Interplay between oncogenic K-Ras and wild-type H-Ras in Caco2 cell transformation

Mutation of RAS genes is one of the most common oncogenic alterations in cancer and acquisition of activating RAS mutations has been demonstrated to cause progression of colorectal adenoma to cancer. The aim of this study was to identify changes in the proteome of the intermediate-stage colorectal cancer cell line Caco2, induced by ectopic expression of two distinct RAS proteins, KRAS(V12) and HRAS(V12), in their mutated, constitutively active form. Using 2D-gel electrophoresis, followed by LC-MS/MS we identified almost 200 differentially expressed proteins in pair-wise comparisons of Caco2 vs Caco2-KRAS(V12) and Caco2 vs Caco2-HRAS(V12). Although many of the affected proteins were unique for each pair, there were also substantial similarities. Interestingly, transformation by the mutant KRAS(V12) gene resulted in elevated expression levels and activity of endogenous H-ras protein. Silencing the latter with a specific RNAi reversed several proteomic changes observed in KRAS(V12)-transformed cells, suggesting that oncogenic K-ras partly exerts its effects through endogenous H-ras activation. Alterations in the expression of cytoskeletal and cell adhesion proteins, caused by HRAS siRNA treatment, correlated with a reduction in the invasive properties of Caco2-KRAS(V12) cells. Our data suggest a novel interplay between K-ras and H-ras, with possible implications for colorectal carcinogenesis.     

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion ¹. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities ². Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab ^3,4 and we have used it in kidney tubular cell lines ^5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay⁸. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.     

Wine Has Activity against Entero-pathogenic Bacteria in Vitro but not in Vivo

We studied the activity of wine against entero-pathogenic bacteria both in vitro and in vivo. The food-borne bacteria were killed in both red and white wine within 30 min. However, the results of a Salmonella infection experiment using mice suggested that wine was not effective in preventing food-borne diseases in vivo.     

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.     

大肠杆菌原核增强子样序列的克隆及其结构与功能的研究

利用氯霉素乙酰转移酶(cat)以及β-半乳糖苷酶(lacZ)基因作为报告基因,从大肠杆菌MC1061株染色体基因组中克隆到3个原核增强子样序列——MC2,MC8,MC9,这3个片段均具有正反向增强活性,对β-半乳糖苷酶基因的增强活性(正向)在2～5.5倍之间。采用体内转录,RNA Dot blot杂交的方法对MC8的功能进行了研究,结果表明,MC8片段对于基因表达的调控发生在转录水平上。用核酸外切酶III末端缺失的方法对MC8的功能区进行了定位。结果显示,MC8的功能区位于距其正向克隆5′端450～950bp长约500bp的区段内。在450～600bp以及840～950bp区段内至少分别含有一个功能位点。序列分析的结果表明,MC8功能区有3个AT丰富区,其中2个分别位于450～600bp以及840～950bp区段内。     

Identification of DK419, a potent inhibitor of Wnt/β-catenin signaling and colorectal cancer growth

The Wnt signaling pathway is critical for normal tissue development and is an underlying mechanism of disease when dysregulated. Previously, we reported that the drug Niclosamide inhibits Wnt/β-catenin signaling by decreasing the cytosolic levels of Dishevelled and β-catenin, and inhibits the growth of colon cancers both in vitro and in vivo. Since the discovery of Niclosamide’s anthelmintic activity, a growing body of literature indicates that Niclosamide is a multifunctional drug. In an effort to identify derivatives of Niclosamide with improved pharmacokinetic properties that maintain the multifunctional drug activity of Niclosamide for clinical evaluation, we designed DK419, a derivative containing a 1H-benzo[d]imidazole-4-carboxamide substructure, using the structure-activity relationships (SAR) of the Niclosamide salicylanilide chemotype. Similar to Niclosamide, we found DK419 inhibited Wnt/β-catenin signaling, altered cellular oxygen consumption rate and induced production of pAMPK. Moreover, we found DK419 inhibited the growth of CRC tumor cells in vitro, had good plasma exposure when dosed orally, and inhibited the growth of patient derived CRC240 tumor explants in mice dosed orally. DK419, a derivative of Niclosamide with multifunctional activity and improved pharmacokinetic properties, is a promising agent to treat colorectal cancer, Wnt-related diseases and other diseases in which Niclosamide has demonstrated functional activity.     

Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagic Escherichia coli strains

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.     

人胰岛素样生长因子Ⅰ在大肠杆菌中的表达研究

胰岛素样生长因子Ⅰ（ＩＧＦ－Ⅰ）是一种多功能细胞增殖调控因子,它对许多疾病有较好的治疗作用．为了获得大量的ＩＧＦ－Ⅰ产品,在测定了ＩＧＦ－Ⅰ全长序列的基础上,构建了ＩＧＦ－Ⅰ表达载体ｐＢＶＩＧＦ,经热诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出７．６ｋＤ的蛋白．研究了不同菌株对ＩＧＦ－Ⅰ表达的影响．ＩＧＦ－Ⅰ的表达水平可达１５ｍｇ／Ｌ．重组蛋白主要以包涵体形式存在,进行了初步纯化和复性研究,Ｗｅｓｔｅｒｎ印迹表明重组蛋白具有ＩＧＦ－Ⅰ的抗原性．并初步建立了ＩＧＦ－Ⅰ生物活性测定方法．     

健康人肠道双歧杆菌和大肠杆菌数量在不同季节的动态观察

本文研究了季节因素对肠道重要菌群双歧杆菌和大肠杆菌的影响。在春、夏、秋、冬四季分别定量检测Ｔ２０名健康青年粪便中的上述两种菌,结果表明：两种细菌数值均有明显的季节变动规律,双歧杆菌秋季显著高于春、夏冬三季;大肠杆菌夏季显著高于冬、春季,而与秋季的差异无显著意义。ｚＬ歧杆菌与大肠杆菌在一年四季中的比值也有明显的季节特征,即秋季明显高于其它三季。     

The Bactericidal Action of Peroxides; An E.P.R. Spin-Trapping Study

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coll) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.