第56位酪氨酸与肿瘤坏死因子细胞毒活性的关系*

选择肿瘤坏死因子分子中间部分保守区第56位酪氨酸,用寡核苷酸诱导的体外基因定点突变法,将其改变为谷氨酰胺。将突变的肿瘤坏死因子基因插入表达载体pBV220,所得表达产物的分子大小及表达量均无明显变化,但其细胞毒活性及比活性均下降数百倍,表弱该区是肿瘤坏死因子细胞因子细胞毒活性的必需区。     

海胆Runx基因保守序列的克隆与初步分析

Runx(Runt box)是Runt结构域转录因子家族的成员之一。马粪海胆Runx基因第663位和728位碱基发生突变,分别为A→T和C→T。第一个突变为错义突变,使密码子编码的氨基酸由天冬氨酸变为酪氨酸,而第二个突变为同义突变,密码子编码的氨基酸未发生变化。海胆Runx基因保守序列的突变可能会导致其表达产物的变化,进而会影响海胆的生理机能。     

一种新型人肿瘤坏死因子突变体在大肠杆菌中的高效表达

采用多位点突变引物和ＰＣＲ方法对合成的人肿瘤坏死因子进行了突变,通过这一突变,人肿瘤坏死因子的第二位精氨酸的密码子ＣＧＴ被变为赖氨酸密码子ＡＡＡ,将突变后的基因插入表达载体ｐＳＢ－９２的ＰＬ启动子下游得到重组质粒ｐＳＢ－ＴＮＦ－Ｋ２,并在大肠杆菌中进行表达。突变体［Ｌｙｓ２］ｈＴＮＦ－α的表达产率达到菌体内总蛋白质的６０％以上,且为一可溶性蛋白质并易于纯化。同野生型ｈＴＮＦ－α相比,［Ｌｙｓ２］ｈＴＮＦ－α具有稍低的毒性,但对于某些人肿瘤细胞株表现出高于数十到数百倍的抑制活性．     

一种新型人TNF表达质粒的构建及在大肠杆菌中的高表达

在详细分析TNF结构及前人有关TNF结构与功能关系研究的基础上,应用PCR技术构建了一种新型人肿瘤坏死因子(TNF)分子的编码基因,并将该编码基因插入表达质粒,转化大肠杆菌,通过温度诱导获得了高表达.活性测定的结果表明,新型人TNF对L929细胞的细胞毒活性较原型人TNF升高了10³数量级.     

膀胱癌H-ras基因点突变和ras癌基因产物P21的表达

应用聚合酶链反应(PCR)技术结合地高辛化寡核甘酸探针杂交以及免疫组织化学方法,检测人膀眈癌组织中H-ras癌基因第12位密码子点突变和基因产物P21蛋白的表达.58例膀胱癌组织中有23例出现H-ras基因的点突变,36例P21蛋白表达阳性,5例正常膀胱组织未见点突变和P21蛋白阳性表达.本组膀胱癌第12位密码子点突变和P21阳性表达水平呈正相关,多发生在肿瘤临床T2-T4期和分化差的肿瘤,并且与膀胱癌的预后有关、提示H-ras癌基因点突变和P21蛋白阳性表达是膀胱癌的晚期表现,并在膀胱癌的发生,发展及预后中起着重要作用.     

重组肿瘤坏死因子-α衍生物的制备及聚乙二醇修饰

应用搭桥PCR技术,将肿瘤坏死因子-α基因的前4位氨基酸的编码序列删除,对hTNF-α的第8/9/10/29/31/157位氨基酸的密码子进行定点突变,将突变后的cDNA插入到pBV220载体中构建重组质粒pBV220-tnf-αD4。将重组质粒转化大肠杆菌DH5α,筛选获得了高效表达TNF-αD4突变体的工程菌,表达的重组蛋白约占菌体蛋白总量的45%左右,经硫酸铵沉淀和阳离子交换层析纯化得到纯度达90%以上的重组目的蛋白,比活性达到8×107。用单甲氧基聚乙二醇-丁醛（mPEG-ButyrALD）对TNF-αD4进行修饰,经阳离子交换层析纯化得到纯的mPEG-TNF-αD4,纯度达85%以上,比活性达到8.6 ×107,系统毒性也有了明显的降低。此项工作通过应用PEG修饰肿瘤坏死因子-α,为降低其毒性,增加其活性进行了有益的尝试,为其进一步研究与开发奠定了基础。     

重组人白细胞介素－6与肿瘤坏死因子突变体融合蛋白的构建及表达

针对肿瘤坏死因子（ＴＮＦ）在肿瘤治疗剂量下产生的严重毒副作用及一些肿瘤细胞上白细胞介素－６（ＩＬ－６）受体明显增高的事实,根据ＴＮＦ结构与功能研究的最新信息,利用ＰＣＲ技术,对人ＴＮＦα基因进行了改造,并将其与人ＩＬ－６成熟肽编码区ｃＤＮＡ通过人工接头进行融合。融合蛋白在大肠杆菌中表达后,Ｗｅｓｔｅｒｎｂｌｏｔ分析表明,分子量约为３７ｋＤ;活性检测结果证实,该融合蛋白兼具有ＴＮＦ抗肿瘤活性和结合ＩＬ－６受体的能力,在高表达ＩＬ－６受体的人骨髓瘤细胞上测得的细胞毒活性较同样位点突变的ＴＮＦ高约３倍。     

