人前列腺癌细胞系PC-3中PTI-1 cDNA的克隆及序列分析

为获取人前列腺癌诱导基因家族新成员 ,以人前列腺癌细胞系PC 3的总RNA为模板 ,用RT PCR方法扩增人前列腺癌诱导基因 1(PTI 1) ,将其亚克隆至克隆载体pUC19,进行测序 .将克隆得到的PTI 1基因片段与国外报道的人PTI 1基因编码区cDNA同源性为 99.3% ,但第 72 6、74 6、116 7、12 70、132 6、144 9、16 94和 16 95位碱基分别由A、A、C、C、A、A、T和C替代了文献中的C、C、G、G、G、C、C和T .其中 6个碱基的不同 ,导致了第 36、183、2 17、2 36、2 77和 35 9位密码子代表的氨基酸分别由Lys替代Gln ,Arg替代Gly ,Ala替代Gly ,Ser替代Gly ,Thr替代Pro和Arg替代Cys .将所获得的基因在GenBank登录 ,登录号为AF3974 0 3.结果提示 ,获得了PTI 1家族新成员 ,并且与EF 1α分子的同源性较已报道的PTI 1高 .     

兔防御素(MCP-1)cDNA的克隆与序列分析

从兔脾脏细胞中分离提取总RNA,经反转录PCP(RT-PCR)扩增出兔巨嗜细胞阳离子多肽(MCP-1)cDNA,插入经EcoRI和XbaI双酶切的pUC19中,构建了克隆质粒pUCDEF.进行了限制性酶切鉴定和序列分析,结果在扩增出的cDNA288个碱基中,在前片段中有一个碱基与发表的兔MCP-1cDNA序列不同,即第157位碱基由G变为A,导致编码的氨基酸由丙氨酸变为苏氨酸.该cDNA全长288bp,编码94个氨基酸,由编码信号肽,前片段及成熟肽片段的序列组成.     

牛生长激素cDNA的分子克隆

我们克隆了与牛生长激素Poly(A)⁺RNA互补的DNA(cDNA)。首先从小牛垂体中提纯总的Pply(A)⁺RNA,用AMV逆转录酶合成单链cDNA,以单链cDNA为模板合成双链cDNA,用多聚G及多聚c谱尾法将双链cDNA克隆到pBR322质粒的Pst I位点上,构建成牛垂体PoIy(A)⁺RNA的cDNA文库。以牛生长激素基因为探针,筛选出7个阳性菌落,经电泳鉴定有两个菌落(1号和2号)含有大于500bp的插入片段。1号克隆经酶切图谱、southeTn blot 杂交及序列分析证实含有牛生长激素的编码序列。     

人乳铁蛋白cDNA的克隆及序列分析

从北京正常人乳腺组织中提取总RNA,用RT-PCR的方法扩增人乳铁蛋白(hLF)的cDNA,将其克隆到pGEM-T载体上并进行DNA序列测定。结果表明,所克隆的hLF cDNA序列全长为2136bp,其DNA序列与GenBank中另外5个hLF cDNA序列相比,有2个碱基与这5个序列不同：1740位这5个序列是G,本文序列是C;1756位这5个序列是T,本文序列是C。其中1740位碱基的变化导致了第580位氨基酸由Glu变为Asp。     

人和小鼠resistin基因的克隆与序列测定

构建人和小鼠resistin基因cDNA克隆,并进行序列分析,为进一步进行resistin表达和生物学活性研究奠定基础。用RT－PCR方法扩增人和小鼠resistin基因cDNA,获得的片段边疆至pGEM-T载体,转化大肠杆菌JM109,并经过敏过鉴定和序列测定。结果成功地构建了人和小鼠resistin基因cDNA克隆,人和小鼠resistin的氨基酸序列具有共同的结构特征CX28CX12CX8CXCX3CX10CXCXCX9CC,克隆的人resistin cDNA在编码序列第133位的A被T代替,导致45位密码子编码的丝氨酸由半胱氨酸代替,小鼠resistin cDNA在第16位的T被C取代,导致第6位密码子编码的苯丙氨酸改变为亮氨酸。密码子发生改变的生物学意义还有待于进一步研究。     

人TRAIL基因cDNA的克隆与序列测定

为研究人TRAIL的基因组结构,生物学性能和用于肿瘤生物治疗的可能性,利用反转录聚合酶链反应(RT-PCR)从人急性早幼粒白血病细胞系HL-60细胞总RNA中扩增出人TRAIL基因编码区cDNA序列,将其克隆至pGEM-T载体中,序列测定表明,克隆片段与文献报道的人TRAIL基因编码区cDNA序列完全一致.     

人凝血因子Ⅶ cDNA基因的克隆与鉴定

目的:克隆并鉴定人凝血因子ⅦcDNA基因。方法:采用反转录聚合酶链反应(RT-PCR)的方法,从人胎肝总RNA中扩增人凝血因子ⅦcDNA基因,将其克隆入pGEM-T载体,对阳性克隆进行序列测定和分析。结果:经RT-PCR扩增和克隆,获得了人凝血因子ⅦcDNA基因,经序列分析表明,所克隆的基因序列正确。结论:本试验成功克隆了人凝血因子ⅦcDNA基因,为重组人凝血因子Ⅶ的研究奠定了基础。     

一种新的人端粒相关锌指蛋白cDNA的克隆及鉴定

为了从早期胚胎寻找与发育分化有关的新基因,本文构建了3周龄人胚cDNA文库,并应用EST技术对该文库中随机挑选的47个低丰度克隆进行测序,结果发现了一个与人亚端粒DNA和锌指基因同源的cDNA克隆(L30),该基因长约3.8kb,5＇端序列有明显的阅读框架(ORF),3＇端序列有加尾信号(AAUAGA)和有39个A组成的Poly(A)尾巴;通过Northern杂交确认在早期人胚胎中有表达,应用地高辛染色体原位杂交技术将其定位于人第12号染色体长臂端部.     

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.     

The cleavage of the urokinase receptor regulates its multiple functions

The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88-92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88-92 (P88-92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88-92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88-92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88-92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions.     

alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)- dependent internalization of the urokinase receptor

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR- associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.     

cDNA transfection of amino-terminal fragment of urokinase efficiently inhibits cancer cell invasion and metastasis.

Focusing of urokinase-type plasminogen activator (uPA) to the cell surface via binding to its specific receptor (uPAR, CD87) is critical for tumor invasion and metastasis. Consequently, the inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy. We examined the effects of cDNA transfection of the human uPA amino-terminal fragment (ATF) on invasion and metastasis of cancer cells. First, a highly metastatic human lung giant-cell carcinoma cell line (PG), used as the target cell for evaluation of this effect, was demonstrated to express both uPA and uPAR. Then, ATF, which contains an intact uPAR binding site but is catalytically inactive, was designed as an antagonist of uPA-uPAR interaction and was transfected into PG cells. [(3)H]-Thymidine incorporation and cell growth curves indicated that expressed ATF did not affect the proliferation of transfected cells. However, analysis by scanning electron microscopy revealed that ATF changed the host cells from the typical invasive phenotype to a noninvasive one. Correspondingly, the modified Boyden chamber test in vitro showed that ATF expression significantly decreased the invasive capacity of transfected cells. Furthermore, in the spontaneous metastasis model, it was confirmed in vivo that expressed ATF remarkably inhibited lung metastasis of implanted ATF-transfected PG cells. In summary, autocrine ATF could act as an antagonist of uPA-uPAR interaction, and ATF cDNA transfection could efficiently inhibit the invasion and metastasis of the cancer cells. Inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy.     

In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.     

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.     

Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.