflect neoplastic transformation or, alternatively, tissue specificity, we examined by immunohistochemical and molecular methods the expression of Haymaker in gynecologic organs with and without tumor. A highly specific, affinity-purified rabbit polyclonal antibody against a 25-mer Haymaker peptide was used for immunohistochemical staining and morphometric analysis of 85 tissue specimens. Immunohistochemical studies demonstrate, for the first time, that Haymaker protein is highly expressed in epithelial cells of the endometrium of the normal uterus and to a somewhat lesser extent in the mucosa of the normal vagina and cervix, but is poorly expressed or absent in cells of the connective tissue and smooth muscle strata of these organs (p < 0.005). Significant differences in Haymaker expression, as assessed by immunohistochemistry, between malignant and normal gynecologic tissues were not observed (p = 0.27). The expression of Haymaker protein does not appear, therefore, to be a marker of malignant transformation of the epithelium of gynecologic organs but rather distinguishes both normal and malignant epithelial cells from normal connective tissue and smooth muscle cells.     

Identification of a cDNA Coding for a Ca-Binding Phosphoprotein (p90), Calnexin, on Melanosomes in Normal and Malignant Human Melanocytes

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca²⁺-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in λ Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M_r 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with β-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca²⁺-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an anti-body against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.     

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.     

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.     

Cellular retinaldehyde-binding protein-like (CRALBPL), a novel human Sec14p-like gene that is upregulated in human hepatocellular carcinomas, may be used as a marker for human hepatocellular carcinomas

Sec14p-like lipid-binding domain (SEC14 domain) is an evolutionarily conserved protein domain often found in secretory proteins, such as Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p, and in lipid-regulated proteins, such as GTPase-activating proteins, guanine nucleotide exchange factors, and neurofibromin. We have cloned a novel human gene, cellular retinaldehyde-binding protein-like (CRALBPL), containing SEC14 domain from the cDNA library of human fetal brain. The RT-PCR expression pattern of 16 adult human tissues indicated that CRALBPL was only expressed in brain, while it was expressed in all of seven human carcinoma cell lines we used, especially in human gastric adenocarcinoma cell line, human rhabdomyoma cell line, human hepatocellular carcinoma (HCC) cell line, and human prostatic carcinoma cell line. Further, we found that CRALBPL has a remarkably more abundant RT-PCR expression pattern in human HCC cell lines than in normal human liver cell line, and the same result was gained when RT-PCR expression patterns between human HCC specimens and normal human liver specimens were compared. We also found that CRALBPL is located mainly in cytoplasm in human liver cell line L-02, which is consistent with the common function of Sec14p-like domain family. Our results show that CRALBPL may be used as a marker for human HCCs.     

蛋白激酶底物p36在肝硬变、肝细胞肝癌中的表达及与HBV、HCV感染的关系

本文应用鼠抗蛋白激酶底物ｐ３６单克隆抗体,采用免疫组织化学法对ｐ３６在５４例肝硬变,７９例肝细胞肝癌中的表达分布进行了研究,同时结合ＨＢＶ、ＨＣＶ感染情况分析其相互关系,结果显示：ｐ３６在肝硬变及肝细胞肝癌中定位于肝细胞或癌细胞胞浆内,在胞浆内弥漫分布,阳性细胞呈灶状或弥漫分布,部分病例癌周肝细胞信号较癌组织为强,ｐ３６在肝硬变、肝细胞癌中的阳性率分别为８８．８％（４８／５４）及８２．３（６５／７９）,ＨＢｘＡｇ在两种组织的阳性率分别为７０．４％及７６％,ＨＣＶ核心抗原在两种组织的阳性率分别为８０％及７８．５％;三者同时阳性分别为５５．５％及５８．２％;ｐ３６、ＨＢｘＡｇ同时阳性分别为６８．５％及６４．５％;ｐ３６、核心抗原同时阳性分别为７４．１％及７０．８％,我们的结果提示,肝硬变、肝细胞肝癌组织中ｐ３６存在高表达,其高表达可能与ＨＢＶ、ＨＣＶ感染密切相关     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Molecular cloning and nucleotide sequence analysis of mRNA for human kidney ornithine aminotransferase. An examination of ornithine aminotransferase isozymes between liver and kidney

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3'- and 5'-untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N-terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln-35 and Gly-36.     