四膜虫eRF1识别终止密码子的功能结构域

原生动物的一些纤毛虫中终止密码子发生重分配现象,将1个或2个终止密码子翻译为氨基酸.目前对这一现象的发生机制仍无合理解释．近年来,对蛋白质合成终止过程中肽链释放因子(eukaryotic polypeptide release factor, eRF)结构和功能的深入研究,为揭示终止密码子的重分配机制提供了重要的线索．本实验以具有终止密码子识别特异性的四膜虫Tt-eRF1为研究对象,将其中与密码子识别有关的GTx、NIKS和Y-C-F关键模体(motif) 引入识别通用终止密码子的酵母Sc=eRF1中,构建成各种嵌合体eRF1．利用双荧光素酶报告系统和细胞活性实验,分析关键模体及其周边的氨基酸对eRF1识别终止密码子性质的影响．结果表明,GTx和NIKS模体一定程度上决定eRF1识别终止密码子第1位碱基U和第2位碱基A;Y-C-F模体决定eRF1识别终止密码子UGA的第2位碱基G．模体内及其相邻氨基酸定点突变分析进一步支持以上结果．本研究推测,eRF1在进化过程中一些关键模体结构的改变决定其识别终止密码子的特异性,只能识别3个终止密码子中的1个或2个．随后,由于tRNA基因的突变产生阻抑性tRNA,促成终止密码子在原生动物纤毛虫中的重新分配．     

基因重组人肿瘤坏死因子-α突变体的构建

在详细分析ＴＮＦ α结构及有关ＴＮＦ α结构与功能关系研究的基础上,设计并人工合成了一对ＴＮＦ α结构基因点突变引物。应用ＰＣＲ和分子克隆技术构建了一种新型人肿瘤坏死因子（ＴＮＦ α）分子的编码基因,将该编码基因插入表达质粒,转化大肠杆菌,通过温度诱导获得了表达蛋白,对突变体克隆进行了ＤＮＡ电泳、酶切位点检测以及基因序列测定,并对突变体蛋白进行了ＳＤＳ ＰＡＧＥ电泳检测。     

外源NF—IL6高表达增强巨噬细胞的细胞毒效应

白介素6核转录因子（NF－IL6）的功能非常广泛,它不仅参与炎症反应和急性期蛋白质基因、细胞因子基因的表达调控,还参与肿瘤的凋亡、抑制和维持巨噬细胞的免疫功能。为探讨NF－IL6基因的表达与巨噬细胞肿瘤杀伤活性的关系,我们用含重组NF－IL6基因编码区的表达质粒pCN,转染小鼠腹腔留居巨噬细胞,并用蛋白质印迹法证实了外源NF－IL6基因在巨噬细胞中的高表达;随后用碱性磷酸酯酶法检测高表达NF－IL6基因的巨噬细胞对肝癌细胞SMMC7721的细胞毒性。结果显示,外源NF－IL6基因的过量表达明显增强小鼠腹腔巨噬细胞对该肝癌细胞的细胞毒活性。这表明人工加强NF－IL6的表达能增强巨噬细胞对肿瘤细胞的杀伤作用。     

Tumor necrosis factor as a pharmacological target

As indicated by its name, tumor necrosis factor (TNF), cloned in 1985, was originally described as a macrophage-derived endogenous mediator that can induce hemorrhagic necrosis of solid tumors and kill some tumor cell lines in vitro. Unfortunately, its promising use as an anticancer agent was biased by its toxicity, which was clear soon from the first clinical trials with TNF in cancer. Almost at the same time TNF was being developed as an anticancer drug, it became clear that TNF was identical to a mediator responsible for cachexia associated with sepsis, which was termed cachectin. This research led to the finding that TNF is, in fact, the main lethal mediator of sepsis and to the publication of a huge number of articles showing that TNF inhibits the toxic effects of bacterial endotoxins, which are now described as systemic inflammatory response. Although the clinical trials with anti-TNF in sepsis have not been successful thus far, undoubtedly as a result of the complexity of this clinical setting, these studies ultimately led to the identification of TNF as a key inflammatory mediator and to the development of anti-TNF molecules (soluble receptors and antibodies) for important diseases including rheumatoid arthritis and Crohn’s disease. On the other side, the mechanisms by which TNF and related molecules induce cell death have been studied in depth, and their knowledge might, in the future, suggest means of improve the therapeutic index of TNF in cancer.     

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.

To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.     

Structure-Function Relationship of Tumor Necrosis Factor (TNF) and Its Receptor Interaction Based on 3D Structural Analysis of a Fully Active TNFR1-Selective TNF Mutant

Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR.Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function.To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.     

Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship.

In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.     

