Caco-2 cells

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Activation of the NF kappa B Pathway Enhances AhR Expression in Intestinal Caco-2 Cells

Activation of the NF kappa B Pathway Enhances AhR Expression in Intestinal Caco-2 Cells

Figure 5: Effect of 8 h exposure to conditioned media from 100 nM PMA-treated Caco-2 cells on AhR mRNA levels (a). Effect of 8 h exposure to conditioned media from 100 nM PMA 24 h treated Caco-2 cells in presence of IL-1𝛽-neutralizing antibodies on AhR mRNA levels (b). Effect of 8 h exposure to conditioned media from 100 nM PMA-treated THP-1 cells on AhR mRNA levels (c). ∗ : 𝑃 < 0.05 versus control. Caco-2 cells express a number of cytokine receptors on their cellular membrane and are also able to secrete proin- flammatory cytokines in response to initial inflammatory signals. These cytokines exert some of their effect through the activation of NF𝜅B. Our results demonstrated that treating Caco-2 cells with a conditioned media derived from PMA- treated cells also leads to an increase of AhR expression, and that this induction involved IL-1𝛽 signaling. Similar results were obtained with human monocytic THP1 cells. These results are consistent with our results from site-directed mutagenesis experiments and the treatments of cells with proteasome inhibitors. Moreover, these data suggested that an autocrine loop could occur and probably generate and amplify a proinflammatory signal. Indeed, environmental exposure to AhR agonists like PAHs has been demonstrated to induce the expression of proinflammatory cytokines such as IL-1𝛽 [ 8 , 9 ] and to activate NF𝜅B [ 16 ]. Our data demonstrated that proinflammatory conditions induced and sustained through AhR expression could, therefore, increase cell susceptibility to PAH-induced inflammation.
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Caco-2: Predicting drug permeability and relationship with COVID-19 infected Caco-2 cells

Caco-2: Predicting drug permeability and relationship with COVID-19 infected Caco-2 cells

Docking BE (TMPRSS2) = 1.67HOMO-LUMO – 15.00 Where R 2 = 0.649, SEE = 0.88, SE(HOMO-LUMO) = 0.62, F=14.77, Significance=0.005 Discussion This study has shown that our LFER methodology based on quantum mechanical derived molecular descriptors can successfully describe: (a) the transport of a wide range of drugs into Caco-2 cells (as described by eqs 3-7) (b) the inhibition of SARS-CoV-2 infected Caco-2 cells (as described by eqs 8-10) and (c) in vitro inhibition and docking binding energies of SARS- CoV-2 of M pro , ACE2, S-RBD, TMPRSS2 (as described by eqs 11-18). These equations were derived from applying the general form equation 2 to a large variety of inhibitors to find which of the five molecular specifiers can describe the transport of drugs into Caco-2 cells, or the inhibition of SARS-CoV-2 infected Caco-2 cells, or the in vitro inhibition or docking binding of inhibitors to the SARS-CoV-2 virus to host cells. It is noted that we have found that ΔG lipo,CDS
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AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells

AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells

In previous studies, it has been shown that epithelial AMPK activation with the commonly used pharmacological AMPK activator, AICAR, was closely associated with enhanced tight junction integrity in MDCK cells during calcium switch [ 11 , 12 ]. The authors suggested that these effects could be mediated by the activation of AMPK. Treatment with AICAR also improved epithelial barrier function in Caco-2 cells as shown by increased TEER and decreased paracellular permeability of FITC-dextran [ 19 , 29 ]. However, the specificity of AICAR has been questioned due to its ability to activate AMP-dependent enzymes and is regarded as a non-specific activator of AMPK [ 30 ]. Here, we analyzed the impact of AMPK activation in Caco-2 cells by using a novel class of direct allosteric AMPK activators, binding to the allosteric drug and metabolite (ADaM) site, located between the α -subunit kinase domain and β-subunit carbohydrate binding module of AMPK complex [ 31 ]. Here, we showed that activation of AMPK with the pan-AMPK activator 991 facilitated the reassembly of tight junctions by increasing TEER and decreasing the paracellular permeability during the calcium switch assay. Compound 991 also induced a faster relocalization of ZO-1 at cell-cell contact and maintained expression levels of ZO-1. Importantly, all the beneficial effects of 991 were lost in AMPK dKO cells showing AMPK-dependent action. Interestingly, compound 991 also had a beneficial effect on the initiation of tight junctions reassembly in calcium-free medium, meaning that the sole AMPK activation is sufficient to induce this process. However, the combination of compound 991 and calcium enables a greater impact on the epithelial barrier recovery associated with enhanced AMPK activation over time.
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Covid-19: Predicting inhibition of SARS-CoV-2 in Caco-2 cells: viral cellular entry

