Danggui, also known as Angelica sinensis (Oliv.) Diels (Apiaceae), has been used in Chinese medicine to treat menstrual disorders. Over 70 compounds have been isolated and identified from Danggui. The main chemical constituents of Angelica roots include ferulic acid, Z-ligustilide, butylidenephthalide and various polysaccharides. Among these compounds, ferulic acid exhibits many bioactivities especially anti-inflammatory and immunostimulatory effects; Z-ligustilide exerts anti-inflammatory, anti-cancer, neuroprotective and anti-hepatotoxic effects; n-butylidenephthalide exerts anti-inflammatory, anti-cancer and anti-cardiovascular effects. Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), the root of which is known in Chinese as Danggui  and has been used as a blood tonic to treat menstrual disorders [Danggui is marketed in various forms worldwide [Danggui, including essential oils such as ligustilide, butylphthalide and senkyunolide A, phthalide dimers, organic acids and their esters such as ferulic acid, coniferyl ferulate, polyacetylenes, vitamins and amino acids. Z-ligustilide (water insoluble and heat stable), among which Z-butylidenephthalide and ferulic acid are thought to be the most biologically active components in AS [Danggui [i Figure , was firisorders . Dangguits in AS  and are [Danggui .Danggui [Ligusticum chuangxiong [Z-ligustilide is the main lipophilic component of the essential oil constituents and a characteristic phthalide component of a number of Umbelliferae plants. Z-ligustilide is considered to be the main active ingredient of many medicinal plants, such as Danggui  and Liguangxiong .Danggui was reported in the literature when the Z-ligustilide dimer E-232 was isolated [Danggui varies between 1.26 and 37.7 mg/g dry weight [Phthalides Figure  consist isolated . The majy weight ,11. Z-liy weight -14. Precy weight .Danggui contains many organic acids. For example, ferulic acid , 70% methanol, 70% ethanol, 50% ethanol or diethyl ether-methanol (20:1) is used as the initial extraction solvent. The amount of ferulic acid in Danggui varies between 0.21 and 1.75 mg/g dry weight [d Figure  isolatedy weight ,16. We ry weight . Abundany weight -21.Danggui is associated with its various polysaccharides [Danggui consist of fucose, galactose, glucose, arabinose, rhamnose and xylose [Danggui contains a neutral polysaccharide and two kinds of acidic polysaccharides [Biochemical and medical researchers have recently been interested in the anti-tumor and immunomodulatory effects of polysaccharides . The effcharides  which ard xylose . Dangguicharides .Danggui [2 production in lipopolysaccharide (LPS)/IFN-γ-stimulated murine primary peritoneal macrophages. Ferulic acid and Z-ligustilide, two major compounds in AS, decrease NF-κB luciferase activity, which may contribute to the anti-inflammatory activity of AS [in vivo study further confirmed that the ethyl acetate extract of AS inhibits the production of inflammatory mediators thereby alleviating acute inflammatory hazards and protecting mice from endotoxic shock [et al. reported that the leukocyte count in the pouch exudate decreases in the BALB/c mice fed with 100 mg/kg body weight of a root extract , accompanied by a decrease in the neutrophil count, IL-6 mRNA level and TNF-α mRNA level in the pouch membrane and by decreased IL-6 and PGE2 concentrations in the pouch fluid and that the concentration of anti-inflammatory PGD2 in the pouch fluid increases as well [et al. reported that n-butylidenephthalide decreases the secretion of IL-6 and TNF-α during LPS stimulated activation of murine dendritic cells 2.4 via the suppression of the NF-κB dependent pathways [Ferulic acid and isoferulic acid inhibit macrophage inflammatory protein-2 (MIP-2) production by murine macropharge RAW 264.7 cells, suggesting that these compounds contribute to the anti-inflammatory activity of AS ,26. Z-liDanggui . AS suppty of AS . Our in ic shock . Using a as well . Fu et apathways .0/G1 in brain tumor cell lines [et al. reported that pretreatment of the PC12 cells with Z-ligustilide attenuates H2O2-induced cell death, attenuates an increase in intracellular reactive oxygen species (ROS) level, decreases Bax expression and cleaves caspase-3 and cytochrome C [via an increased activity in the caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) [AS extract induces apoptosis and causes cell cycle arrest at Gll lines . AS extrll lines . Moreovell lines  and leukll lines . The thrll lines . Yu et achrome C . A novele (PARP) .BALB/c mice spleen cells with AS polysaccharide (100 μg/ml) increases the production of IL-2 and IFN-γ and decreases the production of IL-4 [BALB/c murine peritoneal macrophages to produce higher levels of nitric oxide (NO) via the induction of iNOS gene expression [BALB/c mice from retro-orbital bleeding induced anemia and increases IL-6, granulocyte-macrophages colony stimulating factor (GM-CSF) concentrations in spleen cells [Treatment of  of IL-4 . An acidpression . The AS pression . Four hypression . AS polyen cells .2O2-induced IL-1β, TNF-α, matrix metalloproteinase-1 and matrix metalloproteinase-13 levels and increases SRY-related high mobility group-box gene 9 gene expression in chondrocytes [Listeria monocytogenes-infected BALB/c mice [Ferulic acid, an antioxidant from AS, decreases Hdrocytes . AS indudrocytes . PretreaB/c mice .in vitro and in vivo [Pre-treatment with AS (15 g/kg daily for 4 weeks) decreases doxorubicin-induced (15 mg/kg intravenously) myocardial damage and serum aspartate aminotransferase levels in male ICR mice . Human u in vivo .et al. demonstrated that n-butylidenephthalide is anti-angiogenic and is associated with the activation of the p38 and ERK1/2 signaling pathways [Excess adipose tissue can lead to insulin resistance and increases the risk of type II diabetes and cardiovascular diseases. Water and 95% ethanol extracts of AS effectively decrease fat accumulation in 3T3-L1 adipocytes and reduce triglyceride content . Yeh et pathways .et al. reported that AS extract protects Neuro 2A cell viability against β-amyloid (Aβ) peptide induced oxidative damage by ROS, MDA and glutathione (GSH) and rescues mitochondrial transmembrane potential levels [Z-ligustilide treatment decreases the level of malondialdehyde (MDA) and increases the activities of the antioxidant enzymes glutathione peroxidise (GSH-Px) and superoxide dismutase (SOD) in the ischemic brain tissues in ICR mice; meanwhile there is a decrease in Bax and caspase-3 protein expression . Z-ligusl levels . Z-ligusl levels .1-42 induced neurotoxicity and tau hyperphosphorylation in primary cortical neurons [AS methanol extract significantly attenuates Aβ neurons . AS poly neurons .Panax ginseng, Acanthopanax senitcosus, AS and S. Baicalensis, HT008-1 down regulates COX-2 and OX-42 expression in the penumbra region [A multi-herbal mixture composed of a region .BALB/c murine peritoneal macrophages pretreated with various AS polysaccharide fractions alleviate the decrease in cell survival caused by tert-butylhydroperoxide, with an increased intracellular GSH content [2O2. Acidic polysaccharide fraction also decreases the MDA formation, reduces the decline in SOD activity and inhibits the depletion of GSH in murine peritoneal macrophages caused by H2O2 [Water AS extract can be further purified into various AS polysaccharide fractions, such as a highly acidic polysaccharide fraction consisting of galacturonic acid.  content . Further by H2O2 . Ethanol by H2O2 .et al. reported that water and ethanol extracts of AS strongly increase the CYP2D6 and CYP3A activity in the microsome fraction of male Wistar rat livers [et al. demonstrated that treatment with Danggui Buxue Tang (DBT), which contains the roots of Astragali and AS, induces erythropoietin mRNA expression in a dose-dependent manner in human hepatocellular carcinoma cell line Hep3B [et al. reported that Z-ligustilide targets cysteine residues in human Keap1 protein thereby activating Nrf2 and the transcription of antioxidant response element (ARE) regulated genes and inducing NADPH:quinine oxidoreductase 1 (NQO1) [The liver contains a series of microsome hemoproteins called cytochrome P450s (CYPs). CYPs play an important role in the metabolic oxygenation of a variety of lipophilic chemicals including drugs, pesticides, food additives and environmental pollutants. The most important isoenzyme forms of cytochrome are CYP1A2 (13%), CYP2C (20%), CYP2D6 (2%), CYP2E1 (7%) and CYP3A (29%) . Tang ett livers . Gao et ne Hep3B . Dietz e1 (NQO1) .There has been a 60% increase in the number of people needing treatment for chronic kidney disease between 2001 and 2010. Characterized by an increase in interstitial fibrosis and tubular epithelial cell atrophy, renal tubulointerstitial fibrosis is the common pathogenetic process of chronic kidney disease -67. AngiAstragalus membranaceus var. mongholicus and AS (14 g/kg/day) to Wistar rats increases the constitutive ROS activity in the kidneys. The treatment also enhances NO production via eNOS activation and the scavenging of ROS in the obstructed kidney in Wistar rats after unilateral ureteral obstruction [Astragalus membranaceus var. mongholicus and AS into Sprague Dawley rats, as a unilateral ureteral obstruction model, decreases TGFβ1 levels, fibroblast activation, macrophage accumulation and tubular cell apoptosis [et al. reported that oral administration of Astragalus membranaceus var. mongholicus and AS (12 g/kg/day) shows renoprotective effects, possibly associated with a reduction of proteinuria and up regulation of VEGF. These changes may have reduced the loss of capillaries and improve microstructure dysfunction in nephrectomized rats [Oral administration of truction . In analtruction . Adminispoptosis . Song etzed rats .Centella asiatica, Glycyrrhiza glabra, Scutellaria lateriflora and Olea europaea have strong antioxidant effects in tubular epithelial cells or apoptotic effects on renal mammalian fibroblasts or both [A study on 47 herbs of potential interest in the context of renal or urinary tract pathologies demonstrated that AS,  or both .BALB/c mice after irradiation (4 Gy) [Ethanol extract of AS  prolongs estrus in rats and the estrogenic activity of AS extract is likely due to the presence of Z-ligustilide . A dichln (4 Gy) . ASP alsn (4 Gy) .Danggui extract or its components include anti-inflammatory, anti-cancer, immunomodulatory, anti-cardiovascular, neuroprotective, anti-oxidative, anti-hepatotoxic and renoprotective activities.Bioactive components extracted from AS roots include Z-ligustilide, ferulic acid and AS polysaccharides. Major pharmacological effects of Angelica sinensis; MIP-2: macrophage inflammatory protein-2; LPS: lipopolysaccharide; VEGF: vascular endothelial growth factor; ROS: reactive oxygen species; JNK: c-Jun NH2-terminal kinase; AP-1: activating protein-1; PARP: poly (ADP-ribose) polymerase; GM-CSF: granulocyte-macrophages colony stimulating factor; ERK1/2: extracellular signal-regulated kinase1/2; HUVEC: human umbilical vein endothelial cell; MDA: malondialdehyde; GSH-Px: glutathione peroxidise; SOD: superoxide dismutase; GSH: glutathione; DPPH: 1,1-diphenyl-2-picryl hydrazine; CYP: cytochrome P450; Nrf2: nuclear factor E2-related factor 2; ARE: transcription of antioxidant response element; ACEi: angiotensin converting enzyme inhibitors; TRP3: transient receptor protein 3; BST-1: bone marrow stromal cell antigen 1; PEX6: peroxisomal biogenesis factor 6; XDH: xanthine dehydrogenase; CYP1A1: cytochrome P450 subfamily I member A1; PAI-1: serine/cysteine proteinase inhibitor clade E member 1; FGF23: fibroblast growth factor 23; STAT3/5: signal transducer and activator of transcription 3/5.AS: The authors declare that they have no competing interests.WWC and BFL searched the literature and wrote the manuscript. Both authors read and approved the final version of the manuscript.


