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Effects of Huangqi (Hex) on Inducing cell Differentiation and cell Death in K562 and HEL cells

Acta Biochimica et Biophysica Sinica 2004年第3期36卷 211-217页

作者： Xiao-Dong CHENG1, Chun-Hui HOU1, Xue-Jun ZHANG2, Heng-Yue XIE1, Wei-Ying ZHOU1, Lei YANG2, Shu-Bing ZHANG1, and Ruo-Lan QIAN1 State Key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, the chinese academy of sciences, 320 yueyang road, shanghai 200031, china laboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, the chinese academy of sciences,320 yueyang road shanghai 200031,china the chinese academy of sciences, 320 yueyang road, shanghai 200031, china State Key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Huangqi (Astragalus membranaceus), a traditional chinese medicine, has been used to ameliorate side effects of cancer chemotherapy in china. However, little is known about its molecular mechanisms. Here we show that induction of K562 or HEL cells with 1.5 mg/ml of Huangqi (Hex) (Components extracted from Huangqi) for 3–5 d results in the expression of β-globin gene in both cell lines and leads to terminal differentiation. Moreover, the apoptosis in HEL cells can be induced by increasing concentration of Huangqi (Hex) to 4.5 mg/ml for 3–5 d. Upregulation of Apaf-1, caspase-3 and acetylcholinesterase (AChE) in HEL cells may play a crucial role in the process of apoptosis. The prospect of inducing expression of adult (β) globin gene and apoptosis selectively in cancer cells is obviously attractive from a therapeutic point of view.

关键词： cell death and differentiation Huangqi (Hex) human erythroleukemia cell lines

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Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

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cell Research 2004年第3期14卷 227-233页

作者： Yan REN, Jia Rui WU laboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 yueyang road, shanghai 200031, china. laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chkl checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly dete

关键词： intra-S-phase checkpoint DNA replication DNA damage tripchlorolide.

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Cinnamomin——a Versatile Type Ⅱ Ribosome-inactivating Protein

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 169-176页

作者： Hong XU and Wang-Yi LIU State Key laboratory of molecular biology, the institute of biochemistry and cell biology, shanghai institutes for biological sciences, the chinese academy of sciences, 320 yueyang road, shanghai 200031, china State Key laboratory of molecular biology the Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

关键词： cinnamomin cinphorin ribosome-inactivating protein

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Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies

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cell Research 2001年第2期11卷 85-88页

作者： GENG JIAN Guo institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 Yue Yang road, shanghai 200031, china institute of biochemistry and cell biology Chinese Academy of Sciences Shanghai. jggeng@***

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury the constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial present tation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.

关键词： Directional cell migration leukocytes inflammation therapy.

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Induction of mitochondrion-mediated apoptosis of CHO cells by tripchloro lide

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cell Research 2003年第4期13卷 295-300页

作者： YAN REN, LEI XIONG, JIA RUI WUlaboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 Yue Yang road, shanghai 200031, china 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China

Tripchlorolide (TC) is a potent antitumor reagent purified from a chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TC treated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.

关键词： apoptosis mitochondrial pathway CHO cell.

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Mitosin/CENP-F is a conserved kinetochore protein subjected to cyto plasmic dynein-mediated poleward transport

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cell Research 2003年第4期13卷 275-283页

作者： ZHEN YE YANG, JING GUO, NING LI, MIN QIAN, SHENG NIAN WANG, XUE LIANG ZHUlaboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 yueyang road, shanghai 200031, china 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China 1laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Mitosin/CENP-F is a human nuclear protein transiently associated with the outer kinetochore plate in M phase and is involved in M phase progression. LEK1 and CMF1, which are its murine and chicken orthologs, however, are implicated in muscle differentiation and reportedly not distributed at kinetochores. We therefore conducted several assays to clarify this issue. The typical centromere staining patterns were observed in mitotic cells from both human primary culture and murine, canine, and mink cell lines. A C-terminal portion of LEK1 also conferred centromere localization. Our analysis further suggests conserved kinetochore localization of mammalian mitosin orthologs. Moreover, mitosin was associated preferentially with kinetochores of unaligned chromosomes. It was also constantly transported from kinetochores to spindle poles by cytoplasmic dynein. These properties resemble those of other kinetochore proteins important for the spindle checkpoint, thus implying a role of mitosin in this checkpoint. The

关键词： mitosin LEKl CENP-F kinetochore.

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Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 673-680页

作者： Chun-Hui HOU, Jian HUANG, and Ruo-Lan QIAN* State Key laboratory of molecular biology, 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 State Key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences

The developmental control of the human ε-globin gene expression is mediated by transcrip- tional regulatory elements in the 5’ flanking DNA of this gene. A previously identified negative regulatory element (–3028 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at –3004 bp, –2975 bp and –2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (–177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at –2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at –2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at –2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at –2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε- NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

关键词： ε-globin gene regulatory element NF-κB GATA

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An Effective Method for Raising Antisera Against β-defensins: Double-copy Protein Expression of mBin1b in E. coli

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 571-576页

作者： Li-Qing XIAO, Ai-Hua LIU, and Yong-Lian ZHANG State Key laboratory of molecular biology, 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 State Key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences

Bin1b is a rat epididymis specific β-defensin which may have fertility related functions in addition to its antimicrobial activity. β-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new β-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of β-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the β-defensin gene family.

关键词： Bin1b epididymis β-defensin double-copy protein polyclonal antiserum

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Regulation of mammalian pre-mRNA splicing

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Science china(Life sciences) 2009年第3期52卷 253-260页

作者： HUI JingYi State Key laboratory of molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china 1. State Key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) *** splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct *** genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene ***-regulation of splicing causes a wide range of human *** review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA *** also discusses emerging directions in the field of alternative splicing.

关键词： pre-mRNA splicing alternative splicing splicing regulation signal transduction microRNA

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The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv

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cell Research 2002年第1期12卷 47-54页

作者： LIANG CHEN, GANG LI, LEI TANG, JUE WANG, XI, RUI GE laboratory of cellular Immunology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 320 yueyang road, shanghai 200031, china laboratory of cellular Immunology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences.

A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A -1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

关键词： LC-1 LC-1 ScFv SPC-A-1 pDisplay lung cancer.

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