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WNT signaling enhances breast cancer cell motility and blockade of the WNT pathway by sFRP1 suppresses MDA-MB-231 xenograft growth
DOI: 10.1186/bcr2317
© Matsuda et al.; licensee BioMed Central Ltd. 2009
Received: 19 November 2008
Accepted: 27 May 2009
Published: 27 May 2009
In breast cancer, deregulation of the WNT signaling pathway occurs by autocrine mechanisms. WNT ligands and Frizzled receptors are coexpressed in primary breast tumors and cancer cell lines. Moreover, many breast tumors show hypermethylation of the secreted Frizzled-related protein 1 (sFRP1) promoter region, causing low expression of this WNT antagonist. We have previously shown that the WNT pathway influences proliferation of breast cancer cell lines via activation of canonical signaling and epidermal growth factor receptor transactivation, and that interference with WNT signaling reduces proliferation. Here we examine the role of WNT signaling in breast tumor cell migration and on xenograft outgrowth.
The breast cancer cell line MDA-MB-231 was used to study WNT signaling. We examined the effects of activating or blocking the WNT pathway on cell motility by treatment with WNT ligands or by ectopic sFPR1 expression, respectively. The ability of sFRP1-expressing MDA-MB-231 cells to grow as xenografts was also tested. Microarray analyses were carried out to identify targets with roles in MDA-MB-231/sFRP1 tumor growth inhibition.
We show that WNT stimulates the migratory ability of MDA-MB-231 cells. Furthermore, ectopic expression of sFRP1 in MDA-MB-231 cells blocks canonical WNT signaling and decreases their migratory potential. Moreover, the ability of MDA-MB-231/sFRP1-expressing cells to grow as xenografts in mammary glands and to form lung metastases is dramatically impaired. Microarray analyses led to the identification of two genes, CCND1 and CDKN1A, whose expression level is selectively altered in vivo in sFRP1-expressing tumors. The encoded proteins cyclin D1 and p21Cip1 were downregulated and upregulated, respectively, in sFRP1-expressing tumors, suggesting that they are downstream mediators of WNT signaling.
Our results show that the WNT pathway influences multiple biological properties of MDA-MB-231 breast cancer cells. WNT stimulates tumor cell motility; conversely sFRP1-mediated WNT pathway blockade reduces motility. Moreover, ectopic sFRP1 expression in MDA-MB-231 cells has a strong negative impact on tumor outgrowth and blocked lung metastases. These results suggest that interference with WNT signaling using sFRP1 to block the ligand- receptor interaction may be a valid therapeutic approach in breast cancer.
The WNT signaling network is complex, with 19 WNT ligands, 10 Frizzled (FZD) receptors, as well as the co-receptors, low-density lipoprotein receptor-related protein (LRP) 5 and LRP6. WNT receptor binding stimulates intracellular signaling, promoting stabilization and nuclear translocation of the key effector of the canonical pathway, β-catenin [1, 2]. Intracellular mediators of the WNT pathway are mutated in many human cancers. Inactivating mutations in the APC or AXIN genes, as well as activating CTNNB1 mutations, each causes β-catenin stabilization and nuclear accumulation in the absence of WNT ligands. In the nucleus, β-catenin forms functional complexes with transcription factors of the lymphoid enhancer binding factor-1/T-cell factor (TCF) family, activating expression of target genes with cancer-promoting roles [3]. In addition to activation of the canonical pathway by engagement of FZD and LRP receptors, WNT ligands bind the Ror2 or Ryk receptors to stimulate β-catenin-independent pathways that have been involved with cytoskeletal reorganization and cell migration [2, 4].
In breast cancer, deregulation of WNT signaling appears to occur by autocrine mechanisms [5–7]. Multiple WNT ligands and FZD receptors are expressed in primary human breast tumors and in breast cancer cell lines [3, 7–9]. Furthermore, most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1) and low expression of this negative WNT pathway regulator [10–12]. Interference with autocrine WNT signaling has been shown to block in vitro proliferation of many human breast cancer cell lines [6, 7]. We have extended these studies and show in the present article that blocking the WNT pathway in MDA-MB-231 breast cancer cells not only decreases proliferation, but also impairs the motility of the tumor cells. Furthermore, we show that stable expression of sFRP1 in MDA-MD-231 cells has a dramatic effect on the ability of the cells to grow as tumor xenografts in nude mice. The results presented here provide further evidence supporting approaches to target WNT pathway activity in breast cancer.
Materials and methods
The following primary antibodies were used: c-Myc (9E10, to detect Myc-tagged protein), DVL2 and DVL3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); DVL1 (R&D Systems, Abingdon, UK); active β-catenin (anti-ABC; Upstate, Billerica, VA, USA); α-tubulin (DM1A) (Neomarkers, Fremont, CA, USA); cyclin D1 (SP4) (Cell MARQUE, Rocklin, CA, USA) for immunohistochemistry (IHC); cyclin D1 (Chemicon, Billerica, MA, USA) for western blotting; bromodeoxyuridine (BrdU) (Roche, Basel Switzerland); p21Cip1 (OP64-100UG) (Oncogene Research Products, Cambridge, MA, USA); ERK and P-ERK (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA); total β-catenin and CD31 (BD Pharmingen, Franklin Lakes, CA, USA); and active β1-integrin (Clone HUTS-4, MAB2079Z; Chemicon).
As secondary antibodies we used anti-rabbit and anti-mouse antibodies (GE Healthcare, Little Chalfont, UK; LI-COR Bioscience, Lincoln, NE, USA), anti-rat antibody (GE Healthcare) or anti-goat antibody (DAKO A/S, Glostrup, Denmark) coupled to horseradish peroxidase (HRP) or IRDye 800CW.
For IHC we used Biotin-SP-conjugated affinipure donkey anti-rabbit, anti-mouse, anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA) and goat anti-rat ALEXA 568 (Molecular Probes, Eugene, OR, USA). Recombinant Wnt3a was purchased from R&D Systems. Y27632 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cDNA encoding Myc/His-tagged human sFRP1 in pCDNA was provided by Jeffrey Rubin (NCI, Bethesda, MD, USA) and was recloned into the pBabePuro retroviral vector. Conditioned media (CM) from Wnt1-producing cells, from sFRP1-producing cells, and purified sFRP1 were prepared as previously described [7].
T-cell factor reporter assay
MDA-MB-231/sFRP1-P1 cells and control-P1 cells were seeded on 12-well plates and were transfected with a mixture of Super TOPFlash plasmid and pRL-CMV (Promega, Madison, WI, USA) to assay TCF promoter activity using Fugene6 (Roche) according to the manufacturer's instructions. Luciferase activities were measured 48 hours later using the Dual-Luciferase Reporter Assay System (Promega) and Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany) according to the manufacturer's instructions. The fold activation was normalized against renilla luciferase. Nine wells were used per condition and the average and standard error are shown in the graph.
Cell culture, transfections, retroviral infections, proliferation and anoikis assays
The human breast cancer cell line MDA-MB-231 (ATCC, Manassas, VA, USA) was cultivated in DMEM, 10% heat-inactivated FCS (Amimed, Allschwil, Switzerland) supplemented with penicillin and streptomycin (Sigma-Aldrich). All transfections were performed using FuGENE 6 Transfection Reagent (Roche) following the manufacturer's guidelines. MDA-MB-231 cells were stably transfected with pCDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) encoding Myc/His-tagged human sFRP1 or empty pCDNA3.1(+) as control. After selection with 1 mg/ml G-418, three clones of MDA-MB-231/sFRP1 and three control clones were isolated. Equal cell numbers of these clones were pooled before some experiments (MDA-MB-231/sFRP1-P1 and MDA-MB-231/control-P1). A second pool of sFRP1-expressing MDA-MB-231 cells (MDA-MB-231/sFRP1-P2) and control cells (MDA-MB-231/control-P2), each representing > 100 clones, was generated by infecting the cells with pBabePuro encoding Myc/His-tagged human sFRP1 or empty pBabePuro followed by selection with 2 μg/ml Puromycin (Sigma-Aldrich).
Cell proliferation was measured either by counting cell numbers with a Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA, USA) on selected days after seeding 200,000 cells on six-well plates or using the YOPRO cell viability assay (Invitrogen) 3 days after seeding 1,000 cells on a 96-well plate, according to the manufacturer's instructions. Anoikis was measured by seeding cells in 1% FCS-containing medium on polyHema-coated plates to prevent adhesion. Cells were harvested 24 hours later, stained with propidium iodide. The cell cycle distribution was analyzed with a FACScalibur (Becton Dickinson, San Jose, CA, USA) using the Cellquest software. A representative cell cycle distribution of three MDA-MB-231/sFRP1 clones and three control clones is shown. Unless otherwise noted, P values were calculated using Student's t test.
Protein extraction and western blotting
Cells were lysed in 1% Nonidet P-40, 50 mM Tris pH 7.5, 120 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetra-acetic acid (EGTA), 2 mM sodium vanadate, 20 mM β-glycerophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mM phenylmethanesulphonylfluoride (PMSF), 50 mM NaF, 1 mM dithiothreitol for 5 minutes on ice before collecting lysates. Debris was removed by centrifugation at 4°C and the protein concentration was determined using the Bradford reagent (BioRad, Hercules, CA, USA).