An error was introduced in the preparation of this article for publication. Figure 5 is not complete and not present in its proper place.http://plosone.org/corrections/pone.0067071.g005.cn.tifPlease find a complete version of Figure 5 here: The Figure 5 legend is:pVHL is phosphorylated on Ser72 by Aurora-A in vitroA- Purified recombinant 6xHis-tagged pVHL30 [VHL(His)6] (lane 1) or mutant 6xHis-tagged pVHL30 S72A [VHLSA72(His)6] ( (lane 2) were incubated in the presence of [γ-32P] ATP and recombinant Aurora-A(His)6. Samples were separated by SDS-PAGE and the gel was stained with Coomassie Blue (higher panel). pVHL phosphorylation status was analysed by autoradiography (lower panel).The asterix (*) indicate the pVHL protein.B- Localization and sequence of the putative Aurora-A phosphorylation site on pVHL.


Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies. Lymphocytes originate from hematopoietic stem cells located in the bone marrow. While T-cells complete their development in the thymus, B-cells differentiate in various lymphoid tissues. Lymphoid malignancies emerge in the bone marrow or in secondary hematopoietic organs, acquiring both general and subtype specific mutations including chromosomal rearrangements. Accordingly, subtypes of the diffuse large B-cell lymphoma (DLBCL) differ in mutations and gene activities Deregulated genes in leukemia/lymphoma comprise activated transcription factors (TFs) and signaling components which are either physiologically expressed in early stages of hematopoietic development or ectopically induced. Notable examples include TFs of the basic helix-loop-helix (bHLH) family or constituents of the NOTCH-signaling pathway Homeobox genes encode transcription factors frequently deregulated in cancers, including leukemia/lymphoma, impacting developmental processes during embryogenesis. According to their conserved homeobox sequences, this group of TFs has been classified into several subfamilies On the other hand the clustered HOX genes are usually activated by formation of aberrant chromatin structures in leukemia/lymphoma, although chromosomal aberrations are described in T-ALL Here, we investigate aberrant expression of NKL homeobox gene NKX2-1 in B-cell lymphoma cell line SU-DHL-5. Our data expand the oncogenic role of NKL homeobox genes within the lymphoid system encompassing the B-cell lineage. We demonstrate mechanisms of NKX2-1 activation in addition to examining its downstream effects which include deregulation of cell differentiation in DLBCL.Authenticated mycoplasma-free cell lines were drawn from the DSMZ Cell Lines Bank  and cultivated as described previously www.ncbi.nlm.nih.gov/gds) or from the National Cancer Institute (NCI). The NCI microarray data for SU-DHL-5 are available through the accession numbers SU-DHL-5_SS392729_HG-U133_Plus_2_HCHP-201545_.CEL, SU-DHL-5 _SS392730_HG-U133_Plus_2_HCHP-201546_.CEL, and SU-DHL-5_SS392731_HG-U133_Plus_2_HCHP-201547_.CEL which were combined in this study. Analyses of expression data were performed using Microsoft Excel and online programs. For creation of heat maps we used CLUSTER version 2.11 and TREEVIEW version 1.60 (http://rana.lbl.gov/EisenSoftware.htm). Expression data of 203 DLBCL patient samples were obtained from the NCBI GEO database (accession number GSE11318) as published recently For quantification of gene expression via profiling, we used data obtained by gene chips HG U133 Plus 2.0 from Affymetrix . The datasets were generated at the University of Würzburg and generously provided by Prof. Andreas Rosenwald  or obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (https://cabig.nci.nih.gov/community/caArray_GSKdata/).Genome-wide copy number analysis was performed using the Affymetrix Genotyping Console GTC Software version 4.0 (Affymetrix) and visualized by the Affymetrix GTC-Browser program. The 500K-array dataset for SU-DHL-5 was obtained from the National Cancer Institute , GSK Cancer Cell Line Genomic Profiling Data (Fluorescent in-situ hybridization (FISH) and spectral karyotyping (SKY) analyses were performed as described previously Total RNA from cell lines was extracted using TRIzol reagent . Total human RNA isolated from peripheral blood mononuclear cells (PBMC), bone marrow (BM), lymph nodes (LN), thymus, lung and thyroid was obtained from Clontech , and RNA isolated from CD3-positive T-cells and CD19-positive B-cells from Miltany Biotec . cDNA was subsequently synthesized from 5 µg RNA by random priming, using Superscript II (Invitrogen).Real-time quantitative expression analysis (RQ-PCR) was performed by the 7500 Fast Real-time System, using commercial buffer and primer sets . For normalization of expression levels we used TATA box binding protein (TBP). Quantitative analyses were performed in triplicate and repeated twice. The standard deviations are indicated in the figures as bars.Table S1  and the thermocycler TGradient .Reverse transcription (RT) PCR was performed using taqpol (Qiagen), oligonucleotides as listed in Sodium dodecyl sulphate polyacrylamid gel electrophoresis (SDS-PAGE) was performed in the Mini-Protean 3 system . Gels were either stained with PageBlue  or processed for Western blot analysis via the semi-dry method. Proteins obtained from cell lysates were transferred onto nitrocellulose membranes (Bio-Rad) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: anti-NKX2-1 , anti-NKX3.1 , anti-ERK1 . The secondary antibodies were labelled with peroxidase and detected by Western Lightning-ECL .Table S1 (MWG Eurofins) and thermocycler TGradient (Biometra).ChIP analysis was performed with the ChIP Assay Kit  as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 , anti-H2B , anti-H2Bub1 , anti-H3K4me3  and anti-H3K27me3 . The subsequent ChIP-PCR analysis was performed using taqpol (Qiagen) oligonucleotides as listed in To determine intracellular localizations of proteins we performed immunostaining as described previously Table S1. The PCR products were subcloned into pGEMT-easy  and the inserts sequenced (MWG Eurofins). Sequences of 10 clones were displayed and compared using BiQ Analyzer .SU-DHL-5 cells were treated with 10 µg/ml trichostatin A  or 10 mM 5-Aza-2′-deoxycytidine (Sigma) for 20 h before being examined by RQ-PCR. To identify particular methylated cytidines within the CpG island at the HOPX locus, genomic DNA of SU-DHL-5 and SU-DHL-4 (for control) was subjected to bisulfite-conversion and analyzed as described recently Here, we screened by expression profiling 20 leukemia/lymphoma T- and B-cell lines for aberrant activities of NKL-family homeobox genes which are implicated in T-cell leukemia Quantitative expression analysis of NKX2-1 and NKX3-1 by RQ-PCR confirmed their activation in SU-DHL-5 at the RNA level. While NKX3-1 was also expressed in Hodgkin lymphoma (HL), multiple myeloma (MM), B-cell lymphoma (BCL) and additional DLBCL cell lines, NKX2-1 was nearly undetectable in the same set of 25 lymphoma cell lines Fig. 1A,Protein expression of NKX2-1 and NKX3-1 was analyzed by Western blot. While NKX2-1 protein was clearly displayed in SU-DHL-5, NKX3-1 protein was not detectable in that cell line Fig. 1D,Since deregulated expression of NKL homeobox genes in T-ALL is primarily caused by chromosomal aberrations, we analyzed the karyotype of SU-DHL-5 by SKY, FISH and in a genomic array with respect to the NKX2-1 gene which is located at 14q13 (36.9 Mb). However, copy number data Fig. 1E,Turning to potential transcriptional regulators which might induce aberrant NKX2-1 activity, we compared expression array data of SU-DHL-5 with 3 control DLBCL cell lines - SU-DHL-4, SU-DHL-10, and SU-DHL-16. After inspection of the top 1000 up- and downregulated genes in SU-DHL-5, potential candidates were shortlisted and functionally categorized as shown in Among TFs bHLH factor HEY1 was particularly intriguing (one of the top 10 upregulated genes). RQ-PCR analysis of a panel of cell lines confirmed high expression levels in SU-DHL-5 Fig. 2A.To analyze the regulatory impact of HEY1 on NKX2-1 expression we treated SU-DHL-5 cells with siRNA directed against HEY1. Subsequent quantification of HEY1 and NKX2-1 expression demonstrated reduction of both transcripts as compared to cell samples treated with control siRNA Fig. 2B.Fig. S1), highlighting the role of NKX2-1 in HEY1 regulation.Sequence analysis of the promoter region of HEY1 identified 3 potential binding sites for NKX2-1 Fig. 2C,HEY1 and HES1 are prominent targets of the NOTCH-pathway in lymphopoiesis Expression of Zn-finger homeobox gene 2 (ZHX2) showed elevated levels in SU-DHL-5 as well Table 1.The catalogue of upregulated genes in SU-DHL-5 encoding chromatin components included prominently MLL which is frequently deregulated in leukemia where it activates homeobox genes of the clustered type via H3K4-trimethylation In leukemia MLL is frequently activated by chromosomal aberrations at 11q23 resulting in amplifications or diverse fusion genes , Furthermore, overexpression of histone H3E Table 1 H2B ubiquitinylation levels are regulated by counteracting ubiquitin-transferases  and ubiquitin-specific proteases (USPs) ), showing that HOPX activates NKX2-1 transcription. But this activation was not reciprocal, since reduction of NKX2-1 was unaccompanied by altered HOPX levels . Moreover, examination of DNA methylation of a conspicuous CpG island at the HOPX