Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

Tumor suppressor gene PTEN affects anoikis via both PKB and FAK in human lung carcinoma cell lines

PTEN was identified in 1997 as a new tumor suppressor gene on human chromosome 10q23, deletions and mutations of this gene were associated with a variety of human cancers such as glioblastoma and prostate cancer. In this study Northern blot analysis was carried out and one major band at 2 kb region was observed in all 8 HLCC samples, consistent with previous reports. The result showed that the PTEN gene were expressed and its mRNA level similar in all cell lines tested. To determine whether the PTEN mRNA level reflects the parallel level of protein, the level of PTEN protein was examined by Western blot.PTEN protein level was high in H460 and detectable in A549, A4, A7, L1 cells, not detectable in 95C, 95D, A1     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Apoptosis is a genetically controlled program of cellular self-destruction, which plays a key role in the development and homeostasis of virtually all animals. In recent years, Liu et al.[1] found a novel apoptosis-related gene TFAR19 (TF-1 cells apoptosis-related gene 19). It is conserved in evolution and distributed extensively in tissues. The related study showed that its coding protein is probably very important for the signal transduction in cell nucleus, regulating the cell proliferat…     

Expression and localization of VCX／Y proteins and their possible in-volvement in regulation of ribosome assembly during spermatogenesis

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromo-somes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST)fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated andthe localization of VCY protein in human testis was determined by immunohistochemistry. In the testisseminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium io-dide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainlylocalized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cellswas also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtainedusing yeast two-hybrid system. All the information above indicates that VCX/Y protein family might beinvolved in the regulation of ribosome assembly during spermatogenesis.     

cDNA expression array analysis of gene expression in human hepatocarcinoma Hep3B cells induced by BNIPL-1

Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 （BNIPL-1） is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology （BCH） domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16^INK4, IL-12, TRAIL and the lymphotoxin β gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway（s）.     

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly sug     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.     

TFAR19促进小鼠肝线粒体膜通透性转运孔的开放

TFAR19基因 (TF 1cellapoptosisrelatedgene 19)是北京大学人类疾病基因中心从人白血病细胞株TF 1细胞中克隆到的凋亡相关新基因之一 (GenBank登记号AF0 1495 5 )。初步研究发现 ,该基因在细胞凋亡时高表达 ,并且表达产物具有抑制肿瘤细胞生长和促进凋亡作用。但是其确切的作用机制不明。线粒体膜完整性破坏所导致促凋亡因子 (如细胞色素c等因子 )的释放是细胞凋亡关键性的控制因素。线粒体膜通透性转运孔 (PTP) ,对线粒体膜完整性具有重要的调控作用。研究了重组人TFAR19蛋白在体外条件下 ,对线粒体PTP、跨膜电位 ,以及细胞色素c释放的影响。结果表明 ,TFAR19蛋白使分离的小鼠肝线粒体PTP开放、线粒体跨膜电位下降 ,以及细胞色素c释放。TFAR19对线粒体的上述作用是通过促进PTP开放起作用的。实验结果提示 ,TFAR19对线粒体凋亡信号有正反馈放大作用 ,并进一步揭示了TFAR19促进细胞凋亡的机制     

Soluble expression in Escherichia coli,purification and characterization of a human TF-1 cell apoptosis-related protein TFAR19

A novel human TF-1 cell apoptosis-related protein, TFAR19, cloned from a human leukemia cell line, TF-1, was first overexpressed in Escherichia coli with the sequence Met-Gly-His(6)-Gly-Thr-Asn-Gly, a hexahistidine sequence followed by a hydroxylamine cleavage site attached to its amino terminus. The resulting protein was soluble and single-step purified to homogeneity by metal chelating affinity chromatography. After cleavage of the purified His(6)-tagged TFAR19 sample with hydroxylamine, highly purified untagged TFAR19 protein was then obtained through an FPLC Resource Q column. The structural characteristics and function of the His(6)-tagged and untagged TFAR19 proteins were studied using circular dichroism, intrinsic fluorescence, and ANS-binding fluorescence spectra and apoptosis activity assay. The results show that alpha-helix is the main secondary structure of the proteins and the two forms of TFAR19 protein fold properly, which correspond well to their apoptosis activity expression. The results also indicate that the extra sequence including the His(6)-tag fused to the N-terminus of TFAR19 protein has a minimal effect on its structure and function, suggesting that the His(6)-tagged TFAR19 protein could be further used as an immobilized target for finding potential proteins which interact with TFAR19 from a cDNA library using in vitro ribosome display technique.     

PDCD10，一个新的多功能信号转导调节分子

人程序性细胞死亡分子10(Homo sapiens programmed cell death 10, PDCD10)，最初被称为TFAR15(TF-1 cell apoptosis related gene 15)，是由撤除粒细胞 巨噬细胞集落刺激因子诱导凋亡的人红白血病细胞系TF-1中克隆得到的1个凋亡相关基因. 后来发现它的突变可引起散发性或家族性颅内海绵状血管瘤(cerebral cavernous malformations，CCMs)的发生，为CCMs的第3个致病基因，所以又被叫做CCM3.近年来研究发现,PDCD10能够和GCKⅢ蛋白、γ-PCDH、CCM2、VEGFR2、ERM等众多蛋白相互作用，并能调控ERK/MAPK通路，增加MST4/VEGFR2的稳定性，增强相应的信号转导，促进细胞的增殖、分化和中枢神经系统的发育，与癌症的发生相关，还能调节细胞的凋亡.以上研究证明了PDCD10 的多种生物学效应，并提示其在血管生成、氧化应激、肿瘤中发挥重要作用.     