For western blotting, protein loading buffer was added to 30 to 50 μg total protein and the samples were denatured for 10 minutes at 95°C prior to separation on SDS-polyacrylamide gels and blotting by semi-dry transfer for 90 minutes on PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 10% horse serum in Tris-buffered saline- Tween buffer for 1 hour (0.2 M NaCl, 25 mM Tris, pH 7.5, 0.5 ml/l Tween-20), except for p21Cip detection where PBS- Tween buffer was used instead of TBS-Tween buffer for blocking. Blots were incubated with primary antibodies at room temperature for 1 hour or at 4°C overnight, followed by 30-minute incubation with secondary antibodies. These antibodies were anti-rabbit-HRP, anti-mouse-HRP (1:5000) or anti-goat-HRP (1:5000) for detection of luminescence, which was carried out using ECL (GE Healthcare) according to the manufacturer's instructions and using X-OMAT LS films (Kodak, New York, NY, USA). Secondary antibodies IRDye 800CW goat anti-rabbit-IgG or anti-mouse-IgG (1:10,000; LI-COR Biosciences) were detected with the LI-COR Odyssey system according to the manufacturer's instructions (LI-COR Biosciences). Quantification of protein expression was carried out using Odyssey 2.1 (LI-COR Biosciences).
Wound healing assay
Cells were seeded on six-well plates and grown to confluency. Monolayers were scratched, and in the indicated experiments the media were changed to Wnt1 CM or control CM. When using recombinant Wnt3a and Y27632, 90 minutes before the scratch was made the media were changed to DMEM 10% FBS containing 100 μg/ml Wnt3a (R&D Systems) and/or 5 μM Y27632 (Sigma-Aldrich). Pictures of randomly-chosen nine wound edges per condition were taken at time 0 and at the indicated time points using Nikon DIAPHOT (Nikon, Tokyo, Japan). The recovered area was calculated using ImageQuant TL (GE Healthcare). In some experiments, purified sFRP1 was added to the CM [7].
Fluorescence-activated cell sorting analysis for active β1-integrin
MDA-MB-231/sFRP1-P1 and control-P1 cells (0.3 × 106) were incubated for 30 minutes on ice with an antibody recognizing active β1-integrin (final concentration 20 ng/μl) in HEPES/NaCl buffer. This was followed by incubation for 30 minutes on ice with fluorescein isothiocyanate-conjugated (FITC) donkey anti-mouse IgG secondary antibody (Jackson Laboratories) (diluted 1:250) in Flow PBS (1 × PBS, 2% horse serum, 0.1% sodium azide). Fluorescence was measured using a FACSCalibur machine (Becton Dickinson) and the percentage of gated cells stained with active β1-integrin was calculated.
In vivoexperiments with MDA-MB-231 cells
Statistical analyses were performed with two-way repeated-measures analysis of variance (repeated measures ANOVA (RM ANOVA)).
To assay tumor cell proliferation, 100 μg/g body weight of BrdU (Cell Proliferation Kit II; Roche) was intraperitoneally injected into tumor-bearing mice that were sacrificed 2 hours later. Tumors were excised and washed with PBS before fixation in 4% paraformaldehyde (PFA) at 4°C for 24 hours, and BrdU detection was performed as previously described [13]. To examine experimental metastasis, 1.0 × 106 MDA-MB-231/sFRP1-P2 cells or control-P2 cells were injected into the tail vein of female Balb/c nude mice (5 to 6 mice per group). Fifty-three days after the injection, the mice were sacrificed, lungs were dissected and the total number of surface lung metastases was determined. For western analyses, excised tumors were snap frozen and pulverized in liquid nitrogen and lysed in SDS buffer (100 mM Tris- HCl pH 7.6, 2% SDS, 10 mM dithiothreitol, 2 mM sodium vanadate, 0.5 mM ethylenediamine tetraacetic acid) by incubation at 95°C for 10 minutes.
Immunohistochemistry and functional vessel analysis on tumor sections
To detect functional vessels in tumors, 100 μl of 2 μg/μl solution of fluorescein-labeled Lycopersicon esculentum lectin (Vector Labs, Burlingame, CA, USA) was injected into tail veins of tumor-bearing mice [14], and the mice were sacrificed 5 minutes later. Tumors were excised, fixed in 4% paraformaldehyde in PBS for 48 hours at 4°C, followed by an overnight incubation in 30% sucrose in PBS at 4°C, and then embedded in tissue-Tec O.C.T. Compound 4583 (Sakura, Tokyo, Japan). Frozen sections (9 μm) were subjected to IHC analysis to detect tumor-associated vessels using rat anti-mouse CD31 (diluted 1:100; BD Pharmingen) and goat anti-rat ALEXA 568 (diluted 1:200; Molecular Probes). Staining was performed using Discovery XT (Ventana Medical Systems, Inc., Tucson, AZ, USA). Pictures were taken with a Z1 microscope (Carl Zeiss, Jena, Germany) and were analyzed with IMARIS (Bitplane, Zurich, Switzerland) to calculate the co-localized area. For detection of cyclin D1, frozen tumor sections (9 μm) were subjected to IHC using SP4 (diluted 1:100) and Biotin-SP-conjugated affinipure donkey anti-rabbit IgG (diluted 1:100). Staining was carried out using Discovery XT with sCC1 pretreatment. Pictures were taken with an Eclipse E600 (Nikon) and were analyzed with IMARIS (Bitplane) to calculate the signal intensity.
RNA isolation, real-time PCR and microarray analyses
Cultured cells were collected when plates were 70 to 80% confluent and total RNA was extracted using the RNeasy Mini kit (Qiagen, Venlo, The Netherlands). To extract total RNA from tumors, dissected tumors were put into RNAlater (Qiagen) overnight at 4°C, followed by RNA extraction using TRIzol reagent (Invitrogen) and washing using the RNAeasy Mini kit according to the manufacturer's instructions. RNA from mammary tumors (six MDA-MB-231/sFRP1-P1 tumors and five control tumors) and cultured cells (three MDA-MB-231/sFRP1 clones and three control clones) were individually amplified and labeled using the Ambion MesageAMP III RNA Amplification Kit (Applied Biosystems, Austin, TX, USA). Biotinylated, fragmented cRNA was hybridized to Affymetrix U133 plus 2.0 human GeneChips™ (Affymetrix, Santa Clara, CA, USA).
Expression values were estimated using the GC-RMA implementation found in Genedata's Refiner 4.5 software (Genedata AG, Basel, Switzerland). Quantile normalization and median scaling were performed in order to standardize array signal distributions to facilitate the comparison between in vitro cultured cells and in vivo tumor samples. Probesets showing statistically different expression profiles (one-way analysis of variance with P < 0.01; Benjamini and Hochberg Q values determined to minimize the false discovery rate) and specific pairwise fold changes were clustered by rank correlation with R > 0.8 for the first criterion and R > 0.885 for the second criterion using the Profile Distance Search function of Genedata's Analyst 4.5 tool (Genedata AG, Basel, Switzerland). All of the microarray data are stored in Gene Expression Omnibus [GEO:GSE13806].
For the quantitative real-time PCR, each sample cDNA was made from 2.5 μg RNA using Ready-To-Go™ You-Prime First-Strand Beads (GE Healthcare). Quantitative real-time PCR was performed with ABI Prism 7000 (Applied Biosystems, Austin, TX, USA) using ABsolute SYBR Green ROX Mix (THERMO Scientific, Waltham, MA, USA) following the manufacturer's guidelines. The primer sequences used for quantitative real-time PCR are as follows: human c-Myc forward, 5'-CCTACCCTCTCAACGACAG-3'; human c-Myc reverse, 5'-CTTGTTCCTCCTCAGAGTCG-3'; human β-actin forward, 5'-TGTCCACCTTCCAGCAGATGT-3'; and human β-actin reverse, 5'-CGCAACTAAGTCATAGTCCGCC-3'.
Ectopic sFRP1 expression in MDA-MB-231 cells blocks WNT signaling
WNT pathway activity was examined in the cells using various markers. As a consequence of WNT binding to FZD, cytoplasmic scaffolding proteins of the Dishevelled family (DVL1, DVL2 and DVL3) become phosphorylated on serine and threonine residues. DVL phosphorylation, which is the most proximal signaling event downstream of FZD activation, can be monitored by a decrease in the electrophoretic mobility of p-DVL [16, 17]. DVL1, DVL2 and DVL3, total β-catenin and stabilized active β-catenin were examined by western analysis in the individual clones. In the sFRP1-expressing cells, there was a decrease in the level of the three p-DVLs, compared with the vector control cells. Furthermore, the level of total β-catenin and active β-catenin was reduced in the two clones expressing the highest level of sFRP1 (Figure 1a).
Next we examined transcriptional activity of the canonical WNT signaling pathway following transient expression of the TOPFlash TCF reporter plasmid in the cells. TOPFlash luciferase reporter activity showed a significant 2.7-fold decrease in the sFRP1-expressing cells compared with controls (Figure 1b), confirming that ectopic expression of sFRP1 reduces canonical pathway activity in MDA-MB-231 cells. The phosphorylation status of ERK, another signaling protein that is active in MDA-MB-231 cells, was not altered in the sFRP1-expressing cells (Figure 1a), suggesting that the effects of sFRP1 are specific for the WNT pathway.
For further studies, the three sFRP1-expressing clones were pooled (P1) and a second pool of sFRP1-expressing MDA-MB-231 cells consisting of > 100 clones was generated (P2). Corresponding control pools, control-P1 and control-P2, were also generated. Quantification of a western analysis shows that MDA-MB-231/sFRP1-P1 has 2.8-fold higher levels of sFRP1 than does MDA-MB-231/sFRP1-P2 (Figure 1c).
sFRP1 decreases proliferation of MDA-MB-231 cells