Covid-19: Predicting inhibition of SARS-CoV-2 in Caco-2 cells: viral cellular entry

Abstract A previously described quantum mechanical LFER model can be derived for a wide range of repurposed small molecule drugs which can inhibit SARS-CoV-2 in-vitro in human Caco-2 cells. There is evidence that this model applies to drugs acting as viral S-RBD inhibitors, TMPRSS2 host cell membrane fusion inhibitors and host cell ACE2 inhibitors. A similar LFER involving the lipophilicity, molecular volume and HOMO of the inhibitors can also be derived from available molecular docking binding energies for inhibitor-ACE2 and inhibitor-S-RBD interactions. The HOMO inhibitor à LUMO receptor is the dominant molecular interaction, not the
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Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

immediately snap-frozen and maintained at 280 uC before analysis. For analysis of the TLR4 cascade proteins, Caco-2/TC7 cells and intestinal explants were lysed or homogenized respectively, in cold radioimmunoprotein assay (RIPA: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Na deoxycholate, 1% Triton X- 100) buffer supplemented with 1 mM phenylmethylsulphonyl fluoride, protease inhibitor cocktail (Complete Mini, Roche, Milan) and phosphatase inhibitor cocktail (PhosSTOP, Roche). For analysis of the extracellular Hsp72 and Hsp90, 30 m L of medium collected from basolateral compartment were added to 30 m L of 26RIPA. The cells, explants and basolateral media were centrifuged at 150006g for 20 min and supernatants were recovered. Cell lysates (50 m g total proteins) intestinal homoge- nates (50 m g total proteins) and 50 m L of basolateral media were dissolved in sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 g/L bromophenol blue, 10 mM b-mercapto- ethanol), heated for 5 min, fractionated by SDS-polyacrylamide gel (4–20% gradient) electrophoresis and transferred to 0.2 m m nitrocellulose filters (Trans-Blot Turbo, Biorad, Milan). Mem- branes were incubated with the following primary antibodies: rabbit polyclonal anti-human TLR4, MyD88, IRAK-4, IKKa, IKKb, phospho(P)-IKKa/b, IkBa, P-IkBa, NF-kB p65, P-p65, IRAK-M, Tollip, or mouse monoclonal anti-human Hsp90, Hsp72, a-tubulin antibodies. All primary antibodies were from
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Effect of Foods and β-Cyclodextrin on the Bioaccessibility and the Uptake by Caco-2 Cells of Hydroxytyrosol from Either a Pure Standard or Alperujo

Effect of Foods and β-Cyclodextrin on the Bioaccessibility and the Uptake by Caco-2 Cells of Hydroxytyrosol from Either a Pure Standard or Alperujo

HT = hydroxytyrosol, β-CD = β-cyclodextrin. Figure 2. Bioaccessibility of hydroxytyrosol from standard powder in each digestive compartment Samples were taken at the beginning of the experiment (T0) and at the end of the oral, gastric and duodenal digestion steps.

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Proteomic Analysis of Lipid Droplets from Caco-2/TC7 Enterocytes Identifies Novel Modulators of Lipid Secretion

Proteomic Analysis of Lipid Droplets from Caco-2/TC7 Enterocytes Identifies Novel Modulators of Lipid Secretion