locus (CpG 109) excluded an abnormal configuration , leaving the mechanism of this striking overexpression elusive. HOPX is associated with E2F6 which showed reduced expression levels in SU-DHL-5  2 , and is removed by histone methylase JMJD3 X levels Fig. 4G.The results of comparative expression analysis in SU-DHL-5 indicated deregulation of several signaling components belonging to a variety of pathways, including TNFa/NFkB/PRKC, TGFb/BMP/SMAD, IL4/STAT3 and NOS1/cAMP/cGMP/phosphodiesterases (PDEs) which plausibly contribute to NKX2-1 and/or HEY1 expression Table 1.Treatment of SU-DHL-5 cells with TNFa for 1 h or 4 h resulted in activated transcription of both NKX2-1 and HEY1 Fig. 5A.Treatment of SU-DHL-5 cells with TGFb resulted in enhanced transcription of both NKX2-1 and HEY1 after 16 h Fig. 5D.The impact of additional pathways in expression of NKX2-1 and HEY1 was analyzed by treatment of SU-DHL-5 cells with IL4, BMP4, IL10 and WNT5B for 16 h Fig. 5G.Finally, we analyzed the impact of cAMP/cGMP-signaling in expression of NKX2-1 and HEY1. This pathway stood out due to overexpression of NOS1 (synthesizes nitric oxide activating guanylate cyclase) and reduced expression levels of several PDEs as indicated by comparative profiling data Table 1 In DLBCL cell line SU-DHL-5 we have identified ectopic expression of NKX2-1 which is activated by bHLH TF HEY1, aberrant modifications of the chromatin structure, and particular signaling pathways. NKX2-1 belongs to the NKL family of homeobox genes which is implicated in the tumorigenesis of T-ALL NKX2-1 is physiologically expressed in the developing lung and thyroid but not, as shown here, in hematopoietic cells. In a physiological context NKX2-1 regulates differentiation processes both during embryogenesis and in the adult Our data ruled out aberrant activation of NKX2-1 via chromosomal rearrangements contrasting with the picture of NKL homeo-oncogenes in T-ALL. We identified several (deregulated) genes involved in NKX2-1 expression by comparative profiling and subsequent knockdown and overexpression studies. Among TFs we identified activating HEY1 which underlies NKX2-1 transcription. HEY1 belongs to the inhibitory subgroup of bHLH proteins, deregulation of which promotes the development of leukemia/lymphoma affecting the function of E2A in driving lymphoid development MLL contributes to enhanced expression of NKX2-1 in SU-DHL-5 cells. It is overexpressed in this cell line via chromosomal rearrangements resulting in duplication of the wild type gene. Tandem triplication of MLL has been described in intravascular large B-cell lymphoma suggesting a more widespread oncogenic role in B-cell lymphomas than hitherto supposed Additionally, overexpression of core-histones in SU-DHL-5 coincided with a chromosomal aberration at 6p22 housing the histone gene cluster 1. H2B in particular was shown to be overexpressed and strongly ubiquitinated at position K120. This modification guides and enhances the process of MLL-mediated H3K4-methylation, indicating cooperation of both types of chromosomal rearrangements in NKX2-1 expression in SU-DHL-5 Several enzymes performing histone-modifications were deregulated in SU-DHL-5, contributing to a permissive chromatin structure at the NKX2-1 gene. RNF and USP genes encode ubiquitin-specific transferases and proteases, respectively, regulating ubiquitination of histone H2B As well as TFs and chromatin-modifiers we identified signaling pathways regulating NKX2-1 expression: firstly, TNFa, NFkB and the NFkB-activating kinase PRKCE were involved in activation of both NKX2-1 and HEY1; second, IL4/STAT3-signaling which was primarily engaged in the activation of NKX2-1; and finally, TGFb/BMP4 and SMAD3 which activated transcription of just HEY1. This last named activity may explain both the presence of neighboring binding sites for NKX2-1 and SMAD seen at the HEY1-promoter, and nuclear colocalization of NKX2-1 and SMAD3 in SU-DHL-5, indicating cooperative activation of HEY1. This coactivation may represent a switch-like regulation which stabilizes gene activities Our data plot the emergence of an aberrant gene regulatory network with NKL homeobox gene NKX2-1 occupying a central role Fig. 6. Transdifferentiation or reprogramming of cells is practicable in several cell types including hematopoietic cells and may be effected by forced expression of particular TFs Taken together, we have identified aberrant expression of NKL homeobox gene NKX2-1 in subsets of DLBCL which is mediated by particular factors including TFs, chromatin mediators, and signaling components. This result expands the oncogenic role of this homeobox gene family within the group of lymphoid malignancies. However, diagnostic and/or therapeutic potentials require additional examinations. Nevertheless, our data may also be of interest for analyses and assessment of NKX2-1 in lung and thyroid cancer.Figure S1Copy number and SKY data of HEY1. Copy number analysis by genomic profiling indicates absence of aberrations at the locus of HEY1 at 8q13 in SU-DHL-5. The insert shows an enlargement of chromosome 8 obtained by SKY karyotyping.(TIF)Click here for additional data file.Figure S2Copy number and SKY data of HOPX. Copy number analysis by genomic profiling indicates absence of aberrations at the locus of HOPX at 4q12 in SU-DHL-5. The insert shows an enlargement of chromosome 4 obtained by SKY karyotyping.(TIF)Click here for additional data file.Figure S3Methylation data for HOPX. The locus of HOPX contains a CpG-island (CpG 109) as shown at the UCSC genome browser (above). RQ-PCR analysis of SU-DHL-5 cells treated with histone deacetylase-inhibitor TSA and DNA-methyltransferase-inhibitor 5-Aza-2′-deoxycytidine (AZA) (right). Data obtained by sequence analysis of bisulfite-treated DNA of SU-DHL-5 and SU-DHL-4 show no significant difference, demonstrating absence of HOPX deregulation via demethylated DNA at CpG 109. Each lollipop represents a CpG; filled lollipops represent methylated CpGs.(TIF)Click here for additional data file.Table S1Oligonucleotides used for PCR.(DOC)Click here for additional data file.


Tyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is expressed in numerous cancers and is required for mediating tumorigenesis. Autocrine and paracrine interleukin (IL)-6 signaling is the principal mechanism by which Stat3 is persistently phosphorylated in epithelial tumors including breast, lung, colon and gastric cancer. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. However, non-tyrosine phosphorylated Stat3 was shown to function as a transcriptional activator, localize to the mitochondria and regulate ATP synthesis and mediate cell migration. Here we examined the role of Stat3 in Ras mediated transformation.Ha-rasV12 transformed mammary epithelial cells (MCF10A-Ras) cells were transduced with a Stat3shRNA, IL-6shRNA and/or treated with inhibitors of Janus kinases (JAKs) to examine the role of the IL-6 signaling pathway in Ras mediated migration, invasion and tumorigenesis.Cellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6. Tumors derived from transgenic MMTV-K-Ras mice were also found to express pStat3 and IL-6. MCF10A-Ras cells, when grown in a three-dimensional Matrigel culture system revealed the appearance of the junctional protein E-Cadherin as a consequence of reducing Stat3 levels or inhibiting Stat3 activity. Decreasing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis and reduced cell migration. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture led to a decrease in IL-6 and pStat3 levels with the reappearance of E-Cadherin.The cellular and environmental context can lead to differential IL-6/pStat3 signaling and a dependency on this cytokine and transcription factor for migration, invasion and tumorigenesis. Stat invasion ,8,9. Morinvasion -12.The ras proto-oncogene encodes a guanine nucleotide binding protein that plays an essential role in diverse cellular responses, including cell proliferation and differentiation . AlthougHere we further investigated the role of non-tyrosine phosphorylated Stat3 in Ras-mediated mammary tumorigenesis. Specifically, we examined the consequences of reducing Stat3 levels in Ras transformed mammary epithelial cells. We determined that Stat3 deficient Ras transformed MCF10A cells were less capable of mediating migration, invasion and tumorigenesis than the control MCF10A-Ras cells. Surprisingly, tumors derived from MCF10A-Ras cells expressed high levels of tyrosine phosphorylated Stat 3 (pStat3) as did mammary tumors from MMTV-expressing K-Ras mice. Furthermore, the interleukin-6 ligand (IL-6) which was recently shown to be a principal regulator of Stat3 activation in breast cancer [5'-TAGCCGCCCCACACAGACAG-3'; reverse primer, 5'-GGCTGGCATTTGTGGTTGGG-3'. The β-actin primers were as follows: forward primer, 5'-CGTGCGTGACATTAAGGAGA-3'; reverse primer, 5'-TGATCCACATCTGCTGGAAG-3'. For quantitative PCR 1 ug of total RNA was reverse-transcribed using the Thermoscript RT-PCR system (Invitrogen) at 52°C for one hour. 20 ng of resultant cDNA was used in a Q-PCR reaction using an iCycler (Biorad) and pre-designed TaqMan ABI Gene expression Assays (Hs00985639_m1 for IL-6). Amplification was carried for 40 cycles . To calculate the efficiency of the PCR reaction, and to assess the sensitivity of the assay, we also performed a seven-point standard curve . To obtain normalized qPCR values for IL-6, triplicate cycle threshold values were averaged, amounts of target were interpolated from the standard curves and normalized to HPRT . Antibodies used were: anti-tubulin monoclonal antibody (1:2500) ; Anti-H-Ras polyclonal antibody (1:1000) (Santa Cruz Biotechnology); Anti-Stat 3 and anti-Tyr 705 Stat3 polyclonal antibodies (1:1000)  and Anti-E-cadherin monoclonal antibody (1:1000) .The pBabe-H-RasV12 construct was a gift from P. Sicinski (Dana-Farber Cancer Institute) . Stat3sh4 cells per well (2 × 103/cm2) were plated into six-well dishes. Cells were counted at Days 1, 2, 3, 4, 5 and 6. Each data point represents the mean value from triplicate wells. All transfections were carried out using Superfect (QIAGEN). Retroviral infections were carried out using the RetroMax Retroviral Expression System pCL-Ampho  according to the supplier. Clonal selection was carried out using puromycin (2 ug/ml) (Sigma). Lentiviral infections were performed using lentivirus-based vectors encoding shRNA IRES eGFP targeted to either Stat3 or IL-6 transcripts, and were generated by transient co-transfection of 293T cells with a three-plasmid combination, as described previously [MCF10A and 293T cells were obtained from the American Type Culture Collection. MCF10A cells were cultured as previously described . 