Next we examined effects of sFRP1 on tumor cell proliferation. MDA-MB-231 parental cells were inhibited by treatment with sFRP1 CM (see Additional data file 1). Furthermore, ectopic expression of sFRP1 in MDA-MB-231 cells also caused a decrease in proliferation (Figure 1d, e). One day after plating there was no significant difference between the control cells and the sFRP1-expressing cells. After 3 days, however, we observed a significant difference in the sFRP1-P1 cells and sFRP1-P2 cells compared with control cultures. This effect appears to be dependent on sFRP1 expression levels since, in comparison with controls, there is a 31% and a 16% reduction in proliferation of MDA-MB-231/sFRP1-P1 (Figure 1d) and MDA-MB-231/sFRP1-P2 (Figure 1e) cells at day 3, respectively.
Xenografts of sFRP1-expressing MDA-MB-231 cells show reduced growth in nude mice
MDA-MB-231/sFRP1-P1 cells were tested in two additional experiments that yielded similar results (see Additional data file 2). While there was some variation in the time of tumor onset in the individual experiments, the time to appearance of the first tumor was consistently longer following injection of the MDA-MB-231/sFRP1 cells, in comparison with control cells (Figure 2b; see Additional data file 2). The data pertaining to the number of tumor-free mice were pooled for the three experiments, showing that 68% of the mice injected with MDA-MB-231/sFRP1-P1 cells were tumor-free at day 39, while only 20% of the control animals were free of tumors (Figure 2d). The data on tumor outgrowth kinetics were pooled for two experiments (Figure 2e), yielding a highly significant difference in the outgrowth ability of sFRP1-expressing cells (P < 0.0001, two-way RM ANOVA).
MDA-MB-231/sFRP1-P2 cells, which express 2.8-fold less sFRP1 than do the MDA-MB-231/sFRP1-P1 cells (Figure 1c), also grew more slowly than control cells following injection in the mammary gland (Figure 2f). Although the effect on tumor growth did not reach significance using two-way RM ANOVA and all animals had tumors at the end of the experiment (Figure 2g), tumor onset was delayed significantly in the cohort injected with MDA-MB-231/sFRP1-P2 cells (P = 0.026, log-rank test) (Figure 2g). The in vivo results together with the data on in vitro proliferation (Figure 1d, e) suggest that higher levels of sFRP1 cause a stronger blockade of WNT pathway activity, leading to a more pronounced effect on the time to tumor onset and tumor outgrowth.
MDA-MB-231/sFRP1-expressing cells have a lower migratory ability and form fewer metastases
We also performed a migration assay using the parental MDA-MB-231 cells treated with purified recombinant Wnt3a to confirm the results obtained with Wnt1 CM (recombinant Wnt1 is not commercially available). Wnt3a stimulated the canonical pathway as shown by the increased level of active β-catenin (Figure 3b), and increased the migratory ability of the cells in a wound closure assay (Figure 3c). In summary, in MDA-MB-231 cells, activation of the WNT pathway has a positive effect on cell migration; while sFRP1 lowers the proliferative and migratory ability of the MDA-MB-231 cells.
Mechanisms contributing to the in vivoanti-tumor effects of sFRP1
sFRP1 has been reported to block in vivo neovascularization [20]. We therefore considered the possibility that the density or the functionality of the tumor-associated vessels might be impaired in sFRP1-expressing tumors. Vasculature was visualized by tail vein injection of tumor-bearing mice with FITC-labeled L. esculentum lectin [14] 5 minutes before the animals were sacrificed, which allows only functional vessels to be perfused. Tumor sections were prepared and the associated endothelial cells were stained for CD31, while functional vessels were visualized via the FITC signal. There was no significant difference in the total vessel area or the ratio of FITC-positive/CD31-positive vessels in sFRP1-expressing tumors compared with control tumors (Figure 5d, e), suggesting that sFRP1 does not influence the number or the functionality of tumor-associated blood vessels. In summary, these results suggest that sFRP1-mediated blockade of WNT pathway activity in tumor cells is an important factor contributing to the slower outgrowth of the MDA-MB-231/sFRP1 tumors in mammary glands.
Identification of genes whose expression level is selectively altered in vivoin sFRP1-expressing tumors
To identify target genes that are controlled by sFRP1 expression and might influence proliferation of the MDA-MB-231 cells, we undertook a genome-wide transcriptome analysis using microarrays. RNA isolated from individual tumors arising after injection of MDA-MB-231/sFRP1-P1 cells and control-P1 cells, as well as RNA from in vitro cultured MDA-MB-231/sFRP1-P1 cells and control-P1 cells, was analyzed.
Considering data generated from six sFRP1-expressing tumors and five control tumors, there were 1,753 probesets (1,246 genes) whose signals changed more than 1.5-fold (P < 0.01, Student's t test) in the tumors arising from MDA-MB-231/sFRP1-P1 cells compared with tumors arising from control-P1 cells. The same analysis performed on in vitro cultured samples revealed 428 probesets (332 genes) that had a 1.5-fold difference (P < 0.01, Student's t test). Only 69 probesets (54 genes) overlapped between the two analyses, clearly showing the substantial difference in gene expression between in vivo tumors and in vitro cultured cells.
All of the microarray data were examined for characterized WNT pathway target genes. We identified 16 genes where at least one of the probesets showed a tendency for suppression in MDA-MB-231/sFRP1-expressing cells and tumors (see Additional data files 3 and 4).
Cell cycle regulators are altered in sFPR1-expressing MDA-MB-231 xenografts
The microarray analysis showed that the signal from a probeset for CCND1 (encoding cyclin D1) was downregulated and the probeset for CDKN1A (gene encoding p21Cip1) was upregulated in vivo, in tumors resulting from MDA-MB-231/sFRP1-P1 cell injection (Figure 6c). The CCND1 promoter has a consensus lymphoid enhancer binding factor-1 binding site [21], and in some cancer models its expression is controlled by β-catenin/TCF activation [22]. Our results suggest that CCND1 might also be a direct β-catenin/TCF target in MDA-MB-231 cells.
Considering that c-Myc is a WNT pathway target [23, 24] that regulates cyclin D1 [22] and p21Cip1 expression [25], we also examined c-Myc. There were no significant changes in c-Myc RNA levels in sFRP1-expressing tumors; however, c-Myc protein levels were lower in all of the sFRP1-expressing tumors compared with the control tumors (see Additional data file 7). In summary, the in silico analysis carried out on RNA of tumors arising from the MDA-MB-231/sFRP1 cells revealed altered levels of target genes (CCND1 and CDKN1A) that probably contribute to the anti-proliferative effects of sFRP1 expression. Moreover, our results also show the strong influence that the in vivo tumor environment has, not only on gene expression, but also on c-Myc protein. Decreased c-Myc levels might also contribute to the in vivo activity of sFRP1.
Aberrant activation of WNT signaling plays an important role in many types of human cancer, warranting therapeutic approaches to target the pathway [26]. Wnt1 was the first identified oncogene activated by mouse mammary tumor virus insertional mutagenesis [27], establishing the potential of aberrant WNT expression to promote mammary cancer. Currently, it is well documented that multiple WNT ligands and FZD receptors are expressed in primary human breast tumors and breast cancer cell lines [3, 7–9], making it difficult to identify an individual ligand/receptor complex that could serve as a cancer target. Using broad antagonists – including the cysteine-rich domain of the FZD8 receptor [28] or sFRP1 [6, 7, 29] – to interfere with WNT/FZD binding, however, the potential of targeting WNT binding to FZD as a therapeutic approach in breast cancer [6, 7] and in other cancers [28, 30] has been demonstrated.
Aberrant methylation of the sFRP1 promoter is one of the most consistent alterations in human cancer. In addition to breast tumors that have low sFRP1 levels [10–12, 29, 31], sFRP1 suppression has been described in colon tumors [32], ovarian tumors [33], bladder tumors [34] and prostate tumors [35]. Based on its widespread loss, interest in testing the effects of sFRP1 treatment in tumor models has been high. Indeed, sFRP1 has also been shown to impact on transforming properties of breast cancer cells [6, 29] and cervix cancer cells [36]; while sFRP2 has been shown to block proliferation of gastric cancer cells [37]. We have previously shown that proliferation of the estrogen-receptor-positive MCF7 and T47D, and the ErbB2-overexpressing JIMT-1, SKBR3 and BT474 breast tumor cell lines is decreased following treatment with sFRP1 [7]. In the current study we tested the impact of ectopic sFRP1 expression in the aggressive, basal-like [15] MDA-MB-231 breast tumor cells. The results presented show that ectopic sFRP1 expression in MDA-MB-231 tumor cells blocks the migratory ability and the proliferative potential of the tumor cells, both in vitro and in vivo, supporting the proposal that blockade of WNT signaling with sFRP1 might be a general approach to target not only breast, but also other types of cancer.