中国启东肝癌高发区肝癌中AKR1C2基因异常表达及其在肝癌发生中作用(英文)

已在许多肿瘤中发现AKR1C2基因的异常表达 .为研究启东肝癌中AKR1C2基因异常表达的意义及其在肝癌发生中作用 .通过制备兔抗人AKR1C2多克隆抗体、免疫组化、蛋白质印迹、RT PCR、RNA印迹、原位杂交、cDNA表达芯片、免疫共沉淀、体内外致瘤试验等方法 ,对 68例启东肝癌标本、 8例正常肝组织、QGY 770 3启东肝癌细胞株中AKR1C2表达及作用进行分析 .并研究了AKR 1C2蛋白、mRNA表达与肝癌临床病理特征 ,侵袭性间关系 .研究表明正常及癌旁肝组织中AKR1C2蛋白为膜染色 ,偶见弱的细胞浆染色 .95 3 %肝癌显示胞浆或核染的累积型 .癌及癌旁肝组织中标记指数(LI)分别为 61 4± 2 7 8,10 2± 8 7(P <0 . 0 1) .较高的LI与HCC侵袭性密切相关 .蛋白质印迹显示癌组织中AKR1C2表达升高 .RT PCR显示 ,肝癌中AKR1C2表达指数 (EI)高于癌旁及正常组织 ,而且存在序列差异 .RNA印迹显示 91 2 %为上调表达 .原位杂交显示肝癌细胞胞浆中染色强于癌旁及正常肝 .AKR1C2过表达与肝癌转移潜能有关 .AKR1C2过表达刺激QGY770 3细胞中DNA合成与阻止细胞凋亡 .转染AKR1C2基因的QGY770 3细胞在软琼脂上集落形成能力增强 ,并能促进QGY770 3在裸鼠体内肿瘤形成能力 .cDNA表达芯片显示转染AKR1C2后导致QGY770 3细胞中一些基因表达改变 .AKR     

Rat ceruloplasmin. Molecular cloning and gene expression in liver, choroid plexus, yolk sac, placenta, and testis

A rat ceruloplasmin cDNA clone was isolated from a rat liver cDNA library and identified by partial nucleotide sequence analysis. Rat liver ceruloplasmin mRNA levels were measured during the acute phase response to inflammation by cytoplasmic dot hybridization to ceruloplasmin cDNA. Regulation of ceruloplasmin synthesis appeared to be at the mRNA level, with the concentration of ceruloplasmin mRNA increasing significantly 12 h after induction of inflammation, reaching a maximum of 350% of normal at 36 h and returning to normal levels within 60 h. Using Northern blot analysis, extrahepatic ceruloplasmin gene expression was observed in choroid plexus, yolk sac, placenta, and testis. All these tissues are at the interface between, and possibly involved in maintaining homeostasis in, adjacent extracellular compartments. No ceruloplasmin mRNA was detected in RNA from stomach and small intestine.     

HCRP1, a novel gene that is downregulated in hepatocellular carcinoma, encodes a growth-inhibitory protein

One of the most frequent allelic deletions in hepatocellular carcinoma (HCC) has been found at chromosome 8p21-23. We reported here the identification and characterization of a novel gene for a hepatocellular carcinoma related protein 1 (HCRP1) localized at 8p22, which was isolated by positional candidate cloning. The expression of the gene for HCRP1 was most abundant in normal human liver tissue and significantly reduced or undetected in HCC tissues. The analysis of subcellular distribution showed that HCRP1 diffused in the cytoplasm with a significant fraction accumulated in the nuclei. After introduction of the sense and antisense cDNA of HCRP1 into HCC cell line SMMC-7721, we observed that the overexpression of HCRP1 significantly inhibited both anchorage-dependent and anchorage-independent cell growth in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of HCRP1 gene in the other HCC cell line BEL-7404 resulted in the cell growth greatly enhanced. Moreover, reduction of the HCRP1 gene expression could also elevate the invasive ability of BEL-7404 cells. Our results strongly suggest that HCRP1 might be a growth inhibitory protein and associated with decreasing the invasion of HCC cells.