doi:10.1371/journal.pone.0053017.g005 Figure 6. Gene and protein expression in Caco-2/TC7 cells (TC7), Caco-2/TC7 GFP-CP cells (TC7 GFP-CP) and human jejunum of some proteins identified by LC-MS/MS in lipid droplet fractions. (A) Caco-2/TC7 and Caco-2/TC7 GFP-CP cells were cultured on filters for 17 days then supplied with lipid micelles for 24 h. mRNA levels were measured by quantitative RT-PCR for core (HCV core protein), HSD17B2 (17b- hydroxysteroid dehydrogenase type 2), HSD3B1 (3-beta-hydroxysteroid dehydrogenase), PLIN2 (perilipin-2), MGLL (monoacylglycerol lipase), LPCAT2 (lysophosphatidylcholine acyltransferase 2), C2orf43 (UPF0554 protein C2orf43) and ACSL3 (long-chain-fatty-acid–CoA ligase 3). (B) mRNA levels for the same genes were quantified in human jejunum mRNA samples. (C) Lysates of Caco-2/TC7 and Caco-2/TC7 GFP-CP cells were analyzed by western blot for 17b-hydroxysteroid dehydrogenase type 2 (DHB2) and actin. (D) The immunoblot shown in C was quantified and standardized to actin used as the loading control. Results shown are the means 6 SD from three independent experiments performed in triplicate, except for human jejunum (one sample measured in triplicate). *, p,0.05 compared to control cells.Caco-2 cell line derives from a human epithelial colorectal adenocarcinoma and TC7 is a clone of Caco-2 cells [37]. Although these cells differentiate such that their phenotype resembles absorptive enterocytes of the small intestine, it still remains a cell line with a cancerous origin and thus proteins might be differently expressed in normal cells from human intestine. Therefore, to assess the physiological relevance of these results, we performed similar experiments on mRNA isolated from human small intestine (Fig. 6B). All above-mentioned genes were expressed in human small intestine except HSD3B1. Therefore, since 3BHS1 protein was also not significantly up-regulated in LD isolated from Caco-2/TC7 GFP-CP as compared to Caco-2/TC7 cells, it was not studied further.
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In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2.

In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2.

a b s t r a c t In Burkina Faso, as in most Sahelian countries, the failure to follow good agricultural prac- tices coupled with poor soil and climate conditions in the locust control context lead to high environmental contaminations with pesticide residues. Thus, consumers being orally exposed to a combination of multiple pesticide residues through food and water intake, the digestive tract is a tissue susceptible to be directly exposed to these food contami- nants. The aim of our work was to compare in vitro the impact of five desert locust control pesticides (Deltamethrin DTM, Fenitrothion FNT, Fipronil FPN, Lambda-cyalothrine LCT, and Teflubenzuron TBZ) alone and in combination on the human intestinal Caco-2 cells viability and function. Cells were exposed to 0.1–100 ␮M pesticides for 10 days alone or in mixture (MIX). Our results showed a cytotoxic effect of DTM, FNT, FPN, LCT, and TBZ alone or in combination in human intestinal Caco-2 cells. The most efficient were shown to be FPN and FNT impacting the cell layer integrity and/or barrier function, ALP activity, antioxidant enzyme activity, lipid peroxidation, Akt activation, and apoptosis. The pres- ence of antioxidant reduced lipid peroxidation level and attenuated the pesticides-induced cell toxicity, suggesting that key mechanism of pesticides cytotoxicity may be linked to their pro-oxidative potential. A comparative analysis with the predicted cytotoxic effect of pesticides mixture using mathematical modeling shown that the combination of these pesticides led to synergistic effects rather than to a simple independent or dose addition effect.
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Elucidation of the impact of cell culture conditions of Caco-2 cell monolayer on barrier integrity and intestinal permeability

Elucidation of the impact of cell culture conditions of Caco-2 cell monolayer on barrier integrity and intestinal permeability

11 drug across all Caco-2 monolayers in relation with the TEER measurements (%). It can be observed that the permeability of dextran-FITC was close to zero when membrane supports of 1 µm were used, independently of the Caco-2 cell line. However, the percentage of drug which crossed the monolayer was 9.24% and 4.24% for Caco-2 clone cells seeded on membrane support of 3 µm N and HD, respectively. As can be seen in Figure 5.C, these results were explained by significant differences of the TEER values, as showed previously. Indeed, the high TEER values obtained with Caco-2 cells seeded on membrane with pore size of 1 µm prevented the paracellular transport of the drug. However, the models with lower TEER values allowed the diffusion of a small fraction of drug.
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Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