In addition to testing sFRP1, we also examined the effects of specific Wnt ligands on motility and found that the canonical ligands Wnt1 and Wnt3a stimulate MDA-MB-231 cells in a wound closure assay. These results correlate well with the stimulatory effects of both Wnt ligands in other cellular models [38, 39]. The Wnt/PCP pathway is considered the major mediator of cell motility. Indeed, this pathway stimulates many cytoskeleton regulators, including Rho family GTPases and Rho kinase [40]. Both Wnt1 and Wnt3a have been shown to activate RhoA [38, 39], whereas the non-canonical Wnt5a promotes melanoma migration via RhoB [41]. Furthermore, Rho kinase inhibition has been shown to block the effects of Wnt3a [39]. We have also observed that the Rho kinase inhibitor Y27632 blocks Wnt3a-induced MDA-MB-231 wound closure (data not shown). In contrast to the positive effects of Wnt ligands on motility, we show here that sFRP1-mediated blockade of endogenous WNT signaling not only reduced the basal motility of the MDA-MB-231 cells, but also impaired the ability of the cells to respond to Wnt1 in a wound closure assay. sFRP1 has also been shown to block motility [39] and invasion [36] of other types of tumor cells. Importantly, the negative impact of sFRP1 on MDA-MB231 motility translated, in vivo, to a block in the metastatic potential of these aggressive breast tumor cells. In comparison with control MDA-MB-231 cells, we observed a 20-fold decrease in the number of lung metastasis arising from sFRP1-expressing MDA-MB-231 cells.
MDA-MB-231/sFRP1 cells also proliferated more slowly than control cells; however, the effect of sFRP1 was more striking in vivo than in vitro. Following injection of MDA-MB-231/sFRP1 cells into mammary glands of nude mice, the time to appearance of the tumors was consistently longer than that observed with control MDA-MB-231 cells. Furthermore, tumors generated by the sFRP1-expressing cells not only grew more slowly than control tumors, but there were threefold more tumor-free mice at the end of each experiment in this group. Since sFRP1 is a secreted protein, it could act extrinsically on cells in the tumor environment. We concentrated in particular on tumor-associated vessels based on the reported ability of sFPR1 to block in vivo neovascularization [20]. Neither the vessel number nor their functionality differed, however, in tumors generated by sFRP1-expressing cells in comparison with those of control MDA-MB-231 cells. The impact of sFRP1 on WNT signaling and downstream transcription in the MDA-MB-231 cells might therefore more probably explain the protein's strong in vivo effects. Indeed, there were 3.7-fold more genes whose transcription was altered by sFRP1 expression in vivo compared with in vitro (1,246 vs. 332). Furthermore, only 54 genes overlapped in the two lists. Taken together, these results demonstrate the strong impact of tumor environment on gene expression.
We also performed an in-depth analysis to identify genes that were only affected in vivo, in the sFRP1-expressing tumors, with the intention of finding potential targets that could account for the strong effect of sFRP1 on MDA-MB-231 tumor-forming potential. There were a total of 168 genes (106 downregulated genes and 62 upregulated genes) that were only affected in vivo in sFRP1-expressing tumors (see Additional data files 5 and 6). Changes in expression of two in vivo identified genes, CCND1 and CDKN1A, encoding important cell cycle regulators were confirmed by IHC and immunoblotting on tumor cells. Cyclin D1 and p21Cip1 were found to be downregulated and upregulated, respectively, only in sFRP1-expressing tumors, which might be one reason why the impact of sFRP1 expression is stronger in vivo. These results raise the question as to why CCND1 and CDKN1A were only affected in vivo. While we can only speculate at this time, two explanations are worth discussion. First, c-Myc, which controls expression of both genes, was only downregulated in the sFRP1-expressing tumors; second, in the tumors there were major changes in expression of probesets for extracellular matrix proteins.
MYC is WNT pathway target [23, 24]. We did not detect changes in MYC expression, however, either in the microarrays (see Additional data files 5 and 6) or by quantitative real-time PCR (see Additional data file 7). Interestingly, c-Myc protein was low in all of the sFRP1-expressing tumors (see Additional data file 7). C-Myc is subject to control at many levels [42, 43] and the fact that c-Myc protein only changed in the sFRP-1-expressing tumors could be a reflection of the in vivo environment. Irrespective of the mechanism underlying these results, the fact that c-Myc stimulates cyclin D1 expression [22] and is a repressor for p21Cip1 [25] suggests that lower c-Myc levels could contribute to the altered expression of both cell cycle regulators in sFRP1-expressing tumors.
Turning to the extracellular matrix components, probesets for fibronectin, laminins and collagens were found to be strongly altered (see Additional data file 8, 29 probesets for the extracellular matrix). As expected, FN1 – a WNT pathway target (see Additional data files 3 and 4) – was decreased in sFRP1-expressing cells and tumors. The most striking difference, however, was seen when comparing the signal of the 29 probesets in cultured cells versus tumors. On the one hand, 25 out of the 29 probesets (see Additional data file 8) were strongly increased in vivo in control tumors, showing the impact of the tumor environment on expression of the encoded genes. Moreover, 21 out of these 25 were downregulated in sFRP1-expressing tumors.
Fibronectin, laminin and collagen are ligands for β1-integrin, which was also suppressed in sFRP1-expressing cells (see Additional data file 8). The decrease in β1-integrin RNA was confirmed at the protein level by performing a fluorescence-activated cell sorting analysis on intact cells with an antibody that recognizes active β1-integrin (see Additional data file 9). Integrin engagement is therefore likely to be decreased in sFRP1-expressing MDA-MB-231 cells, which in turn is likely to influence their proliferation. On the one hand fibronectin has been shown to stimulate proliferation of cancer cells, and this was associated with increased cyclin D1 and decreased p21Cip1 levels [44]. Moreover, we have previously shown that siRNA-mediated knockdown of β1-integrin in MDA-MB-231 cells increases p21Cip1 expression and leads to a proliferative decrease [13]. We propose that integrin engagement would be more strongly affected in vivo since not only the receptor but also many of its extracellular matrix binding partners are decreased in vivo.
In summary, the results presented here show that sFRP1-mediated blockade of WNT signaling in MDA-MB-231 breast cancer cells has an impact on the in vitro proliferation and motility of the cancer cells. The in vivo effects of WNT pathway blockade were even more dramatic since we observed a strong decrease in the mammary tumor-forming potential and an impairment of lung metastases. In summary, blockade of the WNT-FZ interaction using sFRP1 has a strong effect on breast tumor growth.
The results presented in this paper showing that sFRP1-mediated WNT pathway blockade strongly blocks the in vivo tumor forming potential of MDA-MB-231 breast cancer cells suggest that interference with WNT signaling at the ligand-receptor level may be a valid therapeutic approach in breast cancer.
- BrdU:
conditioned medium
Dulbecco's modified Eagle's medium
extracellular signal-regulated kinase 1/2
fetal calf serum
fluorescein isothiocyanate
horseradish peroxidase
low-density lipoprotein receptor-related protein
phosphate-buffered saline
polymerase chain reaction
repeated-measures analysis of variance
- sFRP1:
secreted Frizzled-related protein 1
- siRNA:
small interfering RNA
T-cell factor
wingless and integration site growth factor.
The authors acknowledge the contribution of Herbert Angliker in processing the Affymetrix microarrays. YM and TS were supported by a grant from the Swiss Cancer League (OCS-01445-12-2003). The laboratory of NEH is supported by the Friedrich Miescher Institute for Biomedical Research (Basel, Switzerland), part of the Novartis Research Foundation.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text: Posted May 6, 2014 · Report post In light of the recent article on baltimoreravens.com where Ozzie discusses his 2 favorite draft selections (Duane Starks and Jamal Lewis), I thought it might be fun to discuss each of our individual favorite selections. It doesn't have to be the best selection, just your personal favorite. It could be because a certain player was a favorite of yours in college and we nabbed him... Maybe you love the maneuvering that went into a certain pick, or maybe the pick simply became your favorite player. Whatever the reason please explain and have a little fun reminiscing!--My personal favorite pick was Dennis Pitta. It was the first draft that I got reqlly in to and spent a lot of time evaluating prospects beyond the top-rated, big name players. I loved him as a prospect going into the draft. I felt he was a bit under rated at the time partially because of his age and limited experience playing TE. I really wanted the Ravens to draft him and was pretty disappointed when we took Dickson. I thought after the Dickson pick that any chance of getting Pitta was gone especially with Todd Heap still on the roster. Then, we decided to double dip at the position and they took him. Probably because he was one of the first under-the-radar prospects that I invested time in evaluating and was pretty accurate on, because of how badly I wanted him on the team, and the disappointment followed by complete surprise when we picked him after already drafting Dickson - this is my favorite pick. And having recently learned behind the scenes info about another team offering 3 picks to the ravens to try and get Pitta just made it that much cooler to me. --So what's yours? 4 Share this post Link to post Share on other sites