Tumor necrosis factor, cancer and anticancer therapy

Tumor necrosis factor (TNF) is being utilized as an antineoplastic agent for the treatment of patients with locally advanced solid tumors. However, its role in cancer therapy is debated. Although a large body of evidence supports TNF's antineoplastic activity, the cascade of molecular events underlying TNF-mediated tumor regression observed in vivo is still incompletely elucidated. Intriguingly, some pre-clinical findings suggest that TNF may promote cancer development and progression, which has led to propose anti-TNF therapy as a novel approach to malignancies. In the present work, we summarize the molecular biology of TNF with particular regard to its tumor-related properties, and review the experimental and clinical evidence currently available describing the complex and sometime conflicting relationship between this cytokine, cancer and antitumor therapy. Recent insights that might pave the way to further exploitation of the antineoplastic potential of TNF are also discussed.     

Intratumoral tumor necrosis factor induction in tumor-bearing mice by exogenous/endogenous tumor necrosis factor therapy as compared with systemic administration of various biologic response modifiers.

The relationship between the induction of tumor necrosis factor (TNF) (as an indicator of inflammatory reaction) in tumor tissues and its antitumor effect was investigated in tumor-bearing mice by using nine biologic response modifiers (BRMs) and by exogenous/endogenous TNF therapy following a previously reported protocol. Close correlation between the induction of TNF-rich inflammation in tumor tissues and the antitumor effect of BRM were observed. The results of this study suggest that the conditions necessary for exerting antitumor effects of biologic response modifiers may be the induction of TNF (50 to 200 U/g) at the tumor lesions at an early stage after BRM administration and maintenance of the detectable amount of TNF (approximately 10 U/g) for more than 6 hours. Tumor necrosis factor should also be induced in the liver and spleen so that its activity can be maintained in the tumor lesions.     

A role for the non‐receptor tyrosine kinase ACK1 in TNF‐alpha‐mediated apoptosis and proliferation in human intestinal epithelial caco‐2 cells

The roles of tumor necrosis factor alpha (TNF‐alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42‐associated kinase 1 (ACK1) in TNF‐alpha‐mediated apoptosis and proliferation in Caco‐2 cells. ACK1 expression was knocked down using ACK1‐specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin‐dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1‐specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF‐alpha‐mediated anti‐apoptotic effects and proliferation of Caco‐2 cells. Interestingly, TNF‐alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco‐2 cells. ACK1‐Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down‐stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF‐alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor‐кB (NF‐кB) activity, suggesting a correlation between NF‐кB signaling and TNF‐alpha‐mediated apoptosis in Caco‐2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF‐alpha‐induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down‐stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation.     

Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor.

The receptor for granulocyte colony-stimulating factor (G-CSFR) is a hemopoietic growth factor receptor, which mediates proliferation and differentiation signals. The cytoplasmic region of G-CSFR carries four tyrosine residues in its C-terminal half. We constructed mutant receptors in which each tyrosine residue of G-CSFR was mutated to phenylalanine. Two mutant receptors (Tyr703 and Tyr728) neither transduced the growth-inhibitory signal nor induced the neutrophil-specific myeloperoxidase (MPO) gene. The Tyr703 mutant did not induce morphological changes in cells, whereas transformants expressing the Tyr728 mutant adhered to plates with a macrophage-like morphology upon G-CSF stimulation. Mutation of the most distal tyrosine residue (Tyr763) abolished the ability of G-CSFR to stimulate the tyrosine phosphorylation of a cellular protein with an M(r) of 54 kDa. These results indicated that the regions around the three tyrosine residues of G-CSFR play essential and distinct roles in signal transduction.     

Clinical applications of tumour necrosis factor

Tumour necrosis factor (TNF) is a polypeptide hormone produced in vivo by activated macrophages and lymphocytes. TNF has diverse effects in vivo and has a physiological role as an immune modulator, as a mediator of the immune response, both through activation of neutrophils and eosinophils, and also affects the vascular endothelium. TNF also has antiviral activity and causes alterations in lipid metabolism. In disease states excessive production of TNF may have adverse affects. TNF has been implicated as a mediator of endotoxic shock, inflammatory joint disease, immune deficiency states, allograft rejection, and in the cachexia associated with malignant disease and some parasitic infections. When used in pharmacological doses, TNF is cytotoxic to many malignant cells in vitro and in vivo. The mechanisms underlying cytotoxicity are not fully elucidated but involve both a direct toxic effect to the cell and an indirect effect on tumour vasculature. Cytotoxicity is not universal and TNF may act as a differentiating agent or growth factor for some haematological cell types. So far the clinical application of TNF has been as a treatment for cancer in Phase I and II trials in patients with advanced disease and its efficacy here is still unproven. TNF may have potential for clinical application in combination therapy for cancer. There is experimental evidence for its interaction with other biological agents and cytotoxic drugs. The use of specific antibodies to inhibit production of TNF, or other agents to antagonise the toxic effects of TNF may have clinical relevance in counteracting septic shock and the clinical manifestations of TNF in inflammatory and neoplastic disease.