Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs. Project supported in part by the National Natural Science Foundation of China (Grant Nos. 39780019, 39392903) and the Shanghai Life Sciences Research Centre.     

A modified hepatitis B surface antigen carrying both preS1 and preS2 epitopes

ＴｈｅｅｎｖｅｌｏｐｅｐｒｏｔｅｉｎｏｆｈｅｐａｔｉｔｉｓＢｖｉｒｕｓ(ＨＢＶ)ｃｏｎｓｉｓｔｓｏｆｔｈｒｅｅｐｒｏｔｅｉｎｓ:ｓｍａｌｌ(Ｓ),ｍｉｄｄｌｅ(Ｍ)ａｎｄｌａｒｇｅ(Ｌ)[1].ＴｈｅＳｐｒｏｔｅｉｎｃａｒｒｉｅｓａｌｌｔｈｅｉｎｆｏｒｍａｔｉｏｎｒｅｑｕｉｒｅｄｆｏｒｃｅｌｌｕｌａｒｌｉｐｉｄｓｍｏｂｉｌｉｚａｔｉｏｎ,ｓｕｂｖｉｒａｌｐａｒｔｉｃｌｅｆｏｒｍａｔｉｏｎａｎｄｓｅｃｒｅｔｉｏｎ.Ｉｔｈａｓｂｅｅｎｓｕｃｃｅｓｓｆｕｌｌｙｄｅｖｅｌｏｐｅｄａｓａｃａｒｒｉｅｒｔｏｅｘｐｒｅｓｓｆ…     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector. Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission.     

Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter

In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the alpha-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFE0.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LNAFE0.3TK-infected cells. Consistent with this, LN[AFE0.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.     

Transient expression of hepatitis B virus surface antigen (HBsAg) and human erythropoietin (hEPO) genes in milk after direct introduction into ewe

Ｅｘｐｒｅｓｓｉｏｎｏｆｍｉｌｋｐｒｏｔｅｉｎｇｅｎｅｓｉｓｉｎｖｏｌｖｅｄｉｎａｈｕｇｅｎｅｔｗｏｒｋｏｆｒｅｇｕｌａｔｏｒｙｃｉｒｃｕｉｔｓｗｈｉｃｈａｒｅｌｉｎｋｅｄｔｏｔｈｅｉｎｔａｃｔｄｅｖｅｌｏｐｉｎｇｍａｍｍａｒｙｇｌａｎｄ,ａｎｄｈｏｍｅｏｓｔａｓｉｓｄｕｒｉｎｇｐｕｂｅｒｔｙ,ｐｒｅｇｎａｎｃｙ,ｌａｃｔａｔｉｏｎａｎｄｉｎｖｏｌｕｔｉｏｎ.Ａｎａｌｙｓｉｓｏｆｐｕｔａｔｉｖｅｒｅｇｕｌａｔｏｒｙｅｌｅｍｅｎｔｓａｎｄｈｙｂｒｉｄｇｅｎｅｉｎｔｉｓｓｕｅｃｕｌｔｕｒｅｓｙｓｔｅｍｓ…     

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.     

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid^tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.     

Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.     

Methylation of the alpha-fetoprotein gene in isogenic rat hepatoma and liver cell lines

We have found that DNA methylation is inversely correlated with alpha-fetoprotein (AFP) gene expression in a series of isogenic rat hepatoma cell lines. The 5' end of the gene is extensively demethylated in AFP-producing cells and is highly methylated in cell lines which do not produce AFP. Glucocorticoid affects markedly the synthesis of AFP in the hepatoma cells. However, methylation patterns of cell lines which were treated with dexamethasone were not different from those of control cells, indicating that glucocorticoid action on AFP gene expression does not alter DNA methylation in this region of the gene.     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

人IL-2/IFNα2b融合基因在肝癌细胞中靶向表达

 根据细胞因子之间协同作用的特点,采用重组 Ｄ Ｎ Ａ 技术设计并构建了人 Ｉ Ｌ ２ 与 Ｉ Ｆ Ｎα融合基因,并用肝癌组织特异的 Ａ Ｆ Ｐ增强子／ Ａ Ｌ Ｂ启动子调控融合基因在肝癌细胞中的靶向表达．实验结果表明,克隆的 Ｅ Ａ Ｆ Ｐ Ｐ Ａ Ｌ Ｂ联合转录调控序列能调控细胞因子基因在 Ａ Ｆ Ｐ阳性人肝癌细胞中靶向表达, Ｉ Ｌ ２／ Ｉ Ｆ Ｎα２ｂ 融合基因的表达水平与感染肝癌细胞的 Ａ Ｆ Ｐ表达水平呈正相关性．实验证明表达的融合蛋白具有 Ｉ Ｌ ２ 和 Ｉ Ｆ Ｎ 两种生物学活性的细胞因子．这可能为肝癌基因治疗开辟新途径．     

癌基因及癌发育基因在裸小鼠人肝癌移植瘤中表达

本文采用Northern blot,斑点杂交,western Immunoblot,凝集素亲和电泳及聚丙烯酰胺梯度电泳方法分别检测裸小鼠肝癌移植瘤中C-ras基因簇(N-ras,H-ras及K-ras),甲种胎儿蛋白以及γ谷氨酰转肽酶(GGT)基因及其产物的表达。结果提示C-ras基因簇均有不同程度的表达,其中N-ras基因表达水平要比H-ras高8倍,比K-ras高20倍左右,同样AFP及GGT基因均有较高水平的表达。经ras-p21单克隆抗体检测,发现肝癌组织抽提液中p21呈现阳性反应;AFP亚型分析提示AFP分子变异体属肝癌特异性分子亚型;GGT同Ⅰ酶酶谱分析表明组织中仅合成肝癌特异性GGT同Ⅰ酶酶谱(Ⅰ’,Ⅱ’)。上述结果充分表明癌基因及癌发育基因的高表达可能与癌细胞去分化性,分裂旺盛程度及细胞恶性度相关。最后文内还对裸小鼠人肝癌移植瘤中二类基因的相关性作一定的讨论。     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Cloning and biological activity of an anti-tumor peptide of Tumstatin

To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203 amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction, the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC). The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide has the potential to become a novel, potent anti-tumor agent. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报]     

Alpha‐fetoprotein accelerates the progression of hepatocellular carcinoma by promoting Bcl‐2 gene expression through an RA‐RAR signalling pathway

Previous studies have found that alpha‐fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA‐RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA‐induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl‐2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down‐regulation of Bcl‐2; the opposite effect was observed in AFP gene‐transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non‐cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl‐2. Our data reveal a novel mechanism through which AFP regulates Bcl‐2 expression and further suggest that AFP may be used as a novel target for treating HCC.