The effect of C-terminal fragment of JNK2 on the stability of p53 and cell proliferation

The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramatically inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21^waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.     

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.     

pten敲除小鼠中转录上调基因pdd87编码蛋白定位于高尔基体

为了明确pten敲除小鼠中转录上调基因pdd87编码蛋白在细胞中的定位,构建PDD87与绿色荧光蛋白(GFP)的融合蛋白表达载体pEGFP-C1-padS7,利用Lipofectamine2000转染该载体于体外培养的NIH3T3细胞中,采用高尔基体特异探针BODIPY TR C5-Ceramide染色显示高尔基体,激光共聚焦显微镜下观察到PDD87-GFP融合蛋白定位于高尔基体,提示pdd87基因编码的蛋白定位于高尔基体。     

Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.     

Optimized expression of dual reporter genes in transient transfection of purified Toxoplasma gondii using different promoters

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T.?gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.     

Miniaturization of gene transfection assays in 384- and 1536-well microplates

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)–DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35 μl and in 1536-well plates in a total assay volume of 8 μl. A luciferase assay performed in 384-well plates produced a Z′ score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250 cells per well in 384-well plates, with CaPO₄ proving to be 10-fold more potent than PEI.     

Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C

Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.     

利用GFP示踪细胞内源性P53活性检测DNA损伤

ＤＮＡ损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术,将报告基因—绿色荧光蛋白（ＧＦＰ）置于ＳＶ４０基本启动子调控下,构建成对照载体ｐＳＶ－ＧＦＰ。在ＳＶ４０基本启动子上游插入寡核苷酸Ｐ５３ＲＥ,构建成示踪载体ｐ５３ＲＥ－ＧＦＰ。转染ＮＩＨ３Ｔ３细胞,以ＧＦＰ示踪细胞内源性Ｐ５３的转录激活活性。紫外线照射或Ｈ２Ｏ２处理转化细胞使ＤＮＡ损伤,诱导细胞内源性Ｐ５３的表达。用激光扫描共聚焦成像系统（ＬＳＣＩＳ）对细胞进行红、绿、蓝三色光融合成像,并测定ＧＦＰ经４８８ｎｍ激发后发出的绿色荧光光密度,验证ＧＦＰ示踪Ｐ５３的特异性。ｐ５３ＲＥ－ＧＦＰ转化细胞３Ｔ３－ＲＥＧ经紫外线照射或Ｈ２Ｏ２处理后,ＧＦＰ的表达增高,处理后１ｈｒ光密度即达到最高水平,随后逐渐降低。血清“饥饿”—非ＤＮＡ损伤处理的３Ｔ３－ＲＥＧ细胞,以及经紫外和Ｈ２Ｏ２处理的对照载体ｐＳＶ－ＧＦＰ转化细胞３Ｔ３－ＳＶＧ,ＧＦＰ的表达无明显增强。实验表明：ＧＦＰ示踪内源性Ｐ５３转录激活活性用于检测ＤＮＡ损伤有很高的灵敏度和特异性,适宜推广应用。     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism

The immediate-early gene cyclooxygenase 2 (Cox-2) is induced in a variety of hyperplastic pathological conditions, including rheumatoid arthritis and colorectal cancer. Although a causal role for Cox-2 has been proposed, mechanisms by which Cox-2 function contributes to the pathogenesis of hyperplastic disease are not well defined. We constructed a green fluorescent protein-tagged Cox-2 (Cox-2-GFP) to examine its effects on a variety of cell types upon overexpression. Subcellular localization and enzymatic and pharmacological properties of Cox-2-GFP polypeptide were indistinguishable from those of the wild-type Cox-2 polypeptide. Overexpression of the Cox-2-GFP or the Cox-2 polypeptide by transient transfection suppressed the population of cells in the S phase of the cell cycle, with a concomitant increase in G(0)/G(1) population. In contrast, transient overexpression of GFP had no effect on cell cycle distribution, whereas endoplasmic reticulum-retained GFP (GFP-KDEL) overexpression was associated with only a minor decrease of cells in S phase. Interestingly, neither NS-398 (a Cox-2-specific inhibitor) nor indomethacin could reverse the effect of Cox-2-GFP overexpression on cell cycle progression. Furthermore, two mutants of Cox-2, S516Q and S516M, which lack the cyclooxygenase activity, exhibited the same effect as Cox-2-GFP. The cell cycle effect of Cox-2-GFP was observed in ECV-304, NIH 3T3, COS-7, bovine microvascular endothelial cells, and human embryonic kidney 293 cells. These findings suggest that Cox-2 inhibits cell cycle progression in a variety of cell types by a novel mechanism that does not require the synthesis of prostaglandins.     

Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice

BACKGROUND: We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'. METHODS: Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. RESULTS: CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells. CONCLUSIONS: We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.