293T ceeviously . EGFP ex4) were starved for 16 hours in media lacking EGF and were subsequently applied to an 8-μm pore cell culture inserts or in Matrigel Invasion Chambers . MCF10A media containing 2% horse serum and EGF was used as the chemoattractant with or without IL-6 (10 ng/ml). After 16 h of incubation, the cells were stained with crystal violet and counted. Each condition was assayed in triplicate, experiments were performed independently at least three times, and the results were expressed as the number of cells per field.Migration/invasion experiments were performed as described previously ,30. BrieMorphogenesis assays were carried out as previously described . Growth Anchorage-independent growth in triplicate was assessed as previously described . Cells  using previously described methods . Imagingt-test. Significant differences between experimental groups were * P < 0.05 or ** P < 0.01.Data are expressed as means ± standard deviation (SD). The statistical significance of differences was evaluated using an unpaired, non-parametric Student's We examined the role of Stat3 in H-RasV12 mediated cell migration, invasion and cellular transformation using H-RasV12 transformed mammary epithelial cells (MCF10A-Ras). MCF10A cells are a spontaneously immortalized human breast epithelial cell line, mutant in the cdk inhibitor p16, yet has many of the characteristics of normal breast epithelium, do not form tumors in nude mice nor form colonies in soft agar, but undergo transformation upon the introduction of Ha-Ras -35. The Ras transformed cells have increased invasive and migratory potential over control non-transformed cells . We inveThe finding that Stat3 is required for invasion and migration in Ras transformed MCF10A cells led us to determine if Stat3 might also be required for tumorigenesis. Anchorage-independent-growth is a measure of a cells capacity to grow in three dimensions, without contacting a basement membrane. We next determined whether Stat3 expression affects anchorage independent growth of MCF10A-Ras cells. Control cells displayed robust colony formation while Stat3sh cells formed very few colonies (approximately 10-fold less) Figure . TumorigWe next examined the levels of pStat3 in these tumors by immunohistochemical and Western blot analyses. Surprisingly, we observed high pStat3 levels in the control tumors , while the Stat3sh tumors had very low levels of pStat3 Figure . The celIt was recently determined that exogenous sources of IL-6 could enhance autocrine production of IL-6 in models of breast cancer where IL-6/pStat3 levels were very low . Furtherin vivo [To further characterize the requirement for Stat3 in MCF10A-Ras cells, we utilized a Matrigel assay to examine growth in three-dimensions (3-D). The culture of MCF10A mammary epithelial cells on a defined basement membrane (Matrigel) results in the formation of polarized, hollow acini which recapitulates several aspects of glandular architecture in vivo . Furtherin vivo . MCF10A-in vivo ,39. ThisTo investigate the relationship between cell transformation and IL-6 signaling, we introduced an shRNA construct targeting the IL-6 mRNA transcript into Ras transformed MCF10A cells. IL-6 shRNA functionality was confirmed using semi-quantitative RT-PCR analysis on cells stimulated with TNF-α, a ligand known to induce IL-6 mRNA production Figure . IL-6 trOur data indicate that MCF10A-Ras cells when grown in two dimensions 2-D) do not express detectable levels of pStat3 Figure . However-D do notWe sought to determine the role of non-tyrosine phosphorylated Stat3 in tumorigenesis by examining the breast epithelial cell line MCF10A cells transformed with the H-RasV12 oncogene. Non-tyrosine phosphorylated Stat3 can function as a transcription factor in association with NF-kB driving expression of a number of genes involved in tumorigenesis including BCL2A1, Rho GAP6, MRAS, MET, RANTES and Cyclin B1 . We examin vivo growth [integrin β6, tenascinC, twist and liv1 [We examined cell proliferation and observed no differences as a function of Stat3 Figure . Furthero growth ,41. In co growth . Activatand liv1 ,43-46. Iand liv1 ,47. We hIL-6 was shown to be expressed to high levels in numerous Ras-expressing cell lines including kidney, fibroblasts, human mammary epithelial cells and pancreatic cancer derived cell lines when grown in 2-D . In contin vivo [MCF10A cells are immortalized human mammary epithelial cells that undergo a program of apical-basolateral polarization, proliferation, growth arrest and apoptosis leading to acinar formation when grown in matrigel -50. Thesin vivo . Expressin vivo . We previn vivo . Furtherin vivo . Here wein vivo environment which exposes epithelial cells to extracellular matrix proteins but also fibroblasts, endothelial cells and macrophages which produce growth factors capable of mediating IL-6 expression. When MCF10A-Ras cells were cultured on plates coated with matrigel, collagen I, collagen IV, fibronectin and laminin we found that matrigel and laminin could induce modest expression of pStat3  or C/EBPβ and CREB ,53. FurtIn order to determine whether the 3-D environment is required for sustained IL-6 expression by epithelial cells we cultured tumor cells (either from MCF10A-Ras xenografts or MMTV-Ras tumors) and passaged them on plastic dishes (2-D environment). After a few passages in culture, we have an enriched epithelial cell population  which no longer expresses IL-6 nor pStat3 ), plastic coated Matrigel (M), plastic (C), Collagen I (CI), Collagen V(CV), Fibronectin (F) and Laminin (LA) for 16 hours and were analyzed for pStat3 and Tubulin levels.Click here for fileSupplemental figure S2. Stat3 has no effect on acinar formation or cell growth in MCF10A cells.A. Extracts from MCF10A cells expressing control (C) or Stat3 shRNA (S3sh) were analyzed for levels of Stat3, tyrosine phosphorylated (pStat3) and Tubulin by Western blot analysis. B. MCF10A cells (Control) or MCF10A Stat3Sh (S3Sh) were grown on Matrigel and form hollowed structures which were stained for pStat3 (green) and Dapi (blue) by immunofluorescence. C. MCF10A cells expressing control (C) or Stat3 shRNA (S3sh) were plated in six-well dishes and cell numbers were determined daily for seven days. Each data point represents the mean value from triplicate wells.Click here for fileSupplemental figure S3. Jak inhibition does not alter the morphology of MCF10A-Ras cells in Matrigel. MCF10A-Ras cells were grown on Matrigel and structures were stained for Dapi by immunofluorescence treated with DMSO control (Ras-C) or P6 (a pan-Jak inhibitor) for one week.Click here for file


Lupus nephritis (LN) is the major determinant of outcome in pediatric Systemic Lupus Erythematosus (pSLE) and its treatment remains a challenge. Aim. To report the experience of our centre in treating with Rituximab (RTX) SLE patients with severe LN.2 RTX, 2-3 weeks apart. All patients were concurrently on corticosterois (CSs) and mycophenolate mofetil. Patients’ clinical and laboratory findings were recorded at RTX initiation, after each infusion and at 3.4±2.1 month-intervals thereafter. pSLE activity was assessed using the European Consensus Lupus Activity Measurement (ECLAM), while LN activity using 24-hour urine protein excretion and serum cystatin C.Four pSLE patients with, refractory to conventional immunosuppressive treatment, biopsy-proven LN received four doses of 375-500mg/mPatients were followed-up for 6-21 months (median: 16 months). Full Β cell depletion was noticed 2-4 weeks after RTX initiation and lasted 4-7 months. All patients achieved complete LN remission 3.5 months (range: 2-4) after RTX initiation which was retained in 3 patients through the follow-up period. One patient relapsed 15 months after RTX initiation and received one additional RTX dose. ECLAM scores and CSs doses were markedly reduced in all patients, while complement levels increased. No side effects or infections were observed.RTX is an alternative, safe and efficient treatment for refractory LN.


