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Antitumor Effect of a Novel Adeno-associated Virus Vector Targeting to Telomerase Activity in Tumor cells

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 492-500页

作者： Yi-Gang WANG1,2#, Jin-Hui WANG1#, Yan-Hong zhang1, Qing GU2*, and Xin-Yuan LIU1* 1institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 200031, china School of Life Science, Nanchang University, Nanchang 330047, china institute of biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China School of Life Science Nanchang UniversityNanchang 330047 China

Telomerase activity is a wide tumor marker. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated exclusively in about 90% of cancer cells. In this study, we constructed a novel adeno-associated virus (AAV) vector containing the human interferon-β (hIFN-β) gene under the control of hTERT promoter (AAV-hTERT-hIFN-β) and investigated its antitumor effect against various human cancer cells in vitro. AAV-hTERT-hIFN-β displayed cancer-specific hIFN-β expression and cytotoxicity. The cytotoxic ratio was positively correlated with the time length of infection. AAV-hTERT-hIFN-β-mediated apoptotic morphology was observed by transmission electron microscopy. Flow cytometry assay also revealed that the cytotoxicity of AAV-hTERT-hIFN-β was mainly an apoptotic process. These data indicate that AAV in combination with hTERT-mediated therapeutic gene expression may open new possibilities for long-lasting and targeting gene therapy of varieties of cancers.

关键词： adeno-associated virus (AAV) telomerase hTERT promoter human interferon-β cancer

Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

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cell Research 2003年第5期13卷 369-374页

作者： HENG CHUAN XIA, FENG LI, ZHEN LI, Zu CHUAN zhang~* Key Laboratory of Proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 200031, china. State Key Laboratory of Reproductive biology Institute of Zoology Chinese Academy of SciencesBeijing 100080 China Department of Veterinary Pathobiology University of Missouri-Columbia MO 65211 USA

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin （Cucurbita moschata） has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of～29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

关键词： ribosome-inactivating protein (RIP) Moschatin immunotoxin.

The interaction between the human β-globin locus control region and nuclear matrix

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cell Research 2002年第Z2期 411-416页

作者： SHU BING zhang, Ruo LAN QIANState Key Laboratory of Molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 Yueyang Road, shanghai 200031, china

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express humanβ-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681- -10971 bp , HS3 core sequence -14991- -14716 bp and HS4 core sequence -18586- -18306 bp) of DNase I hypersensitive sites in the human β-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of humanβ-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cel

关键词： human β-globin LCR nuclear matrix proteins nuclear matrix HEL cells.

Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells

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Acta Pharmacologica Sinica 2006年第7期27卷 901-910页

作者： Ji-dong FU Hui-mei YU Rong WANG Ji LIANG Huang-tian YANG~2 Key Laboratory of Stem cell biology,institute of Health sciences,shanghai institutes for biological sciences,chinese academy of sciences and shanghai Jiao Tong University School of Medicine,shanghai 200025,china KeyLaboratoryofStemcellbiology InstituteofHealthSciencesShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesandShanghaiJiaoTongUniversitySchoolofMedicineShanghai200025China

Aim:To investigate the developmental regulation of intracellular Ca2+transients,an essential event in excitation-contraction coupling,during ***:Using the embryonic stem(ES)cell in vitro differentia-tion system and pharmacological intervention,we investigated the molecular andfunctional regulation of Ca2+handling proteins on the Ca2+transients at early,intermediate and later differentiation stages of ES cell-derived cardiomyocytes(ESCM).Results:Nifedipine,a selective antagonist of L-type Ca2+channels,totally blocked Ca2+transients even in the condition of field-electric stimulation inESCM at three differentiation *** Ca2+transients of ESCM were alsoinhibited by both ryanodine[an inhibitor of ryanodine receptors(RyRs)]and 2-aminoethoxydipheylborate[2-APB,an inhibitor of inositol-1,4,5-trisphosphatereceptors(IP3Rs)].The inhibitory effect of ryanodine increased with the time ofdifferentiation,while the effect of 2-APB decreased with the ***,an inhibitor of SR Ca2+-pump ATPase,inhibited Ca2+transientsequally at three differentiation stages that matched the expression ***~+free solution,which inhibits Na~+-Ca2+exchanger(NCX)to extrude Ca2+from cytosol,did not affect the amplitude of Ca2+transients of ESCM until the latter differentia-tion stage,but it significantly enhanced the basal Ca2+***:The Ca2+transients in ESCM depend on both the sarcolemmal Ca2+entry viaL-type Ca2+channels and the SR Ca2+release from RyRs and IP3Rs even at theearly differentiation stage;but NCX seems not to regulate the peak of Ca2+tran-sients until the latter differentiation stage.

关键词： Ca2+ transients cardiac differentiation embryonic stem cells Ca2+ handing proteins

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

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cell Research 2001年第2期11卷 89-94页

作者： LIAO JIN HUI, JUN SONG CHEN, MIN QIANG CHAI, SHENG ZHAO, JIAN GUO SONG State Key Laboratory of Molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 Yue Yang Road, Shan 1State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China

We reported in this manuscript that TGF-β1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-β1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-β1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-β1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-β1-mediated gene expression and apoptosis.

关键词： Transforming growth factor β apoptosis p38 hepatocyte signal transduction.