粒细胞-巨噬细胞集落刺激因子/白细胞介素-3融合蛋白的表达研究

利用PCR扩增得到粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)完整基因片段,将其分别克隆至pGEM-T,构建成GMCSF/IL-3融合蛋白基因,DNA序列与设计预期一致。将得到的融合蛋白基因克隆至T7RNA聚合酶表达载体pT7zz,得到表达质粒pFu,经转化至表达宿主E.coli BL21(DE3),在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS-PAGE电泳鉴定扫描分析,目的基因产物表达量占菌体总蛋白量的30%以上,目的基因表达产物以包涵体的形式表达。Westernblot鉴定表明,该表达产物可以与GM-CSF抗体及IL-3抗体特异性结合。目的基因表达产物经过包涵体变性、透析复性及柱层析纯化,用GM-CSF、IL-3依赖细胞株TF-1检测,具有明显的生物学活性。     

人重组GM－CSF／HAS－D3嵌合蛋白（GM－HSA）在大肠杆菌中的表达及其产物稳定性的研究

将人粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人血清白蛋白第三功能区（ＨＡＳ－Ｄ３）的基因串联后,在Ｅ．ｃｏｌｉ中获高效表达,表达量占菌体蛋白的３２．６％．利用ＴＦ－１体外细胞活性测定表明,ＧＭ－ＨＳＡ的活性单位为１．０４×１０~６Ｕ／ｍｇ,虽然其比活性低于ＧＭ－ＣＳＦ,但比后者具有更高的体外热稳定性和储藏稳定性．     

Inhibition of GM-CSF/IL-3/IL-5 signaling by antisense oligodeoxynucleotides targeting the common beta chain of their receptors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 play a key role in allergic inflammation. They mediate their effect via receptors that consist of two distinct subunits, a cytokine-specific alpha subunit and a common beta subunit (betac) that transduces cell signaling. We sought to down-regulate the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Experiments were performed with TF-1 cells (a human erythroleukemia cell line expressing GM-CSF, IL-3, and IL-5 receptors, which proliferates in response to these cytokines), monocytic U937 cells, which require these cytokines for differentiation, and purified human eosinophils. Cells were treated with antisense phosphorothioate oligodeoxynucleotides (ODN) targeting betac mRNA. In contrast to nontreated cells and cells treated by sense or mismatched ODN, antisense ODN inhibited betac mRNA expression and significantly decreased the level of cell surface betac protein expression on TF-1 and U937 cells. Receptor function was also affected. Antisense ODN were able to inhibit TF-1 cell proliferation in vitro in the presence of GM-CSF, IL-3, or IL-5 in the culture medium and eosinophil survival. We suggest that antisense ODN against betac may provide a new therapeutic alternative for the treatment of neoplastic or allergic diseases associated with eosinophilic inflammation.     

PTEN is indispensable for cells to respond to MAPK inhibitors in myeloid leukemia

Constitutively activated MAPK and AKT signaling pathways are often found in solid tumors and leukemias. PTEN is one of the tumor suppressors that are frequently found deficient in patients with late-stage cancers or leukemias. In this study we demonstrate that a MAPK inhibitor, PD98059, inhibits both AKT and ERK phosphorylation in a human myeloid leukemia cell line (TF-1), but not in PTEN-deficient leukemia cells (TF-1a). Ectopic expression of wild-type PTEN in myeloid leukemia cells restored cytokine responsiveness at physiological concentrations of GM-CSF (<0.02 ng/mL) and significantly improved cell sensitivity to MAPK inhibitor. We also found that Early Growth Response 1 (EGR1) was constitutively over-expressed in cytokine-independent TF-1a cells, and ectopic expression of PTEN down-regulated EGR1 expression and restored dynamics of EGR1 expression in response to GM-CSF stimulation. Data from primary bone marrow cells from mice with Pten deletion further supports that PTEN is indispensible for myeloid leukemia cells in response to MAPK inhibitors. Finally, We demonstrate that the absence of EGR1 expression dynamics in response to GM-CSF stimulation is one of the mechanisms underlying drug resistance to MAPK inhibitors in leukemia cells with PTEN deficiency. Our data suggest a novel mechanism of PTEN in regulating expression of EGR1 in hematopoietic cells in response to cytokine stimulation. In conclusion, this study demonstrates that PTEN is dispensable for myeloid leukemia cells in response to MAPK inhibitors, and PTEN regulates EGR1 expression and contributes to the cytokine sensitivity in leukemia cells.     

GM-CSF rescues TF-1 cells from growth factor withdrawal-induced, but not differentiation-induced apoptosis: the role of BCL-2 and MCL-1.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 promote the survival and stimulate the proliferation of haematopoietic cells. Using the GM-CSF-dependent TF-1 myeloid leukaemia cell line, the authors show that the endogenous levels of BCL-2 and MCL-1 are downregulated upon GM-CSF withdrawal, whereas the levels of BCL-x(L)and Bax are unchanged. Re-exposure of growth factor deprived cells to GM-CSF resulted in an early and transient increase in MCL-1 expression, and prolonged induction of BCL-2, which prevented apoptosis. In contrast, the expression of BCL-2 and MCL-1 were not modulated during TPA-induced differentiation of TF-1 cells, which was followed by apoptosis despite the presence of GM-CSF. TF-1 cells overexpressing BCL-2 or MCL-1 underwent delayed apoptosis upon growth factor withdrawal, but displayed no impaired apoptosis in response to TPA. Erythropoietin (Epo) induced the expression of BCL-2 and MCL-1 protein in TF-1 cells, however it did not support their long term proliferation, further demonstrating that upregulation of these anti-apoptotic genes is insufficient for the long term proliferation of TF-1 cells.