subset: commoncrawl


text:
Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Freshwater Journals (http://www.tropicalfishkeeping.com/freshwater-journals/)
- - 20 gal long planted Celestial Pearl Danio tank (http://www.tropicalfishkeeping.com/freshwater-journals/20-gal-long-planted-celestial-pearl-7960/)
20 gal long planted Celestial Pearl Danio tank
i have 10 Celestial Pearl Danio in there. they are spawning. my first fry is almost a adult. i have a few stages of fry in there. i really want to work on breeding these out a little more. i should get another female or two. i will me moving the plecos and cories out soon.
lighting is a T5 36w set up. no co2. plants are thick java moss and guppy grass. i also have one java fern on the driftwood. the pearls hide in the moss and guppy grass. the fry swim on top of the plants darting in if they feel threatened. i hope to get more fry with the other fish moved out.
Nice setup.8)
Lovely tank!
COOOOOL! thats gorgeous!
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text:
|Golden Midget Watermelon Seeds
Developed in 1959 by N.H. University. Golden-yellow skin and salmon pink flesh that's very sweet for an early melon. Leaves are green and yellow so don't be alarmed its normal. When mature, it turns yellow when at its peak ripeness. Black seeded variety. Prompt harvesting will keep golden midget from splitting.
|Organic Cayuse Oats
Cayuse Oats


text:
I'm an RN/RM and I've always said that hospitals need to start pulling their fingers out and offering family friendly hours, it's no wonder people are leaving the profession
If they offered shifts say from9-2 or 3-11pm they would have so many more options. And people would be happier
My workplace do a floater shift frim8pm-2am which is very popular with mums.
But it is hard with family and shifts, especially if you're a single mum.
A lot of the mums I know do nights and weekends. I guess it depends how old your children are etc.
Good luck in whatever you decide


text:
Watch Every Day
"A comedy about a New York City couple in romantic crisis"
Family crises are the straws that stir this love story's drink. This comedic drama is set in New York City and was selected for the 2009 Tribeca Film Festival. This film is worth the while of anyone interested in love stories and great films.
| 2011 | 1 hr 30 min | 10 /
- Cast
- Liev Schreiber, Helen Hunt, Carla Gugino, Ezra Miller, Skyler Fortgang
- Studio
- Image Entertainment
- Director
- Richard Levine
- Language
- English


text:
Supplied air respiratory system. Includes V-300 air-regulating valve, belt, breathing tube, complete helmet assembly with ADF. Welding helmet can provide overhead protection, increased peripheral vision, excellent optical quality and function.
Welding respiratory protection system featuring the V-300 air-regulating valve. Lightweight, belt-mounted valve allows the worker to set the airflow to the most comfortable level. It is strictly a regulator and will not warm or cool supplied air to the worker.
The 3M™ Speedglas™ Welding Helmet 9100 MP features:
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Our goal is to respond to your request within 24 hours. Some questions, however, may need further investigation to provide you with an appropriate response.


text:
Shorter Pieces of Arabian Poetry (Dodo Press)
By (author) Various
Translated by J D Carlyle
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Shorter Pieces of Arabian Poetry (Dodo Press) by Various
Book DescriptionArabic poetry is the earliest work of Arabic literature. The pre-Islamic poetry is commonly referred to as the Jahili poetry, which translates to "The poetry of the period of ignorance." Poetry held an important position in pre-Islamic society with the poet filling the role of historian, soothsayer and propagandist. The poet represented an individual tribe's prestige and importance in the Arabian peninsula and mock battles in poetry would stand in lieu of real wars. 'Ukaz, a market town not far from Mecca, would play host to a regular poetry festival where the craft of the poets would be exhibited. The very best of these early poems were collected in the 8th century as The Mu'allaqat meaning "the hung poems" and The Mufaddaliyat meaning al-Mufaddal's examination or anthology. The former is named "the hung poems" being hung on the Kaaba. It also aimed to be the definitive source of the era's output with only a single example of the work of each of the so-called "seven renowned ones." The Mufaddaliyat on the other hand contains rather a random collection; apparently all that was remembered and perhaps some that was only produced in the 8th century and was not truly pre-Islamic.
Buy Shorter Pieces of Arabian Poetry (Dodo Press) book by Various from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9781409909620
(229mm x 152mm x 5mm)
Pages: 80
Imprint: Dodo Press
Publisher: Dodo Press
Publish Date: 18-Apr-2008
Country of Publication: United Kingdom
Books By Author Various
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The food is very good with a nice selection. Portions are generous. Servers and service is A+. Location here in Slidell is not ideal. Very busy intersection at Gause Blvd and I-10
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Thanks for helping!Share another experience before you go.
The food is very good with a nice selection. Portions are generous. Servers and service is A+. Location here in Slidell is not ideal. Very busy intersection at Gause Blvd and I-10
I love this restaurant! It's been around forever! But it is definitely still one of my favs! Great price and great service!!! Steaks are always cooked correctly and served in a timely manner!
we were hereabout 2month ago. the food was ok, the price was a bit pricey and the service wasn't much. I live in slidell for 16years but only been here twicce.
Traveling with a group from Alabama Baptist Disaster Relief to help Louisiana flood victims we stopped here for supper. We were seated immediately. The food was great. Service was excellent. If I am in the area at meal time again I would stop again. I had the steak and grilled shrimp. Very good.
Stopped by with friends and we had the best experience. Not only was the food great, but the service was outstanding. If you go here ask for Ryan R. He was great. The steaks were all cooked to perfection, so what more can I say. We were seated quickly and the restaurant was clean!
We like this place for the appetizers. The Blooming Onion is a favorite but lately it has been under cooked the last couple of times we have ordered it. Everything else is pretty good and we really just enjoyed each other's company over a light meal.
The night we were there it was especially busy but we got our seating quickly. Service was pretty good considering the crowd and I was very impressed by the Manager coming to the table to make sure everything was going good for us. The selections are great even if you are not in the mood for steak. I found the... More
I love Outback. The food is fresh and taste really good. The manager, Jackie is excellent and willing to help in anyway she can. I like that they are open for lunch. My husband and I go there sometimes. The salad and soup combo is great. The hot bread is my favorite.
Thank goodness the food is good. Customer service is devastating. Never fails, every visit I am cursed with a horrible server. At first I was convinced we were always sat in the beginners section, then after requesting alternate sections, I've learned they're all the same
Hi there, Thanks so much for sharing about your Outback Steakhouse experience. I want to apologize about the issues you've had with your service. Please take a moment to email me directly so I can learn more specifically about the problems you've had. You can email email@example.com with the subject "Tripadvisor Review - Store 1912." Please include your screen name... More
i ordered the handbattered shrimp it was not cooked all the way i told waitress about it when she finnally came back to table she left i told her not to worry about it i didnt want anything my husbands drink stayed emty almost the whole time just not good waitress there at all and anything fried is always way... More
Hi Monique and Robert, Thanks so much for sharing your comments. Guest satisfaction is our utmost importance and on behalf of Outback Steakhouse I want to apologize about the issues you had with your food. Please take a moment to email me directly so I can follow up with you and ensure your comments are addressed. You can email firstname.lastname@example.com... More
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text: The wiki last edited by pikahyper on 01/25/14 05:09PM View full history Dan had it all: no worries, no responsibilities, no hassles, no chance of injury. But now he's got a couple of ankle biters that are counting on him to keep them safe. While he may be tough to hurt, they're not, and a whole lot of sick, twisted people are going to try!