The alkaline phosphatase activity was detected in co-cul- tures by the cytochemistry method. Our results show that the alkaline phosphatase was restricted to the enterocyte- like cell and could then be used as a potential marker for the monitoring of these cells under several co-culture con- ditions. The discrepancy between the alkaline phos- phatase-positive area and the Caco-2 seeding proportion in culture could be explained by the heterogeneity of the cell lineage [32]. Indeed, this cell line differentiates spon- taneously in enterocyte-like cells, but also in other cell types which do not express alkaline phosphatase. Our results underline this heterogeneity, as we observed, by cytochemistry at cellular level, several red-scales (meaning alkaline phosphatase activity) in the same culture. This differentiation seems to be influenced by the Caco-2/ HT29-5M21 co-culture and thus by a cell-cell interaction. In order to collect more data on the proportionate compo- sition of the monolayer, we investigated the expression level of the alkaline phosphatase coding gene and of the MUC5AC gene. Though the two cell lineages were both of an adenocarcinoma origin [16,21,24], we showed that the IAP and MUC5AC gene expressions are restricted, respec- tively, to Caco-2 cells and HT29-5M21 cells. We showed by real-time PCR that the expression level of the IAP and MUC5AC genes in the monolayer is independent of the Alkaline phosphatase specific activity measured in Caco-2/
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Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

cadmium accumulation was significantly higher in cells exposed long-term to cadmium (five weeks) than in cells exposed 1 week. These results are in agreement with a previous study on parental Caco-2 cells (Blais et al. 1999). Moreover, compared to control cells, only intracellular concentrations of copper and zinc increased in Caco-2 cells exposed long-term to cadmium but not the other minerals measured (Table 1). It is well known that zinc interacts with cadmium at the level of absorption and tissue binding (Orlowski & Piotrowski 2003). Exposure to cadmium disturbs zinc levels in the organism, while dietary zinc intake has a marked effect on cadmium absorption, accumulation, and toxicity (Brzoska & Moniuszko-Jakoniuk 2001). Both absorption and metabolism of copper has been shown to be affected by cadmium (Davies & Campbell 1977; Reeves et al. 1996). Moreover, it is generally believed that cadmium decreases copper uptake and/or transport in the gut (Davies & Campbell 1977; Reeves et al. 1996). However, in the present study, there was a significant increase in copper accumulation. Contrary to copper and zinc, increased cadmium accumulation was not correlated with the manganese intracellular con- centration (Table 1). The initial manganese con- centration in the culture medium was low, thus its absorption could not be influenced by the cad- mium accumulation in the medium. These results could by explained by the fact that cadmium, copper, and zinc are well known to combine with metallothionein, an intracellular protein that binds metals, whereas this is not the case with manga- nese (Watanabe et al. 1991).
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Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products

Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products

14 2.5 DNA damage analysis DNA damage in proliferating Caco-2 cells was detected using antibodies against phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks (DSBs) (Bonner et al., 2008; Rogakou et al., 2000). First, 150,000 Caco-2 cells/cm² were seeded on coverslips. After 24 h to allow attachment, the cells were exposed for 1, 6, or 24 h to 0.1, 1, or 3 µg/mL Mesosilver™ or AgC suspension. Exposure to 500 fM calicheamicin was used as a positive control for inducing genotoxicity. At the end of treatment, cells were immediately fixed with 4% paraformaldehyde in PBS. After PBS washing, cells were permeabilized with 0.1% Triton X-100, washed in PBS, and blocked in PBS with 1% BSA overnight. After blocking, cells were incubated with mouse anti-phospho-Histone2AX (Ser139) antibody clone JBW301 at a 1:1,000 dilution, followed by incubation with Alexa Fluor 488 goat anti-mouse IgG antibody at a 1:100 dilution. Coverslips were mounted using ProLong Gold antifade reagent with DAPI to counterstain the nuclei. Slides were visualized under a 40× objective on a Leica TCS SP8 AOBS inverted confocal microscope (Leica Microsystems, Mannheim, Germany). The number of γH2AX positive cells, expressed as the percentage of the total cell number, was calculated, based on the determination of a threshold of γH2AX intensity for untreated cells. The intensity of γH2AX was compared to this threshold for each treated cell, visualized with DAPI staining. A minimum of 50 cells per image and eight images for each condition were analysed in a blinded fashion.
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Toxicological impact of acute exposure to E171 food additive and TiO2 nanoparticles on a co-culture of Caco-2 and HT29-MTX intestinal cells

Toxicological impact of acute exposure to E171 food additive and TiO2 nanoparticles on a co-culture of Caco-2 and HT29-MTX intestinal cells

toxicity of TiO 2 is low, even at high concentration [33-35]. They concur with our previous study, where pure anatase and pure rutile TiO 2 -NPs were proven to induce only minor impact on undifferentiated Caco-2 cells [22]. They induced no cytotoxicity, no DNA strand breaks or alkali-labile sites, but they caused accumulation of ROS, modulation of GSH level and