The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest  drug targets in an animal and identify 40 suppressors of clozapine-induced larval arrest, including the α-like nicotinic acetylcholine receptor (nAChR) acr-7. We validate our RNAi result by showing that an acr-7 knockout suppresses clozapine-induced larval arrest and inhibition of pharyngeal pumping. Expression of a full-length translational acr-7::GFP (Green Fluorescent Protein) construct in the acr-7 mutant rescues suppression of these phenotypes. Clozapine-induced phenotypes are phenocopied by nAChR agonists and blocked by nAChR antagonists. The results suggest that clozapine induces these phenotypes through activation of the ACR-7 receptor. Recent studies have underscored the potential importance of nAChRs in the pathophysiology of schizophrenia. A clearer understanding of APD mechanisms would facilitate the design of improved drugs and may inform our understanding, not only of drug mechanisms, but also of disease pathogenesis.Clozapine is the most effective medication for treatment-refractory schizophrenia but produces toxic side effects such as agranulocytosis, metabolic syndrome, and developmental defects after exposure early in life. However, clozapine's molecular mechanisms of action remain poorly understood. In past studies, we showed that pharmacogenomic experiments in  Treatment of psychotic disorders has been hampered by the limited efficacy of currently available APDs and the toxic side effects of these drugs Caenorhabditis elegans identify novel and important signal transduction pathways through which APDs exert their biological effects C. elegans provide an unbiased approach to discover genetic targets of APDs, and such experiments are not possible in knockout or transgenic mice. Using such an unbiased approach, we identified a potential APD target with homology to human brain receptors.The genetic tools of invertebrate models offer new paradigms for drug discovery in schizophrenia acr-7, which encodes a homolog of the human α-like nAChRs, suppressed both developmental and behavioral effects of clozapine. Our results indicated that clozapine likely activated the ACR-7 receptor, a novel observation that may be relevant to clinical drug effects. No known APDs have been previously demonstrated to activate nAChRs, and whether APDs might act by this mechanism is an important question. Recent studies in humans have underscored the potential importance of nAChRs in the pathophysiology of schizophrenia owing to: (1) the abnormal expression of nAChRs in post-mortem brains of schizophrenic patients; (2) the striking association between smoking (a nicotine delivery system) and schizophrenia; (3) the improved cognition seen in animals and humans with nicotine exposure; (4) the genetic association between the human α7-nAChR and schizophrenia; and (5) the potential efficacy of nAChR agonists as treatments for schizophrenia We found that loss-of-function mutations in C. elegans (scla genes). Mutants that escaped clozapine-induced larval arrest grew to adulthood and were easily detected under a dissecting microscope (rrf-3(pk1426) II strain, which is hypersensitive to RNAi and which improves detection of genes with postembryonic mutant phenotypes, to maximize recovery of neuronal genes To identify novel molecular mechanisms of action of APDs, we conducted a genetic suppressor screen for APD targets in an animal  elegans . APDs incroscope . The expmagi-1, a membrane-associated guanylate kinase gene, and sec-8 , an exocyst complex gene, were each identified as positives twice in the primary screen. Subsequent testing of primary screen positives in triplicate identified 40 candidate suppressors, constituting 0.2% of the total wells screened (acr-7(tm863), cep-1(ep347), cep-1(gk138), cep-1(lg12501), gtl-2(n2618), gtl-2(tm1463), ina-1(gm39), ins-22(ok3616), lron-6(ok1119), magi-1(gk657), puf-6(ok3044), scla-1(tm4534), scla-1(tm4806), and sms-1(ok2399). Suppression of clozapine-induced larval arrest was seen in 70% of the RNAi screen positives when tested by knockout rather than by RNAi knockdown, including acr-7, gtl-2, ina-1, magi-1, puf-6, sms-1, and scla-1. The cep-1, ins-22, and lron-6 knockout strains failed to suppress clozapine-induced larval arrest, suggesting that these suppressors were false positives. Alternatively, suppression may occur in these three cases with partial loss-of-function by RNAi knockdown, but not with complete loss-of-function by knockout.Of the 19,968 wells we tested in the primary screen, 1,375 wells or 6.9% displayed suppression of clozapine-induced larval arrest. screened . To ruleC. elegans. We asked whether genes recovered in our RNAi screen suppress the effects of stressors, including paraquat, DTT, SDS, and tunicamycin sms-1(ok2399), age-1(hx546), scla-1(tm4806), scla-1(tm4534), and acr-7(tm863) mutant strains. None of these mutant strains grew more rapidly in the presence of SDS or paraquat than N2 animals, suggesting that these mutations did not act by suppressing stressor effects . N2 animals grown in the presence of 0.1% DMSO alone reached adulthood in ∼3 days . Consistacr-7(tm863) allele is a 625 bp out-of-frame deletion with a 7 bp insertion, which removes exons 6–7 and most of exons 5 and 8 (acr-7(tm863) is predicted to be a null allele. We backcrossed the acr-7(tm863) allele to the wild-type strain six times to generate the strain EAB200, which, like animals exposed to acr-7(RNAi) (acr-7(tm863) mutant animals reached the L4 and young adult (YA) stages by day 3, compared to 0% of N2 animals (acr-7(tm863) partially suppressed clozapine-induced developmental delay, confirming the knockdown results from our RNAi screen. Transgenic strain acr-7(tm863) II mchEx40, which expresses a full-length translational Pacr-7::acr-7::GFP (Green Fluorescent Protein) fusion construct, was rescued for the Scla phenotype. On 320 µM clozapine, 100% of these transgenic animals were arrested at early larval stages  on day 3, comparable to the delay seen in N2 animals (acr-7(lf) suppression by testing mutations in 21 other C. elegans nAChR subunits and found that only acr-7(lf) suppressed clozapine-induced developmental delay (The  5 and 8  and lack 5 and 8 . Therefo-7(RNAi) , display animals . Therefo animals . We inveal delay .acr-7(tm863) mutation suppressed nicotine-induced developmental delay (acr-7(tm863) II mchEx40 animals . If clozapine does so, then mutations in genes required for Ach release should have suppressed clozapine-induced developmental delay. We tested two alleles of the choline acetyltransferase gene  release .Pacr-7::GFP transcriptional fusion construct or a Pacr-7::acr-7::GFP translational fusion construct. In both cases, GFP was strongly expressed in the pharyngeal muscles of transgenic animals  and acr-7(RNAi) loss-of-function mutations blocked activation by clozapine and thereby suppressed pharyngeal paralysis and larval arrest.What is the mechanism by which clozapine inhibited pharyngeal pumping? The P<0.0001, unpaired t-test). The pharyngeal pumping rate of acr-7(tm863) mutants was slightly decreased compared to wild type. The mean pumping rate of acr-7(tm863) mutants in the absence of drug was 103.1±1.2 pumps/20 sec . That the acr-7(tm863) mutant displays a relatively normal pumping rate argues against a model in which acr-7 is required for pumping and clozapine inhibits this function. However, the acr-7(tm863) deletion suppressed clozapine-induced inhibition of pharyngeal pumping. While the mean pumping rate at 500 µM clozapine was 16.8±2.5 pumps/20 sec for N2 animals, the rate for acr-7(tm863) mutants was 40.9±2.9 pumps/20 sec . Compared to acr-7(tm863) mutants, acr-7(tm863) mchEx40 animals were partially rescued for this suppression with a mean pumping rate of 29.3±2.9 pumps/20 sec at 500 µM clozapine .Clozapine inhibited pharyngeal pumping of wild-type animals in a concentration-dependent manner. On average the pumping rate in the absence of clozapine was 112.7±1.4 pumps/20 sec, whereas the pumping rate in the presence of clozapine was 59.0±4.0 pumps/20 sec (160 µM), 34.4±3.7 pumps/20 sec (320 µM), and 16.8±2.5 pumps/20 sec (500 µM)  mchEx207 animals, expressing the Pmyo-2::acr-7 transgene, were rescued for suppression of clozapine-induced inhibition of pharyngeal pumping with a mean pumping rate of 17.3±1.4 pumps/20 sec at 500 µM clozapine . acr-7(tm863) mchEx207 animals were also rescued for suppression of clozapine-induced developmental delay (acr-7(tm863) mchEx211 animals, expressing the Pmyo-2::acr-7::GFP transgene, displayed strong fluorescence in the pharyngeal muscles and were rescued for both the pharyngeal pumping and developmental phenotypes. Thus, acr-7 was required in pharyngeal muscle for clozapine-induced inhibition of pharyngeal pumping and for clozapine-induced developmental delay.In order to test whether ACR-7 acts specifically in pharyngeal muscle to affect pumping, we generated transgenic lines expressing al delay . acr-7 and 0.0 pumps/20 sec in 300 µM levamisole (data not shown). d-TC alone did not change the pumping rate in wild-type animals, similar to the relatively normal pharyngeal pumping rate seen with acr-7 knockout. However, d-TC did block inhibition of pharyngeal pumping by either clozapine or nicotine. The mean pumping rate of wild-type animals in 500 µM clozapine was 25.3±3.1 pumps/20 sec , compared to 69.5±4.9 pumps/20 sec with co-application of 500 µM d-TC . The mean pumping rate of wild-type animals in 2 mM nicotine was significantly increased to 68.5±3.7 pumps/20 sec with co-application of 500 µM d-TC, compared to wild-type animals in 2 mM nicotine alone . However, d-TC failed to block the residual inhibition of pharyngeal pumping produced by either clozapine or nicotine in acr-7(tm863) mutants, indicating that acr-7 knockout occludes the effect of d-TC  C. elegans for scla genes and identified 40 candidate suppressors of clozapine-induced larval arrest. Several suppressors, such as magi-1, lin-12, and ina-1, have human orthologs implicated in the pathogenesis of schizophrenia but have not been previously shown to mediate the biological effects of an APD Clozapine exposure during adulthood modulates several C. elegans. Several lines of evidence indicate that clozapine-induced larval arrest involves