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Patented locking pushbutton mechanism for fast and easy adjustments. No handle squeezing; works with one-handed downward pressure. Self-gripping jaws for a secure and non-slip grip... View Details
Versatility of a wrench with the convenience of pliers. Push-button adjustment mechanism maintains jaw size setting until reset; great for repetitive work, also saves operator time. Smooth, perfectly parallel jaws will not damage fastener; excellent for chrome or other soft fittings... View Details
Quick set style features a blue colored push-button that allows pliers to be adjusted by moving the handles to the closed position or by pressing the push-button and adjusting the pliers the traditional way. Patented locking push-button adjustment maintains the jaw size setting and does not have to be re-adjusted after each use; saves operator time and is fast and easy to use. Self-gripping jaw design provides a secure, non-slip grip. Does not require handle squeezing; downward pressure on the top handle provides a secure grip and won't round off nuts or bolts... View Details
Patented locking push-button mechanism for fast and easy adjustments. No handle squeezing; works with one-handed downward pressure. Jaw capacity: up to 3-3/4"... View Details
10" Cobra and Alligator pliers jaw protectors. 5 sets of jaw protectors in each package. Jaw protectors increase the versatility of the Alligator and Cobra series pliers by allowing them to be used on softer metal or polished metal applications... View Details
The S-Type jaw on the Swedish Pipe Wrench provides a grip with 3-points of contact to securely handle round workpieces without slipping perfect for tie rod alignments. Gripping capacity of 1 5/8". The Swedish pattern design is fast and easy to adjust - simply put the top jaw on the workpiece slide the bottom jaw up to the workpiece and twist the adjusting nut to tighten... View Details
10" Cobra pliers and 10" diagonal cutters are among our most popular items. Cobra pliers offer a self gripping jaw, fast and easy push button adjustment mechanism, 25 position adjustment and 2 inch jaw capacity. Diagonal cutters offer a high leverage cutting design, induction hardened cutting edges (64 HRC) and require 20% less hand force... View Details
No handle squeezing; works with one-handed downward pressure. Self gripping jaws for a secure, non-slip grip. Specially hardened jaw teeth for longer tool life... View Details
Compact 5" length easily fits into a pocket. Offers the versatility of a wrench with the convenience of pliers. Jaw capacity is up to 7/8" with 11 adjustment positions that assure a precise fit... View Details
PU3SA set contains the following three items: PNC8-8" Long nose pliers, PBDC8-8" Bent high leverage diagonal cutter, PC10-10" Cobra pliers. Long nose pliers feature an integrated cutter and special construction that minimizes jaw tip breakage. Diagonal cutter offers a high leverage design for fast and easy cutting, a forged axle rivet for longer tool life and a 12-degree angled head for greater hand clearance when cutting on a parallel surface... View Details


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- 1GB 128-Bit GDDR5
- Core Clock 875MHz
- 1 x DVI 1 x HDMI 1 x DisplayPort
- 800 Stream Processors
- PCI Express 2.1 x16
Short Life, moderate performer 10/06/2012
This review is from: MSI Radeon HD 5770 DirectX 11 R5770 Hawk 1GB 128-Bit GDDR5 PCI Express 2.1 x16 HDCP Ready CrossFireX Support Video Card
Worked great for about 14 months under moderate use in a very well ventilated box then began to exhibit weakening
Purchased 01/07/2011 Now the fans are loud at start up and the GPU's seem to be weakening. I've got EVGA 8800's that still work today and even earlier cards in Beta test units that are still working. These were advertised with "Military Grade" components - Yea, right.
Other Thoughts:
I've been around the block with MSI on several other pieces of hardware and should have learned my lesson then. Running a Beta test facility all the hardware gets moderate to light usage with short periods of heavy usage in boxes that are maintained weekly (cleaned, tested and so on). If hardware fails in under a couple years use in our facility you now that it weak and was never really stress tested. Guess whose motherboards and Video Cards we won't be using any longer....
Manufacturer Response:
Dear Valued Customer,
We apologize for the issues that you are having with this product.
Please kindly visit the URL below to set up a RMA with the following information.
1. Full name (first, and last)
2. Telephone #
3. Physical address
4. Email address
5. Serial number (located on the box, or the back of VGA card, eg 602-xxxx-xxxBxxxxxxxxxx)
6. Symptoms you are having onto the Special Remark section (as detailed as possible)
Once your information and warranty coverage are verified then we can proceed with this RMA for you.
Our turn-around time is typically about one week and our system will notify you automatically at every stage of your RMA process.
If you have any questions or concerns regarding the product, received the unit back from RMA but are still having issues, please do not hesitate to contact us directly at email@example.com and we will be more than happy to assist you, thank you.
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Nightlife is a 9 piece dance ensemble that is focused on making your next event a success. We strive to offer any type of musical entertainment that a event planner or bridal couple is looking for. Examples are: Classical instrumentalists, Singers, String Quartets, Jazz combos, DJ's, Wedding Bands, Sound/Lighting, and more. Corporate party? Wedding reception? Birthday Bash? Whatever the function,...
Copyright © 2001-2017 OneWed Inc.
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Copyright © 2001-2017 OneWed Inc. All Rights Reserved.