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Daphnanes diterpenes from the latex of Hura crepitans L. and activity against human colorectal cancer cells Caco-2

Daphnanes diterpenes from the latex of Hura crepitans L. and activity against human colorectal cancer cells Caco-2

characterizing a cytostatic activity. Concerning signaling pathways, huratoxin inhibited GSK3 and Akt, two kinases playing a major role in colon carcinogenesis, while TPA inhibited YAP and activated MAPK as previously described, thus revealing different cytostatic mechanisms. Finally, huratoxin has been more potent than TPA to induce the expression of two genes implicated in Caco-2 cells differentiation, which might explain the more pronounced morphological changes induced by huratoxin including the formation of neo-crypts. Works are actually in progress to deeply investigate the signaling pathways modulated by huratoxin, particularly those resulting from an activation of PKC isotypes. Finally, our data revealed that, besides their well-known roles as neurotrophic factors, daphnanes diterpenes could also restore the functions of intestinal epithelium.
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Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode

Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode

Keywords: cancer cell lines; co-culture; fatty acids; gene datasets; signaling pathways; intestinal barrier 1. Introduction In mammals, the gastrointestinal epithelium is the largest surface facing external environment. It is the unique route for nutrients and for many pharmaceuticals to enter the body. Epithelial cells allow the passage of small molecules by several mechanisms: passive transcellular passage, passive paracellular exchanges between cells, and an energy-consuming transport across the epithelium with uptake into coated vesicles (transcytosis) [ 1 ]. In vivo experiments to study intestinal physiology raise numerous questions, which indeed result from animal models, cannot be easily transposed to human and it is difficult to draw mechanistic conclusions. The availability of an in vitro model of intestinal barrier would be extremely useful to test nutrients and drugs absorption. Amongst the in vitro models of intestinal epithelium, the Caco-2 cell line has been extensively used to measure nutrient and drug transports [ 2 , 3 ]. Caco-2 cells derived from a human colorectal adenocarcinoma. After confluence, this cell line differentiates into enterocytes, which represent around 80–85% of the cell population of the intestinal epithelium. However, the use of Caco-2 cells is criticized for several reasons. The
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The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPbeta-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPbeta-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

receptors [26–28]. An increase of C/EBPb mRNA also correlates with metastatic breast cancer and high tumor grade, making it an interesting biomarker for subsets of tumors with a poor prognosis [28–30]. Moreover, our preliminary results have shown that transfection of C/EBPb-2 in MCF-7 cells increases their motility (Figure S1). Similar results were obtained when we increased galectin-7 expression (Figure S2), consistent with our previous findings that high levels of galectin-7 increase metastasis of breast cancer cells to the bone and the lung [9]. In fact, our results may have implications in other types of cancer where gal-7 is expressed, most notably in transformed keratinocytes. Both gal-7 and C/ EBPb-2 have been shown to be involved in the differentiation of keratinocytes [31]. Whether suppression of C/EBPb-2 will necessarily reduces gal-7 is currently unknown but likely since previous studies showing that retinoic acid, which suppresses the expression of C/EBPb-2 target genes in keratinocytes [32–33], also inhibits gal-7 expression [34]. Interestingly, C/EBPb-2 has been shown to be expressed at high levels in most mammary tumors and is the most frequent isoform found in human breast cancer cell lines [35]. Its expression in MCF10A cells has also been shown to induce in vitro epithelial to mesenchymal transition associated with increased invasive properties [17]. However, while C/EBPb-2 is generally considered the most transcriptionally active of all three C/EBPb isoforms, we cannot completely rule out the implication of other isoforms in regulating gal-7 expression. The C/EBPb-3 isoform, for instance, is expressed at high levels in some breast carcinoma, most notably in basal-like breast cancer [36– 37]. Future studies with isoform-specific antibodies will thus be needed to correlate the expression of gal-7 with specific C/EBPb isoforms in breast cancer tissues. It is important to note, however, that other transcription factors can also upregulate gal-7 and may thus compensate for suppression of C/EBPb-2. This is particularly true for breast cancer cells which often express a mutant form of p53, which is capable of inducing gal-7 [38]. Future investigations will thus be needed to determine whether direct suppression of galectin-7 expression or via specific targeting C/EBPb are valuable alternatives to inhibit breast cancer progression.
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Transcriptional Silencing of the TFPI-2 Gene by Promoter Hypermethylation in Choriocarcinoma Cells