multiple genetic pathways. First, the number and diversity of suppressors to emerge from our screen suggest that they may not constitute a single pathway. Second, clozapine's developmental and behavioral effects in C. elegans are partially, not fully, suppressed by acr-7(lf) (C. elegans insulin/IGF receptor ortholog daf-2 and in its downstream effector the phosphatidyl inositol 3-kinase (PI3K) age-1 suppressed clozapine-induced larval arrest. Subsequent work confirmed the discovery that clozapine activates insulin signaling in C. elegans and showed that other APDs do so as well acr-7 and age-1 may act in concert to mediate APD effects in C. elegans, since the α7-nAChR has been shown to transduce signals to PI3K via direct physical association in rat cortical cultures The molecular mechanisms of action of human disease genes underlying psychotic disorders are largely unclear. The identification of candidate APD targets in a genetic model organism with a nervous system afforded us the opportunity to analyze the mechanism of action of one such target in detail. However, elucidating the mechanisms underlying APD modulation of ACR-7 cannot fully explain the effects of APDs on cr-7(lf) . Third, We would also like to know whether particular suppressors mediate clozapine-specific effects or mediate APD effects more generally. To address the question of specificity will require identifying the cellular and molecular mechanisms of suppression for particular mutants and then testing the relevance of those mechanisms for APDs other than clozapine. If the role of a suppressor is clozapine-specific, that target may help explain clozapine's unique therapeutic or toxic effects. If the role is not clozapine-specific, that target may help identify novel mechanisms of action of APDs in general. Importantly then, further characterization of suppressors from our screen may advance the field whether suppression is a clozapine-specific effect or is a characteristic of APDs in general.cha-1(lf) and unc-17(lf) alleles failed to suppress clozapine-induced larval arrest, we hypothesized that clozapine does not stimulate Ach release, but rather that clozapine modulates ACR-7 directly. Particularly in whole-organism studies, direct effects can be pharmacologically complex, involving activation, inhibition, or a combination of the two. For instance, clozapine acts as an agonist at the human muscarinic M1 receptor, while its biologically active metabolite, N-desmethylclozapine, acts as an antagonist C. elegans.Although some RNAi suppressors may act indirectly, some may have human homologs that directly mediate either therapeutic or toxic effects of clozapine. Based on the fact that acr-7 mutants suppressed clozapine-induced developmental delay and that a full-length translational Pacr-7::acr-7::GFP fusion construct rescued this effect. acr-7 was strongly expressed in the C. elegans pharynx, and acr-7 knockout suppressed clozapine-induced inhibition of pharyngeal pumping. Furthermore, a translational Pmyo-2::acr-7 fusion construct rescued suppression of the developmental delay and pharyngeal pumping phenotypes, indicating that ACR-7 acts specifically in pharyngeal muscle cells to mediate these phenotypes. These results suggested that clozapine activates ACR-7 to inhibit pharyngeal pumping and, thereby, produce larval arrest. Consistent with this model, nAChR agonists phenocopied both the developmental and behavioral effects of clozapine. Similarly, nicotine-induced developmental delay required acr-7. However, acr-7(tm863) did not block nicotine-induced inhibition of pharyngeal pumping, suggesting that nicotine may act through multiple nAChRs to inhibit pharyngeal pumping  II mutation in strain TJ1052 had been backcrossed to the N2 strain three times, and we performed another three backcrosses to generate strain EAB100. The presence of the mutation was followed by restriction digest and confirmed by DNA sequencing. We backcrossed the following mutations to the N2 strain six times to generate strains: EAB1 sms-1(ok2399) IV, EAB101 scla-1(tm4806) IV, EAB102 scla-1(tm4534) IV, EAB103 ins-22(ok3616) III, EAB200 acr-7(tm863) II, and EAB201 acr-16(ok789) V from strains RB1854, F22B3.8(tm4806), F22B3.8(tm4534), RB2594, FX863, and RB918, respectively. The presence of the deletions was followed by PCR.We used the rrf-3 animals. These animals were maintained in liquid culture at 20°C on a rotating platform for three days until they reached young adulthood. We then added 1.5 µl of clozapine stock solution (20 mg/ml clozapine powder from Sigma-Aldrich #C6305 in 100% ethanol) to each well for a final concentration of 200 µg/ml (∼600 µM) clozapine. Progeny were scored for suppression of clozapine-induced larval arrest on day 8 (dpy-6 RNAi in the absence of clozapine and blank wells in the presence of clozapine as controls.We employed an RNAi feeding library (Geneservice Ltd), constructed by J. Ahringer's laboratory at the The Wellcome CRC Institute, University of Cambridge, Cambridge, England, to carry out a genome-wide screen in liquid culture for suppressors of clozapine-induced larval arrest on day 8 . Wells con day 8 . Those wdpy-6 RNAi and unc-44 RNAi with and without clozapine as controls. Those wells lacking progeny, lacking a lawn, or containing contamination were retested. Mutants that produced suppression in at least 2/3 wells were taken to be positives, and the strength of suppression was scored: weak (+) - few progeny escaped larval arrest, medium (++) - ∼25% of progeny escaped larval arrest, robust (+++) - most progeny escaped larval arrest, NA - not scored . Each set included triplicate wells of t scored . The idePacr-7::GFP transcriptional fusion construct, we PCR-amplified the putative promoter region, including 1.3-kb upstream of the acr-7 gene, using DNA prepared from adult animals and using primers listed in Pacr-7::acr-7::GFP translational fusion construct, we PCR-amplified the same acr-7 promoter sequence along with 4.3-kb of acr-7 genomic sequence, omitting the native stop codon, using DNA prepared from adult animals and using primers listed in C. elegans Vector Kit (Addgene).To generate a Pmyo-2::acr-7 and Pmyo-2::acr-7::GFP translational fusion constructs, RNA was isolated from mixed-stage worms, and acr-7 cDNA was obtained using SuperScript III First-Strand Synthesis System (Invitrogen) with primers listed in Pmyo-2 constructs were injected into EAB200 animals using the same protocol described below for injection of the Pacr-7::acr-7::GFP construct.To generate the All plasmid constructions were confirmed by sequencing (MGH DNA Sequencing Core).Pacr-7::GFP construct was injected at 25 ng/µl along with the rol-6(su1006) marker (pRF4) at 50 ng/µl into N2 animals. The Pacr-7::acr-7::GFP construct was injected at 25 ng/µl along with the Pmyo-3::DsRed2 marker  at 40 ng/µl into EAB200 animals. Three to four independent extrachromosomal lines were generated for both the Pacr-7::GFP and Pacr-7::acr-7::GFP constructs. ACR-7::GFP expression shown here was studied in the EAB40 strain, but similar results were obtained in studies of the EAB20 - 24, EAB39, and EAB41 strains. Animals were placed on a 2% agarose pad on a slide in M9 with 50 mM sodium azide, then covered with a slip and studied by DIC and fluorescence microscopy using a Zeiss Axio Image A1 microscope. Photographs were taken using a 40× objective on a confocal laser microscope .We generated extrachromosomal transgenic strains by microinjecting DNA into the gonads of young adult animals as described Experiments were performed using Falcon 12-well plates (Becton Dickinson) containing OP50 bacteria on NGM. Clozapine, nicotine, levamisole, DHβE, and d-TC were purchased from Sigma-Aldrich. Olanzapine was purchased from Waterstone Technology. Stock solutions of each drug were prepared in DMSO and then diluted 1∶1000 in water with acetic acid . These diluted solutions were then pipetted onto the agar surrounding the bacterial lawn for final drug concentrations of 40, 80, 160, or 320 µM (0.1% DMSO), and the plates were left to dry overnight. On the next day, ∼30 synchronized L1 larvae were loaded into each well. Animals were then scored as L1, L2, L3, L4, young adult, or gravid adult at 24, 48, 72, 96 hours.For stressor experiments, paraquat dichloride, tunicamycin, and DL-Dithiothreitol (DTT) were purchased from Sigma-Aldrich, and sodium dodecyl sulfate (SDS) was purchased from Fisher Scientific. Stock solutions of 10% SDS, 2 M paraquat, or 0.2 M DTT in water or 2.5 mg/ml tunicamycin in DMSO were diluted in M9 and pipetted onto the agar to achieve a range of drug concentrations. On the next day, 5–10 adult animals were placed in each well of a 12-well plate. Adults were allowed to lay 25–50 eggs and then removed. Plates were scored for developmental delay and lethality each day until the next generation hatched or until all animals died.Drug plates were prepared as described above. Synchronized L1 larvae were grown to the L4 stage and picked 24 hours in advance of the experiments. Five adult animals were transferred into drug wells or control wells . Two hours later, the number of pumps during 20 seconds was scored. Only animals on the bacterial lawn were scored.t-test and one-way ANOVA test where appropriate.Data were expressed as means ± S.E.M. Statistical comparisons were performed using the unpaired, two-tailed Student's Figure S1RNAi screen mutants did not suppress stressor-induced developmental delay. (A) Developmental delay seen with SDS or paraquat after 24 hours. (B) Developmental delay seen with SDS or paraquat after 48 hours. For control conditions, vehicle alone was applied to the agar. Paraquat at 0.5 mM produced significant lethality at 48 hours (data not shown).(PDF)Click here for additional data file.Table S1F22B3.8(tm4806) IV and F22B3.8(tm4534) IV. All other strains used in this work, except strains EAB1 through EAB211, were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). NA, not available.List of mutant strains used in these studies. Dr. L. Chen  provided LH202. Dr. C. Hunter  provided NL4256. Dr. Y. Jin  provided CZ9957. Dr. S. Mitani (NBP-Japan) provided (PDF)Click here for additional data file.Table S2List of primers used for plasmid constructions.(PDF)Click here for additional data file.