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Although I haven't read all of his works, Vladimir Nabokov is one of my personal favorite writers. I love Pale Fire and Lolita, but also like the less tricksy novels, like Laughter in the Dark. (Which was on Lost!) And for the past year(s) I've been planning on reading The Gift and Ada, or Ardor, both of which I'm even more excited to get to after reading about Lila Azam Zanganeh's new book The Enchanter. From The Guardian:
In The Enchanter, Lila Azam Zanganeh attempts to create a new genre: the Bildungs-romance. The book details its author's love affair with Nabokov, and tells how she learned to read books from his novels, using them as doorways to magical worlds.
Rather than attempt conventional biography or literary criticism, she portrays a series of encounters with moments from Nabokov's biography – his last months in Switzerland, his childhood in Russia, his early love affairs. She invents an interview with "VN" about his years in the USA, and provides a whimsical anti-glossary for some of his flamboyantly obscure vocabulary.
All of this is interwoven with Zanganeh's responses to Nabokov's writing, particularly Ada, but also Lolita and Laura, with the theme of happiness always central. By this, Zanganeh explains, she does not mean "platitudinous happy characters", but "deep joyousness", "bliss", or "ecstasy". This is a feeling "connected to the edge, an experience of limits (in its quasi-mathematical sense of an open-end), which in turn becomes one of extreme poetry".
Lila has moderated any number of French-literature related panels that I've attended, always coming off as quite brilliant and composed. (In my mind, her composure is in stark contrast to my shambolic attempts to run a panel. And since we usually go right after one another, her preparedness is even more accentuated . . . ) Which is another reason why I'm excited to get a copy of this. And why I'm excited she's been getting so much attention for this book.
In addition to the above review, the Guardian also ran this interview:
Zanganeh's love of Nabokov led her to want to write about him. Not surprisingly, given her academic background (before going to Harvard she studied at the Sorbonne and the École normale supérieure), her first thought was to write "something serious". "But then I thought, wait a second, Nabokov hated didactic works. How could you say, 'Listen people, Nabokov is a writer of happiness, so let me show you in a serious and well-constructed disquisition'? It would have been boring to death!" She was also put off by reading Nabokov critics, who are obsessed, she says, with questions surrounding the morality of his work. "But the whole point is that his work lies outside the realm of morals, beyond good and evil. He said in the afterword to Lolita that there is no moral in tow, that it is a magnificent game of chess with the reader."
And then in The Daily Beast there's a piece about how she met Dmitri Nabokov in order to get permission to quote from his father's works in her book:
Dmitri himself, now 76 and looking uncannily like his father at about the same age, was seated in a wheelchair, at the head of the dining table. To his left was Ariane, a blond lady with an edgy sense of humor who turned out to be an old friend of Dmitri's, and like him a former opera singer. Dmitri's ice-blue eyes looked dazed and sleepy that night. Ever the gentleman, however, he announced that he, together with Ariane, had read and appreciated the first two chapters. The most conventional ones from a narrative standpoint, I immediately thought to myself, and felt my temperature drop. He had read only a fragment, and everything remained uncertain. I joined them at the table for a three-course meal, and tried to ease into a semblance of small talk. He could yet hate, and kill, the book I had worked on for the last several years. A moment later, Dmitri Nabokov looked up, smiled, and declared he had come up with a good idea. He was too exhausted to read the manuscript, he confessed, so would I kindly read it out loud to him over the span of the next few days? I glanced at Ariane, who nodded in agreement. [. . .]
As the days and nights unfolded, my heart often pounding, I read to Dmitri passages from Lolita, Ada, or Ardor, Speak, Memory, and countless short stories, but also imaginary interviews and fictional accounts of his father's erotic life. The page at times elicited fierce reactions, even fits of anger ("Why, please tell me why you need to invent this? It almost sounds too close to home!"), but also moments of gentleness. His critical ear remained sharp throughout the reading. He not only critiqued and edited, but also corrected grammar ("Is it onto or unto?"), diction ("Vladimir and his mother would never 'argue' over a weak serve! Perhaps you meant 'squabble'?"), and, most precious of all in my sense, pronunciation. Dmitri or I often picked up the dictionary to check British and American variations, and sometimes, to my relief, found we were both right, as in the case of "skein" (pronounced both "skeen" and "skeyn"). Dmitri also taught me to pronounce the names of butterflies (Ly'caeides Samue'lis), myriad plants, and the Latinate shades of many other words I had read and written but never said aloud.
Sounds like a fun experience, and an interesting book. If possible, we'll run a full review of this in the not-too-distant future.
Prose translators will likely disagree, but I believe translating poetry requires a significant level of talent, a commitment to the text, and near mania, all of which suggests that the undertaking is the greatest possible challenge. The task is to. . .
The biggest issues with books like The Subsidiary often have to do with their underpinnings—when we learn that Georges Perec wrote La Disparition without once using the letter E, we are impressed. Imagine such a task! It takes a high. . .
Following The Infatuations, Javier Marías's latest novel seems, like those that have preceded it, an experiment to test fiction's capacity to mesmerize with sombre-sexy atmospheres and ruminative elongated sentences stretched across windowless walls of paragraphs. Thus Bad Begins offers his. . .
Death by Water, Kenzaburo Oe's latest novel to be translated into English, practically begs you to read it as autobiography. Like The Changeling, as well as many other works not yet released in English, Death by Water is narrated in. . .
Jocelyne Saucier's Twenty-One Cardinals is about the type of unique, indestructible, and often tragic loyalty only found in families. For a brief but stunningly mesmerizing 169 pages, Twenty-One Cardinals invited me in to the haunting and intimate world of the. . .
We know so very little; so little that what we think to be knowledge is hardly worth reckoning with at all; instead we ought to settle for being pleasantly surprised if, on the edge of things, against all expectations, our. . .
Many of Virginie Despentes's books revolve around the same central idea: "To be born a woman [is] the worst fate in practically every society." But this message is nearly always packaged in easy-to-read books that fill you with the pleasure. . .
Ilja Leonard Pfeijffer's La Superba is appropriately titled after the Italian city of Genoa, where, after escaping the pressures of fame in his own country, the semi-autobiographical narrator finds himself cataloguing the experiences of its mesmerizing inhabitants with the intention. . .
It took reading 44 pages of Intervenir/Intervene before I began to get a sense of what Dolores Dorantes and Rodrigo Flores Sánchez were up to. Recurring throughout these 44 pages—throughout the entire book—are shovels, shovel smacks to the face, lobelias—aha!. . .
As presaged by its title, contradiction is the theme of Peter Stamm's novel, All Days Are Night. Gillian, a well-known television personality, remains unknowable to herself. And Hubert, a frustrated artist and Gillian's lover, creates art through the process of. . .


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That the talks have been in session since June without reaching consideration of substantive political issues has meant their public credibility has ebbed. With the general election and council elections due, adjournment was seen as inevitable. Their resumption is expected after the polls.
In the meantime, the dangers of a political vacuum were emphasised when it emerged that a bomb left at a Sinn Fein office in the border town of Monaghan had not been a hoax, as first reported.
The bomb, planted on Sunday, contained 26lb of commercial explosives, which could have brought down one or more buildings. In the event only the detonator exploded, causing no significant damage.
It is believed to be the work of the Ulster Volunteer Force. Its significance is that it was the first bombing attack by the group since it declared a ceasefire in October 1994 and throws doubt on the continuing commitment of the loyalist paramilitary groups to political rather than violent activity.
The loyalists had previously feared such acts would lead to expulsion of their political spokesmen from the talks. But with the talks in abeyance this constraint may be lifted.
The immediate view in security circles was that the attackwas probably intended as retaliation for the recent IRA killing of a soldier in south Armagh. As such, sources say, they believe it was a "one-off" rather than the beginning of a concerted campaign.
- More about:
- Bombing
- Irish Politics
- Northern Ireland
- Ulster


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An icon for a social gifting app. With the app user can give his friends discounts for various products and services. Kind of what Wrapp does.
Attached version with white background as well.
Oh, the icon on top of the box is actually app's logo.
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Show and tell for designers
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Mommy Makeover
A mommy makeover is custom tailored for each individual, consisting of a combination of plastic surgery procedures designed to counteract the physical effects of childbearing and restore the aesthetics of the body.
Although the after-effects of pregnancy can seem merely physical, Dr. Harrington, a top female cosmetic surgeon, knows that childbearing can also take a toll on a woman's emotional wellness. She and her caring team of experienced medical professionals work hand-in-hand with each patient to address both their physical and emotional concerns.
This highly personalized approach allows Dr. Harrington to produce the best possible aesthetic results in a safe and comfortable environment.
The most common procedures performed during a mommy makeover include:
- Liposuction
- Tummy tuck
- Breast augmentation (silicone/saline implants)
- Breast reduction
- Breast lift
- Labiaplasty
Is Mommy Makeover Right for Me?
A mommy makeover is designed to help women reclaim the figure that they possessed prior to having children. It is performed to:
- Restore youthfulness
- Eliminate most stretch marks
- Reduce excess fat
- Restore breast volume and perkiness
- Improve self-confidence
- Promote physical and emotional wellness
Mommy Makeover Postoperative Care and Recovery
The recovery time for mommy makeover greatly depends on the number and complexity of the procedures performed. Some mommy makeover procedures can be carried out at one time, while others are performed during multiple visits.
Mommy makeovers are most often performed in our accredited surgery center, which ensures maximum comfort and safety.
The specific risks and the suitability of mommy makeover surgery for a given individual can be determined only at the time of consultation at our Minneapolis plastic surgery office. All surgical procedures have some degree of risk. The information on this Twin Cities plastic surgeons' website is not a substitute for a consultation. Call us at (651) 290-7600 or contact us online to schedule your complimentary personal consultation with Dr. Jennifer Harrington.


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- 1 a person whose job is to visit schools, factories, etc. to check that rules are being obeyed and that standards are acceptable a school/health/safety, etc. inspector see also tax inspector See related entries: People in schools, Professions
- 2 (abbreviation Insp) an officer of middle rank in the police force Inspector Maggie Forbes see also chief inspector See related entries: The police
- 3 (in Britain) a person whose job is to check tickets on a bus or train to make sure that they are valid See related entries: Train and bus travel Word Origin early 17th cent.: from Latin, from inspicere (from in-
nounjump to other results
BrE BrE//ɪnˈspektə(r)//; NAmE NAmE//ɪnˈspektər//People in schools, The police, Train and bus travel, Professions
Check pronunciation: inspector


text: I grew six seedlings from the cross and saw five bloom. The sixth had the bloomstalk frozen out. Three had form like this one, but in different colors. The one I liked best had yellow falls and may be a bloomout since all the fans bloomed. The remaining two looked quite different. At first I didn't think I cared much for them, but changed my mind one morning. That happens. I think this one is now forming a pod. Two of the siblings have pods. The potential bloomout didn't set pods and I tried nearly every bloom, but the pollen worked so there are three pods on another cross using it. Hopefully it will put out some increase and won't disappear. These were all fairly tall plants. Seedlings tend to be taller the first year, so these will probably be about the same as Heart Stealer when they are growing under leaner conditions than first year seedlings. None of them much resembled Heart Stealer or Feeling Sorta Blue. Or anything else I have growing here, for that matter. Go figure!


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Camo Cross Polished Titanium Ring
Camo Cross Polished Titanium Ring
- To keep pushing the limits on design and things that guys would actually want to wear, we have released this new camo ring!
- This is a simple titanium band that features a nice flat profile through the center with a clean beveled edge.
- The ring design has a cross carved into the band through the center portion of the titanium band. Shown here in a 9mm width and a Oak Breakup inlay.
- Sizes 6-14 including Half Sizes
- Light-Weight
- Made to order in just 7 days
CLICK HERE TO SEE THE ENTIRE CAMO LIFE COLLECTION!***Also we offer free engraving up to 15 letters inside of ring. Please leave what you want engraved in the note section. ****
***Note*** Engraving can delay shipping for up to 10 days, so please take that into account if it is time sensitive.
We Also Recommend


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Building off their original 2007 collaboration, Hong Kong-based Know1edge creates a couple of new washes and fits in their joint denim venture with Diamond Supply Co. The outcome features both a distressed wash pair of relaxed fitting joints and a more slim raw denim profile. Both are now available at Karmaloop.