Transcriptional Silencing of the TFPI-2 Gene by Promoter Hypermethylation in Choriocarcinoma Cells

IFR 120 ‘Imagerie et Exploration fonctionnelles’, 2bis Boulevard Tonnellé, F-37032 Tours cedex, France *Corresponding author Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz- type serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the pres- ence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demon- strate that DNA demethylation by 5’-aza-2’-deoxycy- tidine restores TFPI-2 transcription in JAR choriocar- cinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI– 2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor tro- phoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silenc- ing and that the TFPI-2 gene may be a candidate tu- mor suppressor gene.
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Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells.

Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells.

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ASB-2 Inhibits Growth and Promotes Commitment in Myeloid Leukemia Cells*

ASB-2 Inhibits Growth and Promotes Commitment in Myeloid Leukemia Cells*

985/MT cells (Fig. 4B, c), whereas the proportion of PLB-985/ MT-ASB-2 cells with condensed chromatin was markedly in- creased (up to 35% at 60 h) (Fig. 4B, f). This was associated with an increased percentage of cells with a DNA content equivalent of that of S and G 2 /M cells (Fig. 4B, f). A large proportion of PLB-985/MT-ASB-2 cells with condensed chroma- tin had DNA content equivalent of that of S and G 2 /M cells (Fig. 4C, d). In separate measurements, we assessed the percentage of apoptotic cells with DNA strand breaks (terminal dUTP nick-end labeling-positive) (Fig. 4B, e and f) and cleaved poly- (ADP-ribose) polymerase (data not shown). The proportion of apoptotic cells remained below 5% whether PLB-985/MT- ASB-2 and PLB-985/MT were untreated or treated with ZnSO 4 . Thus, in light of the evidence that proliferation of ASB-2 trans- fected cells was suppressed by ZnSO 4 whereas apoptosis was minimal, the observed increase in proportion of S and G 2 /M cells including those cells with condensed chromatin indicates that, although commitment events were induced, cells were arrested in their progression through S and G 2 /M. However, expression of differentiation markers such as CD11b, ICAM-1, MCP-1, interleukin-8, and defensin 1 was not induced in ZnSO 4 -treated PLB-985/MT-ASB-2 cells (data not shown). Al- together, our results strongly suggest that ASB-2 expression is part of the mechanisms controlling growth arrest and chroma- tin condensation known to be associated with early phases of ATRA-induced differentiation of myeloid leukemia cells.
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A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

dC-Mediated anti-HsV protective immunity To assess the capacity of DCs inoculated with mutant HSV viruses to confer protective immunity against later exposure to HSV, we propose performing adoptive transfer experiments that consist on the injection of mutant virus-treated DCs into animals that will then be challenged with virulent HSV. Importantly, this type of assay devises numerous variables that require attention, such as the amount of DCs being transferred into the animals, the route of administration of these cells, and the number of transfers to be performed, among others. Furthermore, DCs from different sources may be used, such as in vitro differentiated DCs from the bone marrow ( 106 , 107 ), DCs isolated from the spleens of naïve mice which then can be inoculated ex vivo with the mutant viruses ( 108 , 109 ) or DCs purified from mice shortly after vac- cination ( 81 ). Indeed, a recent report describes the use of the latter method for obtaining DCs inoculated with a mutant HSV-1 virus, particularly from the spleens 3 days after vaccination. These DCs were then transferred into naïve mice in three separate injec- tions, which conferred protection (increased animal survival) against a challenge with a lethal dose of HSV-1 administered through the intranasal route ( 81 ). Importantly, we consider that for these assays in which virus-inoculated DCs are transferred into animals, an experimental group consisting of animals being transferred with DCs treated with wild-type virulent virus should also be included, as such treatment may confer some degree of protection to the animals because bystander DCs in the recipient animals could capture apoptotic HSV-infected inoculated DCs from the donor and present their antigens to immune cells ( 78 ). Noteworthy, the selection of a particular experimental method will depend, among others on the HSV infection model being evaluated (i.e., genital, skin, nasal or ocular infection, among others). Nevertheless, protective immunity after challenge can be measured, for instance by evaluating viral loads in neuronal and non-neuronal tissues (plaque forming units and viral loads by qPCR) and tissue pathology, among others ( 49 , 50 ).
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