MYCN amplification occurs in about 20–25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas. Although children affected with NB might undergo spontaneous or therapy-induced regression, less than 50% of the high-risk patients experience long-term survival, despite intense multimodal treatment. Together with clinical and pathological features  MYCN amplification (MNA) contributes to the identification of high-risk patients Somehow paradoxically, MYCN overexpression might also lead to apoptosis and/or sensitize cells to death p53 is a master oncosuppressor protein protecting cells from genetic instability and tumor development by inducing cell cycle arrest or apoptosis in response to cellular stress and DNA damage Searching for candidates that could be involved in the apoptosis-sensitive phenotype induced by MYCN, we focused our attention on Galectin-3 . Gal-3 is a ß-galactoside-binding lectin involved in a variety of biological processes including pre-mRNA processing, cell cycle progression, cell adhesion, angiogenesis and apoptosis Here we report that MYCN represses Gal-3 as a part of its apoptosis-sensitive phenotype. In MNA cells, however, along with Gal-3 reduced levels of expression, we uncovered a distinct subcellular localization pattern compatible with the more aggressive behavior of these tumors. Finally, we show that MDM2-p53 antagonists, such as Nut-3, repress Gal-3 and this is required for their full activity on MNA NB tumors.www.iclc.it); SK-N-BE and IMR32 from European Collection of Cell Cultures, ; LAN5 from Deutsche Sammlung Von Mikroorganismen und Zellkulturen . LAN1 cells Human NB cells were obtained as follows. GICAN, ACN, GIMEN, GILIN and IMR5 were acquired from Banca Biologica and Cell Factory , cells were fixed and permeabilized as above, blocked in 5% BSA, 3% goat serum in PBS for 1 hour and incubated ON with the primary antibody (Ab), followed by secondary Ab incubation, counterstained and mounted as above. For all three apoptosis assays, at least 200 cells/sample were counted in duplicate experiments. Each experiment was performed at least three times. Statistical analysis was performed by a standard two-tailed Student’s t test.Subconfluent cells were treated with either bleomycin, Casein Kinase 1 (CK1) inhibitor D4476 , adriamycin, cis-platin, Nut-3  for 24 hours . Cell death was measured by tripan blue exclusion test. For the analysis of nuclear morphology, cells were fixed in 4% formaldehyde/PBS for 10 minutes, permeabilized in 0.25% Triton/PBS for 10 minutes, counterstained with 1 mg/ml Hoechst 33258, and mounted in PBS/50% glycerol. For the immunofluorescent analysis of the p85 cleaved fragment of the PARP protein , cells were treated according to the manufacturer’s instructions.PARP polyclonal Ab , anti-Galectin-3 purified MoAb , MoAb anti-myc 9E10 .Primary Abs: anti-p85Secondary Abs: Alexafluor 488 Goat anti-mouse igG (H+L), Alexafluor 594 Goat anti-rabbit IgG (H+L) , Cy3 conjugated Affini Pure Donkey anti-mouse IgG (H+L) .Total RNA extraction was carried out with TRIzol reagent (Invitrogen). For quantitative reverse transcription-PCR (Q-RT-PCR), total RNA (1 µg) was reverse transcribed using Gene Amp kit  and subjected to PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems) using an ABI Prism 7900 sequence detector (Applied Biosystems) as described 5′-AGCAATGCCTCCTGCACCACCAAC-3′hGAPDH forward, 5′-CCGGAGGGGCCATCCACAGTCT-3′hGAPDH reverse, 5′-TCCACTTTAACCCACGCTTC-3′hGalectin-3 forward, 5′- TCTTCCCTTCCCCAGTTATT-3′hGalectin-3 reverse, All amplification reactions were performed at least in duplicate and averages of threshold cycles were used to interpolate standard curves and calculate transcript amount using the SDS version 2.3 software (Applied Biosystems).g for 15 min at 4°C, the pellets were washed in 5 volumes of lysis buffer containing 250 mM sucrose and further centrifuged at 10,000 g for 15 min at 4°C to reduce cytosolic protein contaminations.Total protein extracts were obtained in RIPA buffer . Fractionation of nuclei and cytoplasm from cultured cells was performed as described PARP (Promega Corporation); mouse anti-p53 (DO-I), mouse anti-MYCN and mouse anti-β-tubulin MoAbs and goat anti-β-actin, and rabbit polyclonal anti-p38 (C20) Ab (Santa Cruz Biotechnology); mouse anti-c-Myc MoAb (Sigma Aldrich); rat anti-Galectin-3 purified monoclonal antibody (Space Import & Export); rabbit anti-HIPK2 polyclonal Ab (kindly provided by Prof. M.L. Schmitz), mouse monoclonal anti-cytochrome c Ab (Pharmingen). Immunoreactive bands were visualized by enhanced chemoluminescence (Perkin Elmer).Total protein extracts (30 µg/sample) and subcellular fractions were separated by SDS-PAGE and blotted onto nitrocellulose membrane . Membranes were blocked with 5% nonfat dry milk and incubated with primary Abs at the appropriate dilutions. Abs were as follows: polyclonal Ab anti-p85The sh-RNA interference pSUPER-HIPK2, the pEGFP-HIPK2 and the pcDNA3.1-galectin-3 vectors were previously described Gal-3 and MYCN knock down was obtained with siRNA interfering oligos  transfected by Dharmafect 2 Reagent  according to the manufacturer’s protocol. siRNA sequences were as follows:Gal-3 sense, 5′-CAGAAUUGCUUUAGAUUUC-3′;Gal-3 antisense 5′-GAAAUCUAAAGCAAUUCUG-3′;Gal-3 no-target control sense, 5′-GCUUCAUUUAAGGUCAAUU -3′;Gal-3 no-target control antisense, 5′-AAUUGACCUUAAAUGAAGC -3′.MYCN sense, 5′-GUAUUAGACUGGAAGUUCA-3′;MYCN antisense 5′-UGAACUUCCAGUCUAAUAC-3′);Control no-target for MYCN was MISSION siRNA Universal Negative Control .Tumor samples from primary lesions were obtained from 25 children with previously untreated neuroblastoma admitted at the Department of Pediatrics, La Sapienza University. Institutional written informed consent was obtained from the patient’s parents or legal guardians and the study underwent review and approval by the Ethics Committee of “Policlinico Umberto I”. Each sample was characterized for MYCN amplification by Southern blot as previously described MYCN sensitizes neuroblastoma cells to apoptosis The analysis of a panel of NB cell lines and tumor samples revealed that Gal-3 mRNA is expressed at easily detectable levels in most samples. However its average expression was substantially lower in MNA compared to MNSC tumor samples and cell lines . ConsistTo test whether Gal-3 repression by MYCN is involved in sensitization to apoptosis we overexpressed or depleted Gal-3 in MYCN expressing SHEP Tet21/N cells or in MYCN-repressed cells, respectively. Interestingly, increased Gal-3 expression protected MYCN expressing cells from bleomycin-induced apoptosis  and Gal-6Gal-3 is a multitalented protein whose activity is strictly linked to its subcellular localization  and primary human tumors (B) by Q-RT-PCR. Asterisks indicate MNA tumor samples.(PDF)Click here for additional data file.Figure S2Gal-3 overexpression in LAN5 MNA cells. Three distinct LAN5 cell clones stably expressing different levels of Gal-3 and three control clones (immunoblot in the upper panel) were chosen to illustrate the effects of Gal-3 overexpression on bleomycin-induced apoptosis shown as percentage of apoptotic nuclei and/or p85PARP positive cells (raw data of experiments shown in (PDF)Click here for additional data file.Figure S3Gal-3 intracellular localization in NB cells. Immunofluorescent analysis showing Gal-3 localization in MNSC (GICAN) and MNA  NB cell lines (A) and in LAN5 stable clone number 6 (C). B, Impairment of Gal-3 nuclear export by inhibition of CK1 with the CK1-inhibitor D4476  sensitizes SHEP cells to bleomycin induced cell death as measured by Tripan blue-exclusion test. Significant differences in cell death fold induction were obtained between bleomycin+CK1i treated samples versus bleomycin-only treated samples (***p<0.0001).(PDF)Click here for additional data file.Figure S4Gal-3 overexpression protects MNA cells from apoptosis. A, The effect of Gal-3 overexpression on apoptosis induced by ADR (0.1 µM), Nut-3 (2 or 10 µM) or combination of Nut-3 (2 µM) with ADR in IMR32 cells is shown as percentage of apoptotic nuclei and p85PARP positive cells. B, Effect of Gal-3 overexpression on apoptosis induced by ADR and Nut-3 in LAN5 stable clones shown as percentage of apoptotic nuclei and p85PARP positive cells.(PDF)Click here for additional data file.


Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H Per) and cryptochrome (Cry), whose protein products accumulate in the cytoplasm where they associate with each other and ultimately translocate to the nucleus. Once in the nucleus, PER and CRY inhibit BMAL1/ CLOCK activity, repressing their own transcription, thus forming the negative limb of the circadian oscillator ,-52,Per2, pathway ,54 likel pathway . Interessurvival . Furthersurvival . Up-regued genes  may alsoCGs, Dbp  was downnduction  59]. Up. UpPer2,nduction . Our datreported . Thus, iectively . Circadig, Mafk) ,64, a tr . A graph with error bar (±SD) showing normalized  and averaged SOD1/2 contents (n = 4) at 8 h post cOS-pulse demonstrates significant differences between (-), WT and S90A: ⋆⋆ (P< 0.01), ⋆⋆⋆ (P< 0.001).(PDF)Click here for additional data file.Table S1Expression profiles of genes belonging to the relevant annotation clusters.Microarray analysis of gene expression in NIH-3T3:Per2-Luc with/without cOS-pulse was performed. AC, ID, name and value of the genes for the heatmap  are shown. Circadian fluctuated genes are highlighted blue (higher expression 32 h post cOS-pulse) or red (higher expression 20 h post cOS-pulse).(PDF)Click here for additional data file.Table S2Expression profiles of genes in cOS-evoked signaling.Microarray analysis of gene expression in NIH-3T3:Per2-Luc with/without cOS-pulse was performed. Gene name and value for the heatmap  are shown. Acutely regulated and circadian fluctuated genes are highlighted with blue (higher expression in the control or at 32 h post cOS-pulse) or red (higher expression at 4 h or 20 h post cOS-pulse).(PDF)Click here for additional data file.Movie S1Per2 rhythms in living single cells.A critical oxidative stress (cOS) for cell survival synchronizes circadian  U2OS:Per2-Luc were cOS-pulsed after 2 days of pre- treatment (S1). Acute surges and circadian rhythms of Per2-Luc (pseudo-color) show synchronization of circadian Per2 rhythms in each cell by cOS-pulse (S2), as monitored via time-lapse bioluminescence imaging analyzed using LV200 . Note that only small parts of cells express Per2-Luc in the confluent cell culture. (MP4)Click here for additional data file.Movie S2Per2 rhythms in living single cells.A critical oxidative stress (cOS) for cell survival synchronizes circadian  U2OS:Per2-Luc were cOS-pulsed after 2 days of pre- treatment (S1). Acute surges and circadian rhythms of Per2-Luc (pseudo-color) show synchronization of circadian Per2 rhythms in each cell by cOS-pulse (S2), as monitored via time-lapse bioluminescence imaging analyzed using LV200 . Note that only small parts of cells express Per2-Luc in the confluent cell culture. (MP4)Click here for additional data file.Movie S3−/− MEFs harboring BMAL1-WT and BMAL1-S90A.Live imaging of Caspase-3/7 active cells after cOS-pulse in BMAL1 The movies show caspase-3/7active cells after cOS-pulse, and BMAL1−/− MEFs harboring BMAL1-WT (S3) and BMAL1-S90A (S4) as monitored via time-lapse imaging fluorescence with CellEvent™ Caspase-3/7 Green (Molecular probes) analyzed using LV200 (Olympus).(MP4)Click here for additional data file.Movie S4−/− MEFs harboring BMAL1-WT and BMAL1-S90A.Live imaging of Caspase-3/7 active cells after cOS-pulse in BMAL1 The movies show caspase-3/7active cells after cOS-pulse, and BMAL1−/− MEFs harboring BMAL1-WT (S3) and BMAL1-S90A (S4) as monitored via time-lapse imaging fluorescence with CellEvent™ Caspase-3/7 Green (Molecular probes) analyzed using LV200 (Olympus).(MP4)Click here for additional data file.