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Dodgers acquire Nolasco for three minor-leaguers
The Dodgers addressed their concerns at the back of their starting rotation Saturday by acquiring right-hander Ricky Nolasco from the Miami Marlins in exchange for three minor-league pitchers.
The Dodgers were one of several teams pursuing Nolasco. Their willingness to take on the remainder of his $11.5 million salary this season (approximately $5.7 million) apparently tipped the scales in their favor.
They also receive an additional $197,000 in international signing bonus money from the Marlins in the deal. The Dodgers also acquired 'slot money' in their trade with the Chicago Cubs for Carlos Marmol earlier this week.
Nolasco, 30, has an 81-72 career record, 4.44 ERA and 1.30 WHIP in eight seasons with the Marlins. In 18 starts this season, he is 5-8 with a 3.85 ERA.
The Dodgers were able to acquire Nolasco without giving up any of their best prospects from a minor-league system very much in rebuilding mode.
The three right-handers sent to Miami were relievers Steven Ames and Josh Wall from Triple-A Albuquerque and Angel Sanchez a starter in Class-A.
Wall, 26, has made 13 relief appearances with the Dodgers this year and in 2012. He was 1-2 with a 5.60 ERA for Albuquerque this season. Ames, 25, was 2-2 with a 3.67 ERA there.
Sanchez, 23, was a combined 2-7 with a 4.67 ERA as a starter with Great Lakes and Rancho Cucamonga.
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Data from Preqin shows that private equity is sitting on the largest amount of dry-powder ever raised over a year-long period in history. The dry-powder, meaning money that is pledged but not invested, has ballooned to $1.074 trillion, overtaking the previous record of $1.067 trillion that was accumulated until the end of 2008. The dry powder near the close of 2013 is up 14% compared to 2012.
Read about Paul Singer's hedge fund raising new capital.
Stagnant deal volume in private equity
Preqin also points out that despite the excessive amount of fundraising, deal volume in private equity industry has more or less stayed the same. This implies that even though the industry has many eager investors, these firms are not finding it as easy to find and invest in attractive opportunities. The aggregate value of buyout investments barely moved in the past three years; it amounted to $264.4 billion in 2011, $263.8 billion in 2012 and $265.8 billion in 2013 YTD. Preqin notes in their analysis,
"As many economies go through periods of stabilization, and experience continued growth, 2014 may present more opportunities for private equity firms to deploy their capital, and we may see an reduction in the level of dry powder."
Preqin also finds that while distressed private equity and growth dry-powder has increased compared to pre-crisis levels, the capital available for exclusive use in buyout opportunities came at $397 billion, down 18% compared to the funds that were available at the end of 2008. Pledged funds for distressed PE are at $74 billion compared to $56 billion in 2008, whereas growth dry powder has increased to $76 billion compared to $46 billion in 2008.
Fortress and KKR ink new deals
Speaking of low deal volume and lack of buyout opportunities, Fortress Investment Group LLC (NYSE:FIG) is closing in on the acquisition of servicing advances from Nationstar Mortgage LLC, a subsidiary of Nationstar Mortgage Holdings Inc (NYSE:NSM). The deal is fronted by FIG's permanent capital vehicle, New Residential Investment Corp and would amount to $3.2 billion. FIG owns 75% shares of Nationstar Mortgage Holdings Inc (NYSE:NSM), RBC Capital Markets' Bluent Oczan expects that the acquisition will add to Fortress' income, as New Residential Corp pays performance and management fees to its parent.
At the same time KKR & Co. L.P. (NYSE:KKR) is finalizing the acquisition of KKR Financial Holdings LLC (NYSE: KFN) in a $2.6 billion buy-out. The deal is expected to bring long-term benefits for the private equity firm with payments from KFN's earnings. Morgan Stanley (NYSE:MS)'s Melissa Bourdon said in a note that this will help KKR & Co. L.P. (NYSE:KKR) in diversifying its portfolio away from private equity and will also provide it with a stronger balance sheet. This would help KKR in launching new funds and pursue further M&A, which will eventually attract new clients and bring in fee-paying AuM.


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|Message view
|« Date » · « Thread »
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|"Mark Harwood (JIRA)" <j...@apache.org>
|[jira] Commented: (LUCENE-1720) TimeLimitedIndexReader and associated utility class
|Mon, 29 Jun 2009 13:59:47 GMT
[ https://issues.apache.org/jira/browse/LUCENE-1720?page=com.atlassian.jira.plugin.system.issuetabpanels:comment-tabpanel&focusedCommentId=12725176#action_12725176 ] Mark Harwood commented on LUCENE-1720: -------------------------------------- bq. Oh, I did not mean to skip this check. But the check is on a variable with a yes/no state. We need to cater for >1 simultaneous timeout error condition in play. With only a boolean it could be hard to know precisely when to clear it, no? bq. Mark here wanted to provide a much more generalized way of stopping any other activity, not just search To be fair I think the use case for IndexWriter is weaker. In reader you have multiple users all expressing different queries and you want them all to share nicely with each other. In index writing it's typically a batch system indexing docs and there's no "fairness" to mediate? Breaking it out into a utility class seems like a good idea anyway. > TimeLimitedIndexReader and associated utility class > --------------------------------------------------- > > Key: LUCENE-1720 > URL: https://issues.apache.org/jira/browse/LUCENE-1720 > Project: Lucene - Java > Issue Type: New Feature > Components: Index > Reporter: Mark Harwood > Assignee: Mark Harwood > Priority: Minor > Attachments: ActivityTimedOutException.java, ActivityTimeMonitor.java, TestTimeLimitedIndexReader.java, TimeLimitedIndexReader.java > > > An alternative to TimeLimitedCollector that has the following advantages: > 1) Any reader activity can be time-limited rather than just single searches e.g. the document retrieve phase. > 2) Times out faster (i.e. runaway queries such as fuzzies detected quickly before last "collect" stage of query processing) > Uses new utility timeout class that is independent of IndexReader. > Initial contribution includes a performance test class but not had time as yet to work up a formal Junit test. > TimeLimitedIndexReader is coded as JDK1.5 but can easily be undone. -- This message is automatically generated by JIRA. - You can reply to this email to add a comment to the issue online. --------------------------------------------------------------------- To unsubscribe, e-mail: email@example.com For additional commands, e-mail: firstname.lastname@example.com
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Valentine's Day video present from Australia
Love is a many-splendored thing, or so the song goes. It's that time of year again, when people get to express their undying devotion to one another. This sometimes takes the form of a card and wilted flowers picked up at the local drug store on the way home. But for the two love birds in this video, true love is a reality, at least in the unreal world of commercials. Unfriendly onlookers and the occasional flying bathtub can't stop … Continue reading


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31st MEU to assist Saipan during typhoon recovery
DVIDS | Aug 10 2015
SAIPAN, Northern Mariana Islands (Aug. 7th, 2015) - Marines and sailors with the 31st Marine Expeditionary Unit arrived in Saipan today to assist with typhoon recovery efforts after the island was hit by a typhoon.
Approximately 600 Marines and sailors of the 31st MEU, aboard the USS Ashland (LSD 48) of the Bonhomme Richard Amphibious Ready Group, will work with the Federal Emergency Management Agency to deliver much needed relief supplies after the island was devastated by Typhoon Soudelor Aug. 2-3.
The MEU has also staged four MV-22B Ospreys in nearby Guam. The aircraft will be on standby to provide aerial support for recovery efforts if needed.
The 31st MEU was in the vicinity of the Mariana Islands for training as part of a regularly scheduled deployment to the Asia-Pacific region. The Marines and sailors were rerouted with orders to assist recovery operations Aug. 5.
"We will work closely with FEMA to bring relief to the people of Saipan as quickly as possible," said Col Romin Dasmalchi, the 31st MEU commanding officer. "As a maritime contingency force, the MEU's ability to quickly respond to a variety of crises across the range of military operations is key, particularly here in the Western Pacific where natural disasters like this are a fairly regular occurrence."
The 31st MEU provides a forward-deployed, flexible sea-based force capable of conducting amphibious operations, crisis response and limited contingency operations in the Asia-Pacific area. The 31st MEU is the only continually forward-deployed MEU and remains the Marine Corps' force-in-readiness in the Asia-Pacific region.
The Defense Video & Imagery Distribution System (DVIDS) is provided as a public service operated by Third Army/U.S. Army Central (ARCENT) on behalf of the Department of the Army in support of all branches of the U.S. military (Navy, Air Force, Marines) and its Coalition partners serving in the U.S. Forces Central Command (CENTCOM) area of responsibility.
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