Neuroprotective effects of cyclooxygenase-2 inhibitor celecoxib against toxicity of LPS-stimulated macrophages toward motor neurons

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Acta Pharmacologica Sinica 2005年第8期26卷 952-958页

作者： Yong HUANG, Jing LIU, Li-zhen WANG, Wei-yu zhang, Xing-zu ZHU~1 Department of Pharmacology, shanghai institute of Materia Medica, shanghai institutes for biological sciences, chinese academy of sciences, Graduate School of chinese academy of sciences, shanghai 201203, china Department of Pharmacology Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of Chinese Academy of Sciences Shanghai 201203 China

Aim: To establish an in vitro injured motor neuronal model and investigate the neuroprotective effects and possible mechanism of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on this model. Methods: After macrophages were stimulated with lipopolysaccharide (LPS)+interferon-γ(IFN-γ) in the pre-sence or absence of celecoxib for 24 h, the cell-free supernatant of LPS-stimulated macrophages was transferred to the culture of NSC34 cells. Viability of NSC34 cells was assessed by MTT assay after a further 24 h and 72 h incubation. After macrophages were stimulated by LPS+IFN-γ for 12 h or 24h, the release of pros-taglandin E2(PGE2), nitric oxide (NO), reactive oxygen species (ROS), tumor necro-sis factor α(TNF-α) and interleukin-1β (IL-1β) from macrophages was measured by radioimmunoassay, Griess assay, fluorescence assay and enzyme-linked immunosorbent assay, respectively. The mRNA levels of COX-2, inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in macrophages were determined by reverse transcription-polymerase chain reaction after macrophages were stimu-lated for 6 h and 12 h. Results: The supernatant of LPS-stimulated mouse mac-rophages induced the death of NSC34 cells and celecoxib protected the NSC34cells against this toxicity. The LPS-induced increases in the release of PGE2, NO,TNF-α and IL-1β from macrophages were attenuated by pre-treatment with celecoxib. However, celecoxib showed no effect on the ROS levels upregulated by LPS+IFN-γin the macrophage supernatant. The mRNA levels of COX-2, iNOS, TNF-α and IL-1β were increased in LPS-activated macrophages and, except COX-2, reduced by pre-treatment with celecoxib. Conclusion: An in vitro injured motor neuronal model was established by using the toxicity of LPS-stimulated mouse macrophages toward motor neuronal NSC34 cells. In this model, celecoxib exerted neuroprotective effects on motor neurons via an inhibition of the neurotoxic secretions from activated macrophages.

关键词： amyotrophic lateral sclerosis celecoxib macrophage NSC34 cell lipopolysaccharide cyclooxygenase-2 prostaglandin E2 nitric oxide reactive oxygen species

Systemic delivery of full-length C/EBPβ / liposome complex suppresses growth of human colon cancer in nude mice

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cell Research 2005年第10期 24-30页

作者： Li SUN, Bei Bei FU, Ding Gan LIU* State Key Laboratory of Molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 200031, china

C/EBPβ (CCAAT/enhancer-binding protein β) is an important transcription factor involved in cellular proliferation and differentiation. Overexpression of the full-length C/EBPβ protein results in cellular growth arrest and apoptosis. Using a nonviral liposome as carrier, we delivered the full-length C/EBPβ expression plasmid, pCN, into nude mice bearing CW-2 human colon cancer tumors via tail vein. Southern blots revealed that the major organs and tumors were transfected. Experimental gene therapy showed that a strong suppression of tumor growth was observed in the pCN- treated mice, and such suppression was due to the overexpression of C/EBPβ, leading to the increased apoptosis in tumors of pCN-treated mice. No apparent toxic effects of pCN/liposome complex were observed in the animals. Thus, C/EBPβ has tumor suppression effect in vivo and may be used in gene therapy for cancers.

关键词： tumor supression C/EBPβ colon cancer apoptosis.

Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in Insect cells

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Acta Biochimica et Biophysica Sinica 2004年第11期36卷 754-758页

作者： Ai-Xia REN, You-Hua XIE, Yu-Ying KONG, Guan-Zhen YANG, Yao-Zhou zhang,Yuan WANG, and Xiang-Fu WUinstitute of biochemistry and Biotechnology, College of Life Science, Zhejiang University, Hangzhou 310029, china State Key Laboratory of Molecular biology, shanghai institute of biochemistry and cell biology, shanghai institutes for biological sciences,chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 200031, china institute of biochemistry, Zhejiang University of sciences, Hangzhou 310018, china institute of biochemistry and Biotechnology College of Life Science Zhejiang University Hangzhou 310029 China.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus

关键词： severe acute respiratory syndrome (SARS) SARS coronavirus nucleocapsid protein baculovirus insect cells

Identification and Characterization of GH Receptor and Serum GH-binding Protein in chinese Sturgeon (Acipenser sinensis)

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 811-816页

作者： Zhi-Yong LIAO1,2 and Shang-Quan ZHU2* 1College of Life sciences, Jiangxi Normal University, Nanchang 330027, china 2Key Laboratory of Proteomics biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 200031, china College of Life sciences Jiangxi Normal UniversityNanchang 330027 China Key Laboratory of Proteomics biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

chinese sturgeon, a kind of cartilage ganoid, has a history of over one billion years and it is called the living fossil of aquatic biology since it keeps some evolutionary trace. Here, we characterized the growth hormone receptor (GHR) and serum growth hormone binding protein (GHBP) of chinese sturgeon. It was shown that GHR was expressed in various tissues, mainly in hepatic, kidney and intestine tissues. GHR on the hepatic membrane has high and specific affinity for bream GH (brGH) and Scatchard analysis of the binding data showed a single class of high affinity binding site with an association constant Ka of 3.1×109 M–1. A specific band around 94 kD was detected by SDS-PAGE in cross-linking studies of membrane receptors. After incubation of chinese sturgeon serum with 125I-brGH, a 125I-brGH-GHBP complex was identified by Sephadex G-75, indicating that in the serum exists GHBP specially binding to brGH.

关键词： chinese sturgeon growth hormone receptor growth hormone binding protein ligand binding