The CdII atom is coordinated by two N atoms from two cis-positioned bridging 1,2-bis(imidazol-1-yl)ethane (bime) ligands and four N atoms from four azide anions. Each azide ligand acts in an end-on bridging coordination mode. The azide ligands and CdII atoms form a one-dimensional zigzag chain constructed from four-membered [Cd(N3)2]n metallacycles extending along the a axis. These inorganic chains are connected with four other chains via bridging bime ligands to form a three-dimensional coordination network.In the title three-dimensional coordination polymer, [Cd(N DOI: 10.1107/S1600536812004370/gk2436Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


IL-6-/- mice with KrasG12D mutant mice, which develop lung tumors after activation of mutant KrasG12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. KrasG12D; IL-6-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than KrasG12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated KrasG12D; p53flox/flox; IL-6-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than KrasG12D; p53flox/flox mice. Tumors from KrasG12D; IL-6-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than KrasG12D mice; however, these changes were not present between tumors from KrasG12D; p53flox/flox; IL-6-/- and KrasG12D; p53flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in KrasG12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed  Accumulating evidence indicates that inflammation contributes to tumorigenesis, tumor progression, and metastasis ,2. Oncogin vivo [IL-6 knockdown, genetic ablation, or treatment with a neutralizing IL-6 antibody inhibits tumor growth in vivo [IL-6 expression can be detected in lung tumors  and in 5in vivo ,26,32. T in vivo ,33. Conv in vivo ,35, whil in vivo . An IL-6 monoclonal antibody therapy would be predicted to inhibit the inflammatory microenvironment in lung cancer. One such therapy, ALD518, has undergone preclinical and Phase I and II clinical trials. It appears to be well tolerated and ameliorates NSCLC-related anemia and cachexia , althougin vivo, we crossed IL-6-/- mice with mutant KrasG12D mice because IL-6 is a downstream effector of oncogenic Ras to promote tumorigenesis[p53 are found in at least 50% of NSCLC cases [KrasG12D activation accompanied by p53 deletion can cause lung tumor metastasis [KrasG12D; p53flox/flox; IL-6-/- mice..To assess the contribution of IL-6 signaling inhibition on tumor progression and survival time rigenesis. NSCLC irigenesis. InactivLC cases , and Kratastasis . To studIL-6-/- mice were purchased from The Jackson Laboratory and maintained in sterile housing [Lox–Stop–Lox KrasG12D (hereafter referred to as KrasG12D) mice [p53flox/flox mice [KrasG12D and KrasG12D; lL-6-/- mice were inoculated with 5 × 106 PFU of adenoviral Cre (adeno-Cre) by intranasal inhalation to activate oncogenic KrasG12D in the lungs. KrasG12D; p53flox/flox and KrasG12D; p53flox/flox;IL-6-/- mice were inoculated with 5 × 105 PFU of adeno-Cre. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee at Dana-Farber Cancer Institute (permit number 04-094). All surgeries were performed under Avertin anesthesia to minimize suffering. After euthanasia, organs, including heart, liver, spleen, kidney, stomach, intestine, spine, brain, breast, skin, and testis or ovary, were undergone gross inspection for metastases. Lung tumors adhered to the pleura were considered parietal pleural metastases. Suspected metastases were harvested and confirmed by histological features. housing . Conditi2D) mice  and p53lox mice  were desAfter euthanasia, the lungs were removed and fixed in 10% neutral buffered formalin overnight before embedding in paraffin. Five-micrometer sections of mouse lung tissues were cut. Some sections were stained with H&E. For immunohistochemistry, heat treatment with citrate solution  in a decloaking chamber  unmasked antigens for phosphorylated ERK (pERK), BrdU, Ki67, Endomucin and Caspase-3 staining. Whole lung tissue sections were incubated overnight at 4°C with primary antibodies: pERK  at 1:100; BrdU  at 1:200; Ki67  at 1:200; Endomucin  at 1:100; cleaved Caspase-3  at 1:300. Digest-All 2B Trypsin (Invitrogen) was used to retrieve the antigen for MAC2  staining at 1:5000. At 400X magnification, all MAC2-positive macrophages in tumors were counted within 3 microscope fields with the most MAC2-positive macrophages after review of the whole lung section. Three mice per genotype were analyzed.At 20 weeks post-infection, mice were injected intraperitoneally with 10 μL of 10 mM BrdU in PBS per gram of body weight and euthanized after 2 hours. Whole lungs were harvested and processed as described above. At 400X magnification, all BrdU-positive tumor cell nuclei were counted within 3 microscope fields with the most BrdU-positive nuclei after review of the whole lung section. Four mice per genotype were analyzed. Same method was used to calculate Ki67-labeled tumor cells on sections from mice 28 weeks post-infection with adeno-Cre.KrasG12D and KrasG12D; lL-6-/- mice 32 weeks post-infection and from KrasG12D; p53flox/flox and KrasG12D; p53flox/flox; IL-6-/- mice 15 weeks post-infection for Western blot analysis. Tumors were lysed with a homogenizer in RIPA buffer  containing complete mini protease inhibitors (Roche) and phosphatase inhibitors . Nuclear and Cytoplasmic Extraction Kit  was used to extract cytoplasmic (C) and nuclear (N) fractions from tumors. Lysates (20 μg per lane) were separated on 10% polyacrylamide gels, transferred to PVDF filters, and incubated overnight at 4°C with antibodies to β-actin , pERK , total-ERK , pAKT , total-AKT , pSTAT3 , STAT3 , p65 , PARP , GAPDH , or β-catenin . Western blots were exposed to X-ray films or scanned with an ImageQuant LAS 4000mini . Lung tumors were harvested from KrasG12D and KrasG12D; lL-6-/- mice 32 weeks post-infection and KrasG12D; p53flox/flox and KrasG12D; p53flox/flox; IL-6-/- mice around 16 weeks post-infection for analysis. 2μg total RNA was reverse transcribed to cDNA using SuperRT cDNA synthesis kit . Real-time PCR was performed using the BioRad iQ5 Realtime PCR system and StepOnePlus Realtime PCR system (ABI) with Realtime PCR Master Mix containing SYBR Green  and unique primers  (n = 3), both KrasG12D and KrasG12D; IL-6-/- mice had early lung lesions. At 4 weeks post-infection, KrasG12D; IL-6-/- mice had more early lung lesions than KrasG12D mice  . Mice we12D mice . These lKrasG12D; IL-6-/- mice were modestly smaller and less dense than those in KrasG12D mice    despite nfection . BrdU stnfection , and pERnfection ; no statKrasG12D; p53flox/flox; IL-6-/- mice and 19 KrasG12D; p53flox/flox mice were analyzed for metastases around 16 weeks post-infection with adeno-Cre  and 10 of 19 KrasG12D; p53flox/flox mice (52.6%), although this difference was not significant (P=0.19). Metastatic lesions to the parietal pleura, thymus  . Oncogenr groups . KrasG12D; IL-6-/- tumors , a C-C motif chemokine, is overexpressed in pancreatic carcinoma cells and stimulates growth of tumor cells . CCL-19 nfection , but incnfection . These dp53 deletion more dramatically affected KrasG12D-induced lung cancer than IL-6 deletion. To a large extent, p53 deletion attenuated the effects of IL-6 deletion on delayed tumor growth and prolonged survival. p53 deletion enhanced pSTAT3 expression   . These rIL-6 deficiency promotes lung tumorigenesis, but suppresses tumor progression and elongates survival in vivo. However, these effects can be attenuated by p53 deletion.The involvement of inflammation in tumorigenesis, progression, and metastasis is widely accepted; however, whether IL-6-targeted therapies will prolong the survival time of lung cancer patients remains uncertain. Our results indicate anti-IL-6 therapies may have some success in clinical trials. For example, when NSCLC has not metastasized, IL-6 inhibition may prolong survival but increase the risk of further tumorigenesis; if metastasized, IL-6 inhibition may only moderately impact metastasis but may lengthen survival time. Further studies are needed to elucidate these possibilities. In summary, our results provide evidence that Figure S1Kras allelic recombination.PCR analysis of  A 500 bp PCR product represents the floxed, unrecombined KrasG12D allele; a 622 bp fragment represents the wildtype Kras allele; and a 650 bp fragment represents a recombined KrasG12D allele after removal of floxed stop cassette by adeno-Cre. K, KI, KP, and KPI mice were treated with adeno-Cre and the 650 bp recombined band revealed. Abbreviations: WT= wildtype lungs. Floxed=floxed KrasG12D, without adeno-Cre treatment. K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIF)Click here for additional data file.Figure S2IL-6 deletion attenuates tumor proliferation determined by Ki67 staining.(A and B) Representative images of Ki67-stained lung tissue sections from (A) K and (B) KI mice 28 weeks post-infection with adeno-Cre. (C) Quantification of Ki67-positive tumor cells in lung tissue sections of K and KI mice (n=3). **P<0.01. Scale bar indicates 50 μm. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. (TIF)Click here for additional data file.Figure S3p53 allelic recombination.PCR analysis of  A 212 bp PCR product represents the floxed, unrecombined p53 allele; a 168 bp fragment represents the recombined allele after inoculation with adeno-Cre; and a 130 bp fragment represents the wildtype p53 allele. K, KI, KP, and KPI mice were treated with adeno-Cre. The 168 bp recombined band was showed in KP and KPI mice and 212 bp fragment remained due to tumor stromal cells. Abbreviations: WT=wildtype lungs. Floxed=floxed p53, without adeno-Cre treatment. K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIFF)Click here for additional data file.Figure S4KP and KPI mice develop metastatic lesions.(A and B) Representative images of metastatic lesions to the (A) pleura, (B) thymus in KPI mice 15 weeks post-infection with adeno-Cre. Dotted lines in the images indicate metastatic tumor edges. Asterisks indicate center of metastatic tumors. (C) Representative image of heart metastases in KP mice 14 weeks post-infection with adeno-Cre. Metastatic lesions in the heart are left of the dotted line. Scale bar indicates 200 μm (A) or 100 μm (B and C). Abbreviations: KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIF)Click here for additional data file.Figure S5Real-time PCR screen of changes in inflammatory cytokines levels.. P<0.05 vs. K tumors. # P<0.05 vs. KP tumors. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.Three tumors from each genotype were analyzed by real-time PCR without replicate for expression of the indicated cytokine. Gene expression was normalized to β-actin mRNA. *(TIF)Click here for additional data file.Figure S6Nuclear localization of p65 and β-catenin are unchanged.Tumors from each mouse genotype were lysed to obtain cytoplasmic (C) and nuclear (N) fractions. Lysates were analyzed for the presence of nuclear p65 and β-catenin by Western blot. Fraction purity was determined by GAPDH (cytoplasmic) and PARP (nuclear) blots. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIFF)Click here for additional data file.Table S1Primers for real-time PCR analysis of gene expression.(DOCX)Click here for additional data file.